key: cord-020097-eh5deunk authors: nan title: cumulative author index for 2006 (volumes 115–122) date: 2006-10-27 journal: virus res doi: 10.1016/s0168-1702(06)00318-2 sha: doc_id: 20097 cord_uid: eh5deunk nan domingues, h.g., see spilki, f.r. (116) distribution and heterogeneity of small ruminant lentivirus envelope subtypes in naturally infected french sheep p1-and vpg-transgenic plants show similar resistance to potato virus a and may compromise long distance movement of the virus in plant sections expressing rna silencing-based resistance identification of novel foot-and-mouth disease virus specific t-cell epitopes in c/c and d/d haplotype miniature swine modulation of pkr activity in cells infected by bovine viral diarrhea virus genetic analysis of the function of the plum pox virus ci rna helicase in virus movement nidovirales: evolving the largest rna virus genome characterization of epstein-barr virus type i variants based on linked polymorphism among ebna3a, -3b, and -3c genes phloem specific promoter from a satellite associated with a dna virus various 30 and 69 bp deletion variants of the epstein-barr virus lmp1 may arise by homologous recombination in nasopharyngeal carcinoma of tunisian patients complete genome analysis of rflp 184 isolates of porcine reproductive and respiratory syndrome virus hepatitis c virus e2 links soluble human cd81 and sr-b1 protein specific binding of heat shock protein 70 with hn-protein inhibits the hn-protein assembly in sendai virus-infected vero cells protecting crops from non-persistently aphid-transmitted viruses: a review on the use of barrier plants as a management tool ppv long-distance movement is occasionally permitted in resistant apricot hosts evolutionary genomics of nucleo-cytoplasmic large dna viruses heat shock enhances the susceptibility of bhk cells to rotavirus infection through the facilitation of entry and post-entry virus replication steps adenoviral vectors-how to use them in cancer gene therapy inhibition of anatid herpes virus-1 replication by small interfering rnas in cell culture system prevalence of mutations in hepatitis c virus core protein associated with alteration of nf-b activation molecular epidemiological study of arctic rabies virus isolates from greenland and comparison with isolates from throughout the arctic and baltic regions asian prunus viruses: new related members of the family flexiviridae in prunus germplasm of asian origin the impact of the use of col-1492, a nonoxynol-9 vaginal gel, on the presence of cervical human papillomavirus in female sex workers microglial cells initiate vigorous yet non-protective immune responses during hsv-1 brain infection evolution of orf5 of spanish porcine reproductive and respiratory syndrome virus strains from identification of an interferon antagonist protein encoded by segment 7 of infectious salmon anaemia virus topics in herpesvirus genomics and evolution comparative analysis of genome sequences of three isolates of orf virus reveals unexpected sequence variation activities of membrane bound phosphatases, transaminases and mitochondrial enzymes in white spot syndrome virus infected tissues of fenneropenaeus indicus white spot syndrome virus infection decreases the activity of antioxidant enzymes in fenneropenaeus indicus phosphorylation and dephosphorylation events that regulate viral mrna translation identification of two amino acid residues on ebola virus glycoprotein 1 critical for cell entry sindbis virus infection of two model insect cell systems-a comparative study the amino-terminal residue of glycoprotein b is critical for neutralization of bovine herpesvirus an endornavirus from a hypovirulent strain of the violet root rot fungus importance of the extracellular and cytoplasmic/transmembrane domains of the haemagglutinin protein of rinderpest virus for recovery of viable virus from cdna copies envelope gene capture and insect retrovirus evolution: the relationship between errantivirus and baculovirus envelope proteins multiclonal pattern of jaagsiekte sheep retrovirus integration sites in ovine pulmonary adenocarcinoma venezuelan equine encephalitis virus complex-specific monoclonal antibody provides broad protection, in murine models, against airborne challenge with viruses from serogroups i, ii and iii patterns of sequence evolution at epitopes for host antibodies and cytotoxic t-lymphocytes in human immunodeficiency virus type phylogenetic analysis of the gag region encoding the matrix protein of small ruminant lentiviruses: comparative analysis and molecular epidemiological applications gene expression array analyses predict increased proto-oncogene expression in mmtv induced mammary tumors evolutionary genomics of archaeal viruses: unique viral genomes in the third domain of life hiv-l and the microrna-guided silencing pathway: an intricate and multifaceted encounter a modified viral satellite dna-based gene silencing vector is effective in association with heterologous begomoviruses tatabinding protein and tbp-associated factors during herpes simplex virus type 1 infection: localization at viral dna replication sites immunohistochemical examination of the role of fas ligand and lymphocytes in the pathogenesis of human liver yellow fever complete sequence and organization of the human adenovirus serotype 46 genome molecular characterization and phylogenetic study of maedi visna and caprine arthritis encephalitis viral sequences in sheep lymphocytopathogenic activity in vitro correlates with high virulence in vivo for bvdv type 2 strains: criteria for a third biotype of bvdv inactivation of white spot syndrome virus (wssv) by normal rabbit serum: implications for the role of the envelope protein vp28 in wssv infection of shrimp low prevalence of primary antiretroviral resistance mutations and predominance of hiv-1 clade c at polymerase gene in newly diagnosed individuals from south brazil characterization of a highly virulent feline calicivirus and attenuation of this virus interaction of the hepatitis b virus protein hbx with the human transcription regulatory protein p120e4f in vitro translation reinitiation and leaky scanning in plant viruses phylogenetic analysis of recent isolates of classical swine fever virus from colombia inhibition of severe acute respiratory syndrome-associated coronavirus (sars-cov) infectivity by peptides analogous to the viral spike protein rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways translational control during virus infection epstein-barr virus immunossuppression of innate immunity mediated by phagocytes binding of shrimp cellular proteins to taura syndrome viral capsid proteins vp1 collaborative study to evaluate a new elisa test to monitor the effectiveness of rabies vaccination in domestic carnivores efficient inhibition of hepatitis b virus replication by small interfering rnas targeted to the viral x gene in mice preparation and characterization of a novel monoclonal antibody specific to severe acute respiratory syndrome-coronavirus nucleocapsid protein the kinetics of proinflammatory cytokines in murine peritoneal macrophages infected with envelope protein-glycosylated or non-glycosylated west nile virus members of adenovirus species b utilize cd80 and cd86 as cellular attachment receptors exogenous nitric oxide inhibits crimean congo hemorrhagic fever virus structural and antigenic analysis of the yellow head virus nucleocapsid protein p20 efficient expression of the 15-kda form of infectious pancreatic necrosis virus vp5 by suppression of a uga codon phylogenetic relationships of brazilian bovine respiratory syncytial virus isolates and molecular homology modeling of attachment glycoprotein genetic manipulation of two fowlpox virus late transcriptional regulatory elements influences their ability to direct expression of foreign genes a new rna virus found in black tiger shrimp penaeus monodon from thailand conformational maturation of the nucleoprotein synthesized in influenza c virus-infected cells hiv-1-mediated syncytium formation promotes cell-to-cell transfer of tax protein and htlv-i gene expression lack of a mechanism for faithful partition and maintenance of the kshv genome studies on the activity of a bidirectional promoter of mungbean yellow mosaic india virus by agroinfiltration expression, purification, and in vitro activity of an arterivirus main proteinase genetic characterization of the capra hircus papillomavirus: a novel close-to-root artiodactyl papillomavirus transient expression of human papillomavirus type 16 l1 protein in nicotiana benthamiana using an infectious tobamovirus vector a deletion and point mutation study of the human papillomavirus type 16 major capsid gene hearsnpv orf83 encodes a late, nonstructural protein with an active chitin-binding domain molecular epidemiology of bluetongue virus in northern colorado amino acid sequence of the amur tiger prion protein involvement of endoplasmic reticulum in hepatitis b virus replication systemic antiviral silencing in plants sequencing and comparative analysis of a pig bovine viral diarrhea virus genome esirnas inhibit hepatitis b virus replication in mice model more efficiently than synthesized sirnas chronological and geographical variations in the small rna segment of the teratogenic akabane virus hcv ns2 protein inhibits cell proliferation and induces cell cycle arrest in the s-phase in mammalian cells through down-regulation of cyclin a expression pepper mild mottle virus pathogenicity determinants and cross protection effect of attenuated mutants in pepper hepatitis e virus genotyping based on full-length genome and partial genomic regions location and phylogenetic analysis of the region immediately upstream of the granulin gene of the clostera anachoreta granulovirus molecular characterization of rabies virus isolates in china during proteolytic cleavage and shedding of the bovine prion protein in two cell culture systems antigenic structure analysis of glycosylated protein 3 of porcine reproductive and respiratory syndrome virus generation of virus-like particles consisting of the major capsid protein vp1 of goose hemorrhagic polyomavirus and their application in serological tests amino acid changes in the recombinant dengue 3 envelope domain iii determine its antigenicity and immunogenicity in mice key: cord-020101-5rib7pe8 authors: nan title: cumulative author index for 2008 date: 2008-11-17 journal: virus res doi: 10.1016/s0168-1702(08)00367-5 sha: doc_id: 20101 cord_uid: 5rib7pe8 nan dettori, g., see medici, m.c. (137) 163 devi, s., see osman, o. (135) murine leukemia virus reverse transcriptase: structural comparison with hiv-1 reverse transcriptase the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus detection of ovine herpesvirus 2 major capsid gene transcripts as an indicator of virus replication in shedding sheep and clinically affected animals genetic characterization of equine influenza viruses isolated in italy between a new living cell-based assay system for monitoring genome-length hepatitis c virus rna replication unraveling the puzzle of human anellovirus infections by comparison with avian infections with the chicken anemia virus the contribution of feathers in the spread of chicken anemia virus cloning and subcellular localization of the phosphoprotein and nucleocapsid proteins of potato yellow dwarf virus, type species of the genus nucleorhabdovirus the p26 gene of the autographa californica nucleopolyhedrovirus: timing of transcription, and cellular localization and dimerization of product complete genomic sequence of turkey coronavirus recombinant l and p protein complex of rinderpest virus catalyses mrna synthesis in vitro molecular divergence of grapevine virus a (gva) variants associated with shiraz disease in south africa sequence analysis of a reovirus isolated from the winter moth operophtera brumata (lepidoptera: geometridae) and its parasitoid wasp phobocampe tempestiva (hymenoptera: ichneumonidae sars coronavirus replicase proteins in pathogenesis virus-induced gene silencing in medicago truncatula and lathyrus odorata evaluating the 3c-like protease activity of sars-coronavirus: recommendations for standardized assays for drug discovery hbx modulates iron regulatory protein 1-mediated iron metabolism via reactive oxygen species pathogenetic mechanisms of severe acute respiratory syndrome detection of a novel circovirus in mute swans (cygnus olor) by using nested broadspectrum pcr chimaeric hiv-1 subtype c gag molecules with large in-frame c-terminal polypeptide fusions form virus-like particles cross-species recombination in the haemagglutinin gene of canine distemper virus sapovirus-like particles derived from polyprotein cauliflower mosaic virus gene vi product n-terminus contains regions involved in resistance-breakage, self-association and interactions with movement protein adenovirus vector induced innate immune responses: impact upon efficacy and toxicity in gene therapy and vaccine applications interfering with cellular signaling pathways enhances sensitization to combined sodium butyrate and gcv treatment in ebv-positive tumor cells evidence for recombination between pcv2a and pcv2b in the field retroviral reverse transcriptases (other than those of hiv-1 and murine leukemia virus): a comparison of their molecular and biochemical properties mitochondrial plasmids of sugar beet amplified via rolling circle method detected during curtovirus screening appearance of intratypic recombination of enterovirus 71 in taiwan from the circulation of subgenogroups b5 and c5 of enterovirus 71 in taiwan from in vitro replication of bamboo mosaic virus satellite characterization of the interaction of domain iii of the envelope protein of dengue virus with putative receptors from cho cells intrahost evolution of envelope glycoprotein and orfa sequences after experimental infection of cats with a molecular clone and a biological isolate of feline immunodeficiency virus the sars-coronavirus plnc domain of nsp3 as a replication/ transcription scaffolding protein limited compatibility between the rna polymerase components of influenza virus type a and b serotype-specificity of recombinant fusion proteins containing domain iii of dengue virus very virulent infectious bursal disease virus isolated from wild birds in korea: epidemiological implications genetic analysis and evaluation of the reassortment of influenza b viruses isolated in taiwan during the enhanced immune responses of mice inoculated recombinant adenoviruses expressing gp5 by fusion with gp3 and/or gp4 of prrs virus effect of antiviral treatment and host susceptibility on positive selection in hepatitis c virus novel hiv-1 reverse transcriptase inhibitors synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast saccharomyces cerevisiae evolutionary analyses of european h1n2 swine influenza a virus by placing timestamps on the multiple reassortment events hepatitis b pregenomic rna splicing-the products, the regulatory mechanisms and its biological significance human papillomavirus 16 e6, l1, l2 and e2 gene variants in cervical lesion progression pathology and hematology of the caribbean spiny lobster experimentally infected with panulirus argus virus tomato leaf curl virus satellite dna as a gene silencing vector activated by helper virus infection down-regulation of sclerotinia sclerotiorum gene expression in response to infection with sclerotinia sclerotiorum debilitation-associated rna virus presence of p1b and absence of hc-pro in squash vein yellowing virus suggests a general feature of the genus ipomovirus in the family potyviridae the truncated virus-like particles of c6/36 cell densovirus: implications for the assembly mechanism of brevidensovirus tubulovesicular structures are a consistent (and unexplained) finding in the brains of humans with prion diseases interferon antagonist function of japanese encephalitis virus ns4a and its interaction with dead-box rna helicase identification of novel viral interleukin-10 isoforms of human cytomegalovirus ad169 cross-reactive and serospecific epitopes of nucleocapsid proteins of three hantaviruses: prospects for new diagnostic tools genetic diversity of the vp1 gene of duck hepatitis virus type i (dhv-i) isolates from southeast china is related to isolate attenuation the major tegument structural protein vp22 targets areas of dispersed nucleolin and marginalized chromatin during productive herpes simplex virus 1 infection mutational events during the primary propagation and consecutive passages of hepatitis e virus strain je03-1760f in cell culture the n protein of tomato spotted wilt virus (tswv) is associated with the induction of programmed cell death (pcd) in capsicum chinense plants evolutionary relationships of virus species belonging to a distinct lineage within the ampelovirus genus a novel genomic constellation (g10p[3]) of group a rotavirus detected from buffalo calves in northern india phylogeny of lagos bat virus: challenges for lyssavirus taxonomy dc-sign enhances infection of cells with glycosylated west nile virus in vitro and virus replication in human dendritic cells induces production of different modulation of cellular transcription by adenovirus 5, ⌬e1/e3 adenovirus and helper-dependent vectors involvement of cytoskeleton in junín virus entry hiv-1 reverse transcriptase inhibitor resistance mutations and fitness: a view from the clinic and ex vivo genomic characterization of novel marine vesiviruses from steller sea lions restricted quasispecies variation following infection with the gb virus b molecular characterization of vp4, vp6 and vp7 genes of a rare g8p[14] rotavirus strain detected in an infant with gastroenteritis in italy retroviral reverse transcription mechanisms of resistance to nucleoside analogue inhibitors of hiv-1 reverse transcriptase truncation of cytoplasmic tail of eiav env increases the pathogenic necrosis characterization of russian rabies virus vaccine strain rv-97 bacteriophage preparation inhibition of reactive oxygen species generation by endotoxin inhibition of autographa californica nucleopolyhedrovirus (acnpv) polyhedrin gene expression by dnazyme knockout of its serine/threonine kinase (pk1) gene serine/threonine kinase (pk-1) is a component of autographa californica multiple nucleopolyhedrovirus (acmnpv) very late gene transcription complex and it phosphorylates a 102 kda polypeptide of the complex amino acid at position 95 of the matrix protein is a cytopathic determinant of rabies virus phosphorylation of the tgbp1 movement protein of potato virus x by a nicotiana tabacum ck2-like activity comparative genomics of serotype asia 1 foot-and-mouth disease virus isolates from india sampled over the last two decades low dna htlv-2 proviral load among women in são paulo city antiviral potentials of medicinal plants a molecular epidemiological study of rabies in puerto rico the latent membrane protein 1 (lmp1) encoded by epstein-barr virus induces expression of the putative oncogene bcl-3 sars coronavirus accessory proteins hepatitis b viruses: reverse transcription a different way increase in proto-oncogene mrna transcript levels in bovine lymphoid cells infected with a cytopathic type 2 bovine viral diarrhea virus plantibody-mediated inhibition of the potato leafroll virus p1 protein reduces virus accumulation interferon ␤1-a and selective anti-5ht 2a receptor antagonists inhibit infection of human glial cells by jc virus the begomoviruses honeysuckle yellow vein mosaic virus and tobacco leaf curl japan virus with dna␤ satellites cause yellow dwarf disease of tomato genetic structure of a population of potato virus y inducing potato tuber necrotic ringspot disease in japan molecular characterisation and phylogenetic analysis of chronic bee paralysis virus inhibition of west nile virus replication in cells stably transfected with vector-based shrna expression system complete genome sequence analysis of dengue virus type 2 isolated in detecting molecular adaptation at individual codons in the glycoprotein gene of the geographically diversified infectious hematopoietic necrosis virus positive natural selection in the evolution of human metapneumovirus attachment glycoprotein sphingomyelin induces structural alteration in canine parvovirus capsid late steps of parvoviral infection induce changes in cell morphology molecular cloning and sequence analysis of the duck enteritis virus ul5 gene the interaction between kshv rta and cellular rbp-j and their subsequent dna binding are not sufficient for activation of modulation of hepatitis b virus replication by expression of polymerasesurface fusion protein through splicing: implications for viral persistence seroprevalence and genetic evolutions of swine influenza viruses under vaccination pressure in korean swine herds the cycle for a siphoviridae-like phage (vhs1) of vibrio harveyi is dependent on the physiological state of the host expression and biochemical characterization of nsp2 cysteine protease of chikungunya virus effect of sirna mediated suppression of signaling lymphocyte activation molecule on replication of peste des petits ruminants virus in vitro prevalence and molecular characterization of wu/ki polyomaviruses isolated from pediatric patients with respiratory disease in functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association genetic variation of hepatitis c virus in a cohort of injection heroin users in wuhan identification of a conserved linear b-cell epitope at the n-terminus of the e2 glycoprotein of classical swine fever virus by phage-displayed random peptide library lópez-galíndez, an hiv-1 215v mutant shows increased phenotypic resistance to d4t hiv-1 p17 binds heparan sulfate proteoglycans to activated cd4 ϩ t cells up-regulation of murid herpesvirus 4 orf50 by hypoxia: possible implication for virus reactivation from latency effective inhibition of japanese encephalitis virus replication by small interfering rnas targeting the ns5 gene size reversion of a truncated dna␤ associated with tobacco curly shoot virus f gene recombination between genotype ii and vii newcastle disease virus growth of tick-borne encephalitis virus (european subtype) in cell lines from vector and non-vector ticks dna recognition properties of the cell-to-cell movement protein (mp) of soybean isolate of mungbean yellow mosaic india virus emerging g9 rotavirus strains in the northwest of china implications of recombination for hiv diversity induction of apoptosis in vero cells by newcastle disease virus requires viral replication, de-novo protein synthesis and caspase activation subcellular localization of the triple gene block proteins encoded by a foveavirus infecting grapevines phylogenetic analysis of the ns5 gene of dengue viruses structural basis for drug resistance mechanisms for non-nucleoside inhibitors of hiv reverse transcriptase importance of cholesterol for infection of cells by transmissible gastroenteritis virus animal models and vaccines for sars-cov infection comparative full genome analysis revealed e1: a226v shift in oropouche virus entry into hela cells involves clathrin and requires endosomal acidification molecular evidence for polyphyletic origin of human immunodeficiency virus type 1 subtype c in transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells rnase h activity: structure, specificity, and function in reverse transcription a single nucleotide change in hop stunt viroid modulates citrus cachexia symptoms sequence analysis of mrna transcripts encoding jembrana disease virus tat-1 in vivo isolation of a type 3 vaccine-derived poliovirus (vdpv) from an iranian child with x-linked agammaglobulinemia a review of studies on animal reservoirs of the sars coronavirus genetic analysis of dengue 3 virus subtype iii 5ј and 3ј non-coding regions allopurinol, an inhibitor of purine catabolism, enhances susceptibility of tobacco to tobacco mosaic virus effects of acp26 on in vitro and in vivo productivity, pathogenesis and virulence of autographa californica multiple nucleopolyhedrovirus sars-cov replication and pathogenesis in an in vitro model of the human conducting airway epithelium rna transcription analysis and completion of the genome sequence of yellow head nidovirus mechanisms of inhibition of hiv replication by non-nucleoside reverse transcriptase inhibitors acute non-cytopathic bovine viral diarrhea virus infection induces pronounced type i interferon response in pregnant cows and fetuses extracellular vesicles containing virus-encoded membrane proteins are a byproduct of infection with modified vaccinia virus ankara analysis of jembrana disease virus mrna transcripts produced during acute infection demonstrates a complex transcription pattern complete sequence of the genome of avian paramyxovirus type 2 (strain yucaipa) and comparison with other paramyxoviruses cloning and sequencing of capsid protein of indian isolate of extra small virus from macrobrachium rosenbergii a member of a new genus in the potyviridae infects rubus molecular epidemiology of rabies in indonesia key: cord-295187-konm26x5 authors: decaro, nicola; mari, viviana; elia, gabriella; lanave, gianvito; dowgier, giulia; colaianni, maria loredana; martella, vito; buonavoglia, canio title: full-length genome analysis of canine coronavirus type i date: 2015-12-02 journal: virus res doi: 10.1016/j.virusres.2015.07.018 sha: doc_id: 295187 cord_uid: konm26x5 canine coronavirus types i (ccov-i) and ii (ccov-ii) are usually responsible for mild enteritis in dogs. while the ccov-ii genome has been completely sequenced, to date there are no complete genomic sequence data available publicly for ccov-i. thus, the aim of the present study was to analyze the full-length genome of a ccov-i prototype strain that had been recovered from a dog with diarrhea in italy. ccov-i strain 23/03 has a genome of 30,000 nucleotides, excluding the 3′ poly(a) tail, displaying the typical alphacoronavirus-1 organization and the highest genetic relatedness to ccov-ii. however, two distinct features were observed in the ccov-i genome: (i) the presence of an additional orf between the spike (s) protein gene and orf3a; (ii) the diversity of the s protein, which is more closely related to that of feline coronavirus type i and presents a furin cleavage site. the present study may contribute to a better understanding of the alphacoronavirus-1 evolutionary pattern and may be paradigmatic of how coronaviruses evolve through gene losses, acquisition and exchanges among different members. coronaviruses (covs) are large, single-stranded, positive-sense rna viruses, which are responsible for enteric and/or respiratory disease in mammals and birds. canine coronavirus (ccov) is usually responsible for mild enteritis in young dogs buonavoglia, 2008, 2011) , although fatal disease has been associated to a pantropic variant of the virus (decaro et al., , 2010a marinaro et al., 2010; zicola et al., 2012; ntafis et al., 2012) . based on the genetic distance encountered in the spike (s) protein gene (pratelli et al., 2003) , two ccov genotypes are known, ccov type i (ccov-i) and type ii (ccov-ii), which are variously distributed worldwide (decaro et al., 2005 mcelligott et al., 2011; ntafis et al., 2013; licitra et al., 2014; cavalli et al., 2014; costa et al., 2014) . ccov-ii has been found to exist in two different subtypes, ccov-iia and ccov-iib, the latter being the result of homologous recombination with transmissible gastroenteritis virus of swine (tgev) (decaro et al., 2009 (decaro et al., , 2010b . intermediate viruses between ccov-i and ccov-ii have been also detected . ccov-i and ccov-ii form a unique viral species, alphacoronavirus-1 (family coronaviridae, genus alphacoronavirus), along with feline coronavirus types i (fcov-i) and ii (fcov-ii), tgev and porcine respiratory coronavirus (prcov) ). an additional orf, named orf3, was found in the ccov-i genome, whereas only its remnants were evident in the genomes of ccov-ii and tgev, revealing an intriguing evolutionary history within the alphacoronavirus-1 species . while the full-length genomes of several strains of ccov-ii have been determined (decaro et al., 2015) , to date there are no complete genomic sequence data available publicly for ccov-i. thus, the aim of the present study was to analyze the full-length genome of a ccov-i prototype strain that had been recovered from a dog with diarrhea in italy. strain 23/03 was detected during an epidemiological survey for ccov in italian dogs with diarrhea . the ill dog, a male german shepherd of 6 weeks of age, belonged to a kennel located in the apulia region, southern italy. the feces were collected by a vet directly from the rectal ampulla into a sterile container during the clinical examination of the dog. ccov-i rna detection in the specimen was obtained by means of genotype-specific pcr and real-time rt-pcr (decaro et al., 2005) . virus isolation attempts using different cell lines of canine and feline origin were unsuccessful, since ccov-i has not been adapted to the in vitro growth buonavoglia, 2008, 2011) . the original fecal sample was aliquoted and stored at −70 • c until rna extraction. an aliquot of the original fecal specimen was clarified by centrifuging at 2500 × g for 10 min. one-hundred-forty microliters of the supernatant were then used for rna extraction by means of qiaamp ® viral rna mini kit (qiagen s.p.a., milan, italy), following the manufacturer's protocol and the rna template was stored at −70 • c until its use. the rna extract was subjected to a previously-established taqman-based real-time rt-pcr assay for rapid detection and quantification of ccov rna , with minor modifications. briefly, a one-step method was adopted using superscript ® iii platinum ® one-step qrt-pcr kit (life technologies srl, milan, italy) and the following 50-l mixture: 25 l of master mix, 1 l of superscript ® iii rt/platinum taq mix, 300 nm of primers ccov-for and ccov-rev, 200 nm of probe ccov-pb and 10 l of template rna. duplicates of log 10 dilutions of standard rna were analyzed simultaneously in order to obtain a standard curve for absolute quantification. the thermal profile consisted of reverse transcription at 50 • c for 15 min and activation of platinum taq dna polymerase at 95 • c for 2 min, followed by 45 cycles of denaturation at 95 • c for 15 s, annealing at 48 • c for 30 s and extension at 60 • c for 30 s. ccov genotyping was achieved by means of two distinct genotype-specific assays (decaro et al., 2005) performed by using superscript ® iii platinum ® one-step qrt-pcr kit (life technologies srl) and the following oligonucleotide sets (final concentrations were 600 and 200 nm for primers and probes, respectively): primer pair ccovi-f/ccovi-r and probe ccovi-pb for ccov-i and ccovii-f/ccovii-r and probe ccovii-pb (decaro et al., 2005) for ccov-ii. the thermal protocol was as described for ccov detection except for different annealing temperatures (53 • c and 48 • c for ccov-i and ccov-ii, respectively). overlapping fragments of the genome of ccov-i strain 23/03 were obtained through rt-pcr reaction carried out using primer sets designed based on the genome sequence of other alphacoronaviruses and the kit superscript tm one-step rt-pcr for long templates (life technologies srl). additional rt-pcr assays and subsequent sequencing attempts were performed to close gaps between assembled contigs and to sequence unresolved genomic regions using primers designed on the alignment of the reference alphacoronavirus strains. the very 5 and 3 ends were amplified using 5 and 3 race system for rapid amplification of cdna ends (invitrogen), respectively, following the manufacturer's instructions. the pcr products were detected by electrophoresis through a 1.5% agarose gel and visualization under uv light after ethidium bromide staining. rt-pcr products were subjected to direct sequencing at the baseclear b.v. (leiden, the netherlands). the sequences were manually edited and analyzed using the geneious platform (http:// www.geneious.com) and the ncbi's (htttp://www.ncbi.nlm.nih. gov) and embl's (http://www.ebi.ac.uk) analysis tools. nucleotide (nt) sequences of the different orfs were converted into amino acid (aa) sequences and comparative sequence analysis with reference coronavirus sequences was carried out in the full-length genome and encoded structural and nonstructural proteins. phylogenetic and molecular evolutionary analyses were conducted using mega4.1 beta (tamura et al., 2007) . in order to include in the analysis ccov-iib, whose genome has not been completely sequenced, pylogenetic trees were elaborated on a 22,366 genomic sequence (encompassing from the 3 end of orf 1a to the 3 utr) and on the amino acid (aa) sequences of s, membrane (m), and nucleocapsid (n) proteins using both parsimony and neighbor-joining methods, supplying a statistical support with bootstrapping over 1000 replicates. the following alphacoronavirus reference strains were used for phylogeny ( the full-length genome of ccov-i strain 23/03 was deposited in genbank under accession number kp849472. by the real-time rt-pcr panels, the fecal sample was confirmed to contain a ccov-i strain, whose titer was calculated as 6.73 × 10 6 rna copies/l of template. the specimen had no traces of ccov-ii rna. the genome of ccov-i strain 23/03 has a size of 30,000 nt, excluding the 3 poly(a) tail, and shows typical alphacoronavirus-1 organization (table 1 and fig. 1 ). the 5 utr consists of 313 nt including the leader sequence (l, nt 1-94) and the conserved core 5 -cuaaac-3 (nt 95-100) of the transcription regulatory sequence (trs), which controls the mrna synthesis through interaction with the viral polymerase during the discontinuous transcription of the negative strand subgenomic rna of the nidovirales members (enjuanes et al., 2000) . similar trs signals precede each of the 8 putative mrna encoding for the structural and nonstructural proteins ( table 1) . the 3 end of the viral genome consists of a 274-nt 3 utr that is followed by the poly(a) tail. sequence analysis showed intact structural and non-structural proteins with respect to reference ccov-ii, fcov-i and fcov-ii genomes. about two-thirds of the viral genome is occupied by the replicase gene encoding for two large polyproteins (pp), pp1a and pp1ab, the latter being synthesized through ribosomal slippage at position 12,327. the polyproteins of the replicase complex are processed by viral proteinases, resulting in several products with different size and function. sequence comparison with other alphacoronavirus-1 genomes led to the detection of three putative papain-like proteinase cleavage sites and 11 putative 3c-like proteinase cleavage sites, producing 16 nonstructural proteins (table 2) . four structural proteins were detected downstream of the replicase gene, namely the spike (s), small envelope (e), membrane (m) and nucleocapsid (n) proteins. the s protein has a size of 1481 aa, thus being longer than the analogous protein of glycosylated asn residues. the n protein of strain ccov-i 23/03 is 380-aa long product with three potential n-glycosylation sites. analogously to ccov-ii and fcov-i/ii, some accessory genes were detected between orfs 2 (s-protein gene) and 4 (e-protein gene) and downstream of orf6 (n-protein gene). the s-e intergenic region contains the canonical three orfs 3a, 3b and 3c, encoding for products with sizes of 78, 71 and 251 aa, respectively, plus an additional accessory protein gene, orf3, encoding for a putative 206 aa protein, which has been found to be unique to the ccov-i genome . the 3 end accessory genes were orfs 7a and 7b that encodes for 101-aa and 213-aa long proteins, respectively. alignment of complete genome sequences of ccov-i strain 23/03 and reference alphacoronaviruses showed the closest genetic relatedness with ccov-iia isolates (83.82-84.98% nt identity), followed by tgev (82.81%) and . no comparison was possible with ccov-iib since there are no full-length genomes available in the genbank database for this virus. when the spike protein was analyzed, ccov-i displayed a higher aa identity to fcov-i (73.09%) than to ccov-iia/iib (42.74-43.71%), fcov-ii (43.05%) and tgev (42.83%). among extant ccov strains, the closest identity was observed with isolate a76, which has been proven to have a ccov-i/ii recombinant s protein (regan et al., 2012) . the e, m and n proteins of strain 23/03 were all more closely related to the analogous products of ccov-ii and porcine cov reference strains (table 3) . in order to include in the analysis the nt sequences available for ccov-iib, phylogeny was first constructed on a 23,106-nt fragment spanning from the 3 end of orf1a to the very 3 end of the viral genomes. in the neighbor-joining tree elaborated using these sequences, strain ccov-i 23/03 clustered separately from extant ccov/fcov isolates leading to the formation of an outlier branch ( fig. 2a) . the prototype ccov-i strain formed a separate branch from other analyzed alphacoronaviruses also in the trees elaborated using the m (fig. 2c) and n (fig. 2d) proteins, whereas the s protein revealed a clustering with fcov-i (fig. 2d) . the same tree topologies were obtained through phylogenetic analysis using the maximum-parsimony method (data not shown). ccov has progressively emerged as being responsible for moderate to severe enteritis in dogs, with different genotypes and subgenotypes being detected in recent years buonavoglia, 2008, 2011) . ccov-ii is the oldest genotype, which has been known since 1971 (binn et al., 1974) , whereas ccov-i was discovered only 30 years later thanks to molecular methods, since this virus has not been adapted to grow in vitro (pratelli et al., 2003) . more recently, two ccov-ii subtypes have been recognized, namely ccov-iia and ccov-iib, on the basis on the relatedness of the spike protein of the latter virus to that of tgev (decaro et al., 2009 (decaro et al., , 2010b . in addition, a virulent ccov-iia biotype, strain cb/05, has been proven to cause fatal infections and long-lasting lymphopenia in naturally (buonavoglia et al., 2006; decaro et al., 2012; ntafis et al., 2012; zicola et al., 2012; pinto et al., 2014) and experimentally marinaro et al., 2010) infected dogs. to date, while the ccov-ii genome has been fully determined (decaro et al., 2015) , only fragments of the 3 genomic end are available for ccov-i. therefore, in the present study we have carried out the complete genome sequence analysis for this canine pathogen. the results showed that the ccov-i genome displays some distinct features with respect to ccov-ii and other alphacoronaviruses. the first finding is the presence of an additional accessory protein gene, orf3, which was located between the s protein gene and orf3a. this additional gene, which has been recently characterized, was found to be unique to the ccov-i genome, whereas ccov-ii and tgev exhibit only 5 and 3 end remnants . however, fcov strains harboring different forms of table 3 percent (%) identities of ccov-i 23/03 to alphacoronavirus-1 reference strains in the complete genomic sequence (nucleotide, nt) and structural proteins (amino acid, aa). orf3 and a ccov-i n protein gene have been detected, thus proving the circulation of fcov-i/ccov-i recombinant viruses . another finding of the ccov-i genome is that the s protein gene is closely related to that of fcov-i, whereas the rest of the genome displays a higher relatedness to ccov-ii. in addition, a furin cleavage site leads to the potential generation of two subunits, s1 and s2. a similar basic motif is present, approximately in the same position, in most beta-and gammacoronaviruses. cleavage of the cov s protein has been correlated to cell-fusion activity in vitro but the potential implications in viral pathobiology have not been fully determined (hingley et al., 1998) . in addition to ccov-i, furin cleavage motifs have been detected in fcov-i, but even in this case the biological consequences were not completely understood (de haan et al., 2008) . unlike ccov-ii and fcov-ii that display a similar s protein as a consequence of homologous recombination, ccov-i does not grow in cell cultures and only few fcov-i strains have been adapted to grow in vitro. the cov s protein interacts with cell receptors, thus being responsible for binding of virions to the cell surface (enjuanes et al., 2000) . thus, the different s proteins between ccov-i/fcov-i and ccov-ii/fcov-ii are likely to be responsible for the diverse biological behaviors in cell cultures. the cell receptor for most alphacoronavirus-1 isolates is the cell surface glycoprotein aminopeptidase n (apn), but there is no evidence for this receptor being used by ccov-i/fcov-i (de haan et al., 2008) . on the basis of the most recent findings, the evolutionary history of alphacoronavirus-1 members has been tentatively reconstructed, suggesting that ccov-i and fcov-i are the ancestral viruses from which tgev, ccov-ii and fcov-ii have originated through gene losses and recombination events . our findings corroborate the hypothesis that ccov-i is the ancestor for ccov-ii, since these viruses exhibit high identity in the entire genome with the exception of the s protein gene, which is markedly diverse, and orf3, of which only remants are present in the ccov-ii genome. in conclusion, the full-length sequencing of the ccov-i genome may contribute to a better understanding of the alphacoronavirus-1 evolutionary pattern and may be paradigmatic of how covs evolve through gene losses, acquisition and exchanges among different members. recovery and characterization of a coronavirus from military dogs with diarrhea canine coronavirus highly pathogenic for dogs detection and genetic characterization of canine parvovirus and canine coronavirus strains circulating in district of tirana in albania 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canine parvoviruses and coronaviruses in southern ireland molecular characterization of a canine coronavirus na/09 strain detected in a dog's organs canine coronavirus, greece. molecular analysis and genetic diversity characterization characterization of pantropic canine coronavirus from brazil genetic diversity of a canine coronavirus detected in pups with diarrhoea in italy two genotypes of canine coronavirus simultaneously detected in fecal samples of dogs with diarrhea characterization of a recombinant canine coronavirus with a distinct receptor-binding (s1) domain molecular characterization of a virulent canine coronavirus bgf strain mega4: molecular evolutionary genetics analysis (mega) software version 4.0 characterization of a recombinant canine coronavirus with a distinct receptor-binding (s1) domain fatal outbreaks in dogs associated with pantropic canine coronavirus in france and belgium the authors are grateful to carlo armenise, arturo gentile and donato narcisi for their technical assistance. key: cord-287324-ecpicv5v authors: qiu, yuan; chen, ji-ming; wang, tong; hou, guang-yu; zhuang, qing-ye; wu, run; wang, kai-cheng title: detection of viromes of rna viruses using the next generation sequencing libraries prepared by three methods date: 2017-06-02 journal: virus res doi: 10.1016/j.virusres.2017.05.003 sha: doc_id: 287324 cord_uid: ecpicv5v virome (viral megagenomics) detection using next generation sequencing has been widely applied in virology, but its methods remain complicated and need optimization. in this study, we detected the viromes of rna viruses of one mock sample, one pooled duck feces sample and one pooled mink feces sample on the personal genome machine platform using the sequencing libraries prepared by three methods. the sequencing primers were added through random hybridization and ligation to fragmented viral rna using a rna-seq kit in method 1, through random reverse transcription (rt) and polymerase chain reaction (pcr) in method 2 which was developed in our laboratory, and through hybridization and ligation to fragmented amplicons of random rt-pcr using a single primer in method 3. although the results of these three samples (nine libraries) all showed that more classified viral families and genera were identified using methods 2 and 3 than using method 1, and more classified viral families and genera were identified using method 2 than using method 3, most of the differences were of no statistical significance. moreover, 11 mammalian viral genera in minks were possibly identified for the first time through this study. many human and animal infectious diseases, such as influenza, rabies, mumps, acquired immune deficiency syndrome, classical swine fever, foot-and-mouth disease and newcastle disease, are all caused by rna viruses (holmes, 2009; woolhouse et al., 2016) . moreover, most of emerging infectious diseases, such as ebola, nipah virus infection, severe acute respiratory syndrome, middle east respiratory syndrome, zoonotic h7n9 avian influenza and zika virus infection, are also caused by rna viruses (aziz et al., 2017; eaton et al., 2006; gao et al., 2013; li et al., 2005; nyakarahuka et al., 2016; zaki et al., 2012) . rapid detection of rna viruses is critical for the diagnosis, treatment, control and prevention of human and animal infectious diseases caused by rna viruses (drosten et al., 2002; torok and cooke, 2009) . traditionally, rapid detection of rna viruses has relied on nucleic acid-based techniques like polymerase chain reaction (pcr) using specific primers or probes, and/or serology-based techniques like enzyme linked immunosorbent assay (aziz et al., 2017) . in recent years, the development of next-generation sequencing (ngs) technologies has innovated methods for rna virus detection (capobianchi et al., 2013; shan et al., 2011; yu et al., 2013; zhang et al., 2014) . ngs can reveal a huge number sequences of nucleic acids (dna or rna) within specimens through some random modes, and can detect various kinds of rna viruses simultaneously thereby. virome detection has been widely applied to identification of novel rna viruses and research of rna viruses (masson et al., 2014; paez-espino et al., 2016; webster et al., 2015) . viral rna in clinical specimens is usually limited in quantity and prone to degradation, and should be transformed into ngs libraries for sequencing. efficient library preparation is critical for virome detection of rna viruses and remains challenging. in this study, we detected the viromes of rna viruses of one mock sample and two pooled authentic samples, using the libraries prepared by the three methods on the personal genome machine (pgm) platform, with the aim to generate data of significance for virome detection of rna viruses and characterize the viromes of rna viruses in ducks and minks. this study was conducted according to the animal welfare guidelines of the world organization for animal health and approved by the animal welfare committee of china animal health and epidemiology center. fecal samples were collected with permission given by multiple relevant parties, including china animal health and epidemiology center, the relevant farm owners. sample 1 was a mock sample mixed from the allantoic fluids of three chicken embryonated eggs (1 ml for each egg) which contained h9n2 subtype avian influenza virus (aiv), a strain of newcastle disease virus (ndv) and a strain of infectious bronchitis virus (ibv) with unknown viral load, respectively. sample 2 (approximately 50 ml) was a pool of 300 pieces of fresh duck feces collected from three live poultry markets in july 2015. sample 3 (approximately 50 ml) was a pool of 1000 pieces of fresh mink feces collected from 13 mink farms in may 2016. the fecal samples were stored in 200 ml phosphate buffered solution (pbs, ph 7.2) containing 10% glycerol at 4°c. viral rna of each sample was extracted within one day after collection. sample 1 was diluted in 9 ml pbs (ph 7.2) containing 10% glycerol and then filtered through a 0.22-μm filter (millipore, usa). samples 2 and 3 were suspended and then clarified by centrifugation at 12,000 rpm for 10 min. the supernatant was filtered through 0.22-μm filters to remove bacteria. the filtered solution was precipitated using 1/10 vol of 50% (w/v) polyethylene glycol 6000 at 4°c for 2 h. then the solution was centrifuged at 12,000 rpm at 4°c for 1 h. the precipitate was suspended in 1 ml pbs solution, and then treated with a mixture of 0.5 μl recombinant dnase i (rnase free, 5 u/μl) (takara, japan) and 0.5 μl ribonuclease a (rnase a, 10 mg/ml) (takara, japan) at 37°c for 30 min to digest nucleic acids out of cells and viruses. thereafter, viral nucleic acids were extracted using the qiaamp viral rna mini kit (qiagen, germany) according to the manufacturer's instructions. method 1 for the library preparation was based on the ion total rna-seq kit v2 (life technologies, usa) according to the manufacturer's instructions. briefly, the extracted viral rna was quantified, and then digested using rnase iii. the fragmented rna was ligated with random adaptors, and this was followed by reverse transcription. the cdna was amplified through pcr, and the amplicons around 450 bp were collected with the e-gel ® sizeselect™ agarose gel (life technologies, usa). method 2 for the library preparation was based on a procedure developed by ourselves. the details of the method development will be published elsewhere. it began with random rt reaction: 8 μl viral rna, 1 μl 100 μm primer a 15 n 6 (table 1), and 2 μl nuclease-free water were mixed and incubated at 72°c for 5 min. then the rna/primer mixture was placed on ice for at least 3 min. then the mixture was added with 4 μl 5 × first-strand buffer, 1 μl dntp (100 μm), 2 μl dtt (0.1 m), 1 μl rnaseout™ recombinant ribonuclease inhibitor (40 u/μl) and 1 μl superscript ® iii reverse transcriptase (200 u/μl) (invitrogen, usa), and incubated at 25°c for 15 min and 42°c for 30 min. the reaction was terminated at 70°c for 15 min. then the reaction system was added with 1 μl rnase h (takara, japan) and further incubated at 37°c for 20 min. after purification using dynamag™-2 magnet and agencourt ® ampure ® xp reagent (beckman coulter, usa), the purified first-strand cdna was used for the synthesis of the second-strand cdna with primer b 15 n 6 (table 1) at 70°c for 5 min. then the mixture was added with 1 μl klenow fragment (5 u) (neb, usa), 5 μl 10 × nebuffer 2, 2 μl dntp (100 μm) and 1 μl dtt (0.1 m), and then incubated at 37°c for 30 min. this was followed by pcr amplification using a system containing the double-stranded cdna template, 1 × phusion high-fidelity buffer, 10 μm primers a 30 and b 30 (table 1) , 0.5 u phusion high-fidelity dna polymerase (neb, usa). the pcr was performed as follows: 2 cycles of 94°c for 30 s, 50°c for 30 s and 72°c for 30 s, followed by 14 cycles of 94°c for 30 s, 65°c for 30 s and 72°c for 30 s, with a final extension at 72°c for 5 min. the library amplicons around 450 bp were collected with the e-gel ® sizeselect™ agarose gel. method 3 for the library preparation was modified from a procedure reported previously as sequence independent single primer amplification (sispa) (allander et al., 2001; cheng et al., 2010; djikeng et al., 2008; palacios et al., 2007) (fig. 1) . briefly, the extracted viral rna was reversely transcribed using the random primer spn 8 (table 1) , and then the rt system was denaturated at 94°c for 5 min and on ice for 3 min. the second-strand cdna synthesis was modified using the denaturated rt system which was added with 1 μl klenow fragment (5 u) (neb, usa) and 2.33 μl 10 × nebuffer, and the synthesis was performed at 37°c for 60 min and 75°c for 20 min. this was followed with pcr using the primer sp (table 1 ). the pcr amplicons were sheared and then ligated with sequencing adaptors. after that, the ligation products around 450 bp were collected and amplified. the libraries were sequenced through the ion torrent pgm platform (life technologies, usa) with the ion pgm™ sequencing 400 kit (life technologies, usa) (merriman et al., 2012) . each library was sequenced separately on an ion 318™ chip (life technologies, usa). obtained short reads were deposited in the sequence read archives of ncbi (accession numbers: srr5078294, srr5078297, srr5078298, srr5078299, srr5078300, srr5078301, srr5078288, srr4051861 and srr4051862). the primers spn 8 and sp were trimmed from the reads before assembly for method 3. the reads were assembled using the clc genomics workbench 8.5.1 (qiagen, germany). assembled contigs shorter than 100 bp were removed, and the remaining contigs were compared to the non-redundant nucleotide databases using the standalone ncbi blastn tool (mcginnis and madden, 2004) . the evalue of 10 −3 was used as the cutoff value for significant hits. the blastn results were parsed using the metagenome analyzer (megan vesion 5.10.5) with the default lca parameters (huson et al., 2007) , and the taxonomic assignment was based on the first hit. sequences placed in the roots in megan diagrams were taken into account. all the contig hits of viruses excluding phages were verified manually through online blast at ncbi web station. the sequencing depth (corresponding to the number of hits obtained for a given nucleotide position) and table 1 the details of all the primers used in the paper. primer sequence coverage ( . the reads of sample 1 were mapped to the reference sequences using the software tool blat (v 34) (kent, 2002) . the total length of the contigs (the sum of the lengths of all the contigs) plus the sequencing coverage and depth were evaluated using the software tool soap.coverage (2.7.7) (li et al., 2008) . as shown in table 2 , the numbers of reads from the nine sequence libraries varied greatly among the three samples and the three methods for preparing the libraries. the table further showed that the reads using method 1 were shorter than their counterparts using methods 2 and 3 by 20.54%-64.75%. the contigs using method 1 were less than their counterparts using methods 2 and 3 by 47.87%-94.28%, and the total length of the contigs using method 1 was shorter than its counterparts using methods 2 and 3 by 70.43%-99.12%. the contigs using method 1 mapped to viruses including phages were less than their counterparts using methods 2 and 3 by 62.50%-94.77%, and the contigs using method 1 mapped to viruses excluding phages were less than their counterparts using methods 2 and 3 by 46.86%-78.95%. sample 1 was a mock sample containing three known viruses aiv, ndv and ibv. the aiv and ndv were identified by all the three methods, while the ibv was identified only by methods 2 and 3 ( fig. s1 and table 3 ). the viral load of ibv in the sample was possible lower than the other two known viruses. the sequencing mean depth of method 1 was only approximately 0.00%-10.19% of its counterparts of methods 2 and 3, and the sequencing coverage of method 1 was only approximately 0.00%-88.51% of its counterparts sequenced by methods 2 and 3, in the sequencing of the three known viruses (fig. s1 and table 3 ). sample 2 was a pool of duck feces collected from a live bird market. as shown in table 2 , from this sample, the classified families of viruses excluding phages identified through method 1 were approximately 50.00% − 61.54% of those identified through method 2 or 3, and the classified genera of viruses excluding phages identified through method 1 were approximately 34.78%-42.11% of those identified through method 2 or 3. taken the results of the three methods together, 17 classified families and 27 classified genera of viruses, as well as some unclassified viruses, were detected in sample 2 (table s1 ). as shown in fig. s2 , 41.18% of the above 17 classified families and 25.93% of the above 27 classified genera of viruses were identified by all the three methods, and some other viral families and genera were identified only by one or two of the three methods. among (table s1 ). regarding some other viral genera (aparavirus, cripavirus, iflavirus, potyvirus, carmovirus, tobamovirus, ambidensovirus and iteradensovirus), they were assumed to be from other sources (e.g., duck feed or environment), as similar viruses had been identified from plants, insects or protozoa previously (table s1 ). sample 3 was a pool of mink feces collected from 13 mink farms. as shown in table 2 , from this sample, the classified families and genera of viruses excluding phages of identified through method 1 were only one less than those identified through method 3, and four or five less than those identified through method 2. taken the results of the three methods together, 20 classified families and 29 classified genera of viruses, as well as some unclassified viruses, were detected in sample 3 (table s2 ). as shown in fig. s3 , 45.45% of the above 20 classified families and 44.83% of the above 29 classified genera of viruses were identified by all the three methods, and some other viral families and genera were identified only by one or two of the three methods. among the 29 viral genera, mamastrovirus, norovirus, sapovirus, vesivirus, hepevirus, alphacoronavirus, cardiovirus, kobuvirus, picobirnavirus, rotavirus, orthoreovirus, gyrovirus, ambidensovirus, amdoparvovirus and bocaparvovirus, were assumed to mammalian viruses, because similar mammalian viruses (e.g. aleutian mink disease virus within amdoparvovirus) had been identified previously (table s2 ). through investigation of the mink farmers, we knew that the mink in the farms had not been fed with any mammalian meat. therefore, we assumed that all mammalian viruses identified in the mink feces sample were possibly mink viruses. of these mammalian viral genera, mamastrovirus, norovirus, sapovirus, vesivirus, cardiovirus, picobirnavirus, rotavirus, orthoreovirus, gyrovirus, ambidensovirus and bocaparvovirus from mink had not been reported previously. some other viral genera, like betanodavirus, cripavirus, chlorovirus, trichovirus, marafivirus, tobamovirus, giardiavirus and pelamoviroid, were assumed to be from other sources (e.g., mink feed or environment), as similar viruses from plants, fishes, insects or unicellular eukaryotic organisms had been identified previously (table s2 ). some avian viruses in the genera of influenzavirus a, avastrovirus, gammacoronavirus and avibirnavirus which were identified in sample 3 might be from mink feed which contained fresh meat, giblets and eggs of chickens and ducks. it should be mentioned that avian influenza virus can replicate in mink. various ngs platforms, such as illumina hiseq, miseq and novaseq, ion torrent pgm, proton and s5, bgi bgiseq-500, have been commercially available (quail et al., 2012) . among these platforms, ion torrent pgm is competitive for detection of viruses and bacteria with respect to instrumental price, sequencing cost and simplicity of operation, although its sequencing throughput is lower than miseq and proton. due to high cost for comparing these three methods for library preparation, only three samples (one mock sample and two authentic samples) were detected using the pgm platform in this study. the results of these three samples (nine libraries) all showed that more classified viral families and genera were identified using methods 2 and 3 than using method 1, and largely more classified viral families and genera were identified using methods 2 than using method 3. however, all of the differences were of no statistical significance by the chisquare test (p > 0.05), except that significantly more viral families and genera were identified by method 2 than by method 1 for samples 2 and 3 (p < 0.05). methods 1 and 3 require specific commercial kits for fragmentation of nucleic acids and ligation of sequencing adaptors. therefore, they are more costly than method 2 which does not require any commercial kits. moreover, method 1 is of great technical difficulty as it requires rna fragmentation whose quality is difficult to control. theoretically, methods 2 and 3 can be started from viral rna of a concentration below rna quantification limits, while method 1 should be started from viral rna above the rna quantification limits. since most clinical samples collected from a single person or animal contains viral rna below the rna quantification limit, method 1 is not suitable for most clinical samples, although it may be suitable for other applications, e.g., transcriptome detection. we selected the three samples containing much amount of viral rna in this study in order to obtain ngs data using all the three methods. in the future, it is of significance to compare methods 2 and 3 in detecting viromes of rna viruses in some clinical samples containing limited viral rna. although all the three methods were designed for detection of viromes of rna viruses, some dna viruses were also identified using the three methods. this may be attributed to that the sequencing primers could be added to viral dna at one or more steps for preparing the libraries (e.g. the step of rt or pcr amplification of method 2). nevertheless, most (96.65%) hits of viruses excluding phages detected in this study were from rna viruses (tables s1 and s2). detection of viromes of duck guts has been reported recently and 18 classified viral families were identified (fawaz et al., 2016) . seven of these 18 families (circoviridae, dicistroviridae, iflaviridae, parvoviridae, picornaviridae, phycodnaviridae, and virgaviridae) were also identified in the duck feces sample through this study. hits of mimiviridae and retroviridae were also identified in the duck feces sample through this study, but they were assumed to be unreliable through our online blast analysis. the remaining nine families including baculoviridae, herpesviridae, iridoviridae, marseilleviridae, nodaviridae, papillomaviridae, partitiviridae, poxviridae and totiviridae were not identified through this study, while ten families including paramyxoviridae, orthomyxoviridae, astroviridae, coronaviridae, potyviridae, tombusviridae, picobirnaviridae, reoviridae, adenoviridae and avsunviroidae identified in this study were not identified in the recent report. the differences can be attributed to that the samples were collected at different sites, regions and time and detected using different methods. it is of value to conduct further studies to make clear whether some viruses identified in the duck feces belong to new viral species. detection of viromes of minks has not been reported previously, and a total of 11 mammalian viral genera in minks might be reported for the first time through this study. it is of value to conduct further studies to make clear clinical significance and taxonomic status of these viruses. ducks and minks are economically important for their feather, fur, egg and/or meat. detection of viromes of ducks and minks increases our understanding of the viral diversity in the animals, and provides novel clues for further studies regarding diagnosis of infectious diseases, identification of novel viruses and research of host-virus relationships. conflict of interest: kai-cheng wang declares that she has no conflict of interest. run wu declares that he has no conflict of interest. yuan qiu declares that he has no conflict of interest. ji-ming chen declares that he has no conflict of interest. tong wang declares that he has no conflict of interest. qing-ye zhuang declares that she has no conflict of interest. guang-yu hou declares that he has no conflict of interest. ethical approval: all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species zika virus: global health 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supported with the innovation fund of cahec (number: 2015if-0004ff). supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2017.05.003. key: cord-261388-d56ci0hl authors: tibbles, k.w; cavanagh, d; brown, t.d.k title: activity of a purified his-tagged 3c-like proteinase from the coronavirus infectious bronchitis virus date: 1999-05-28 journal: virus res doi: 10.1016/s0168-1702(99)00011-8 sha: doc_id: 261388 cord_uid: d56ci0hl previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (ibv) large open reading frame (orf1), confirmed the activity of a predicted 3c-like proteinase (3clp) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kda protein, 3clpro, capable of further cleavages in trans. in order to identify such cleavages within the orf1 polyprotein mediated by 3clpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3clp domain as targets. the proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3clpro. a histidine (his(6)) tag was introduced close to the c-terminus of the proteinase to aid purification. processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. from these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. this tentatively completes the processing map for the orf1 region with respect to 3clpro. the coronavirus infectious bronchitis virus (ibv) encodes the major portion of its non-struc-tural proteins within two large open reading frames, orfs 1a and 1b, situated at the 5' end and occupying the major portion of the 27.6 kb single-stranded positive-sense rna genome . the orfs overlap slightly with orf1b accessed via programmed ribosomal frameshifting (brierley et al., 1989) resulting in orf1a and orf1a/1b (a fusion) trans-lation products. the large size of the orfs suggests that the translation products are polyproteins that are processed to generate a set of mature non-structural proteins. in common with all other coronaviruses so far examined, ibv encodes a 3c-like proteinase domain which is located towards the c-terminus of the orf1a polyprotein and approximately centrally in the orf1a/1b fusion product. the 3clp domain was first identified as a possible component of ibv polyproteins (gorbalenya et al., 1989) due to sequence similarity to picornavirus 3c proteinases (reviewed in ryan and flint, 1997) . in the same study, predictions were made as to likely target dipeptide cleavage sites for a 3c-like proteinase within the orf1 polyproteins, the large number suggested implicating this proteinase in a pivotal role in the maturation of the polyproteins. similar domains were subsequently identified in mouse hepatitis virus (mhv) (lee et al., 1991) and human coronavirus (hcv 229e) (herold et al., 1993) . there are no cellular counterparts for the activities of the 3c-like proteinases, therefore they are essential to the successful maturation of their respective polyproteins and subsequent replication events. this, and the comparatively high degree of conservation (at functional level), makes the 3clike proteinases attractive targets for antiviral intervention. given the essential role played by the 3c-like proteinase of the coronaviruses in maturation of the 'polymerase' polyproteins, there has been considerable interest in characterising the enzyme, with initial emphasis on defining the processing events involving the proteinase. such studies are already at an advanced stage for both ibv (liu et al., 1994 and hcv 229e orf1b regions (grotzinger et al., 1996; heusipp et al., 1997a,b) . 3c-like proteinase activities have been confirmed recently for three representatives of the coronavirus group, mhv (lu et al., 1995) , hcv 229e (ziebuhr et al., 1995) and ibv (tibbles et al., 1996) and the protein has been identified in vivo during viral infection in the case of hcv (ziebuhr et al., 1995) and mhv (lu et al., 1996) . in addition, the proteinases from these two viruses have been purified and partially characterised (seybert et al., 1997; ziebuhr et al., 1997) . the identification of the ibv 3clpro was first realised in vitro when a cloned fragment of the polyprotein was expressed in reticulocyte lysate (tibbles et al., 1996) . in the presence of membranes, upon which activity was vitally dependent, processing of the region containing the protease domain was observed with the release of a 35 kda protein (p35). this product was capable of trans processing, but was inefficient and of limited use for mapping studies. we undertook the identification of the remaining ibv 3clpro cleavage targets within the orf1 polyprotein using bacterial expression to obtain significant quantities of 3clpro. the bacterial expression system described utilises the minimum processing unit identified in our previous studies which established that, in addition to the 3clp domain, mp2 protein sequence between the q/s 4 cleavage target and a point delimited by an ncoi restriction site (ntd position 10118) was that minimally required for processing. a bamhi fragment released from pkt205 (see below) was introduced into bamhi digested pet3xa (novagen) to yield pet205 ( fig. 1 ). this construct would allow expression to be monitored through the synthesis of the strongly expressed t7 gene 10 protein, which composes the major insoluble fraction component upon extraction. expression beyond the ncoi site was restricted by digestion of pet205 with ncoi followed by end repair and religation. this manipulation introduces a frame-shift in the sequence which results in a translation stop signal being encountered. this plasmid was then used to create an internal deletion between the vector initiator atg triplet and the ibv sequence by digestion with nhei and bamhi. the newly generated plasmid, petdn205dc nco , was modified to include a his-6 tag approximately 35 aminoacid residues from the c-terminus of 3clpro to aid in purification. a mutation which replaced the amino-acid sequence vknsqw (single letter code) with six histidine residues near the c-terminus of the 3clp domain in pkt205 (ntds 9679fig. 1 . schematic representation of the ibv orf1a/1b polyprotein with positions of predicted cleavage dipeptide targets shown as solid vertical bars, those with arrowheads have been experimentally confirmed. predicted catalytic domains are also indicated (mp1/2; membrane proteins, 3clp; 3c-like proteinase, pol; polymerase, zn; zinc finger, hel; helicase). the derivation of the eukaryotic and bacterial expression clones and the principal cloning vectors used are depicted. the principal restriction sites used in cloning manipulations (adjacent solid bars), promoters (t7, sp6 and ptac) and translation (tln) start sites for the plasmids are also shown. 9696) was made, giving rise to pkt205his. the positioning of this tag sequence was determined by consideration of the possible amino-acid sequences involved in cleavage at the q/s 4 site and potential for interaction with the affinity matrix based on predicted hydrophilicity (kyte and doolittle, 1982) of the proteinase sequence. this modified sequence was transferred to the 205bearing pet plasmid via replacement of a nsii fragment. the processing properties of the modified proteinase were tested extensively, using a variety of systems, and were found to be indistinguishable from wild-type (data not shown). therefore the appropriate pet plasmids (studier et al., 1990) were introduced into e.coli bl21 plyss cells (studier, 1991) . cultures were grown for approximately 3 h until the od 600 was between 0.6 and 0.9 and expression induced by the introduction of iptg (at 100 mg/ml) into the growth medium. the cells were harvested after a further 3 h. cells were lysed, clarified and the supernatant applied to a chelating agarose column charged with cobalt chloride. the use of cobalt instead of the usual nickel resulted in lower binding of background material (data not shown). alternatively, cultures were induced after 1.5 -2 h (od 600 0.3 approx.) and rifampicin was added (to 200 mg/ml) after a further hour and incubation continued for 3 h. this resulted in a substantial inhibition of cellular protein expression without affecting ongoing t7-dependent expression. although this resulted in a lower overall protein yield, there was a higher proportion of 3clpro and background binding for the metal chelate column selection was significantly reduced. typical results are shown in fig. 2 where the appearance of a novel 35 kda protein was observed after induction (compare lanes 1 and 2, panel a). clarification of the crude lysate (lane 3) by centrifugation revealed that the major portion of the protein of interest (i.e. 35 kda) remained in the supernatant (lane 5, compare to lane 4, pellet fraction). comparison of the total applied protein and the unbound fraction (lanes 5 and 6 respectively) revealed that over 90% of the novel band seen after induction was retained on the column. this putative 3clproh6 bound with relatively low affinity and was seen to elute at 60mm imidazole (lane 8), conditions normally associated with removing background binding. no observable protein remained to be eluted with 1m imidazole (lane 9). the background contaminating proteins could be significantly reduced by expression in the presence of rifampicin. while this resulted in a reduction in the total protein yield of some 80%, the proportion that was 3clproh6 was greater and resulted in less background in the purified product (panel b, compare products under normal conditions, lane 1, with those obtained in the presence of rifampicin, lane 2, after single step purification on a cobalt column). only fractions seen to contain the putative 3clproh6 were found to cleave target polypeptide targets in a trans assay which supports the identity of the induced product as 3clproh6 (data not shown). from comparison with known concentration protein standards (bsa) we estimated the yield to be approximately 2-4 mg/l of culture (0.5 -1 mg/l in the presence of rifampicin), which compares favourably with that obtained by ziebuhr et al., 1997 . different regions of the orf1a/1b polyprotein were expressed in reticulocyte lysate (tnt, promega) or bacterial cell extract (s30, promega) in the presence of [ 35 s] methionine using the following expression plasmids (see fig. 1 ). construction of the pkt205 and pktbp5 plasmids (encoding ibv sequence from nucleotides (ntd) 8693 -10927 and 10752 -12600 respectively) has been described elsewhere (liu et al., 1994) . these encode predicted cleavage sites q/s 3 to q/g 6 and q/s 7 to q/s 9 , respectively, within the c-terminal region of orf1a. ibv sequence between ntd 16785 and 23060 (boursnell et al., 1987) , amplified by rt-pcr using a first-strand cdna synthesis kit (pharmacia) and pfu polymerase (stratagene), was cloned into pcrscript (stratagene). this plasmid, pcrf4, was digested with espi and psti (following end repair of espi site with dna polymerase) and the fragment introduced into ecorv-psti digested pkt0/ns1 vector to produce pkt1bc (encoding nucleotides 16841 -20926). the polyprotein fragment encoded contains the three predicted 3clpro cleavage sites q/g 11 , q/s 12 and q/s 13 from the c-terminal region of orf1b. cleavage dipeptides were mutated (to g/s or a/s) by oligonucleotide-directed mutagenesis as described previously (tibbles et al., 1996) . this region was transferred as a spei fragment to pgex4t2 (pharmacia) and deletions made to express portions containing each target dipeptide individually. the n-terminal half of the orf1a region (up to nucleotide position 6500 approx.) is encoded by the plasmid pkt1a3, constructed as described previously (liu et al., 1995) . analysis of the q/g 5 site was made using a kpni deletion of the pet205 plasmid, pet205d kpn . a hincii fragment (ntd 10438-10902) bearing the q/g 6 target from pkt205 was introduced into pgex4t2 to yield pgexmp2c (resulting in the production of a fusion with glutathione-s-transferrase (gst), smith and johnson, 1988) . the region encoding the mp1 region and q/g 2 was derived from pcr amplification and a fragment (ntds 6919-8693) cloned upstream of the 205 sequence in pkt205/ bp5. purified proteinase was added in varying amounts to the radiolabelled target fragments and incubated for up to 16 h at room temperature. cleavage patterns were analysed following polyacrylamide gel electrophoresis and an example of these analyses is shown in fig. 3 . complete digestion conditions for the fragment corresponding to the bp5 region of the polyprotein revealed a cleavage pattern that was consistent with the pattern expected from the cleavage predictions made by gorbalenya et al., 1989 . this target contained the predicted cleavage sites of q/s 7 , q/s 8 and q/s 9 , all of which, along with an additional unpredicted q/n site, have been experimentally confirmed (liu et al., 1997; ng and liu, 1998) . cleavage within bp5 would be expected to yield processed products of approximate sizes of 6, 24, 10, 17 and 3 kda (as they lie on the polyprotein) assuming cleavage at q/s 7 , q/n, q/s 8 and q/s 9 . in fig. 3 the effect of processing by the addition of 3clproh6 can be seen. the uncleaved target (lane 1) consisted of two species (of approximately 60 and 70 kda) because the sequence contained the frame-shift signal, followed by approximately 300 nucleotides from orf 1b. only the two largest cleavage products were resolved in this experiment (lane 2) which correspond to cleavage reinforced by comparing them with those produced after incorporation of [ 35 s] cysteine. the relative intensity of the two bands is reversed reflecting the inverse ratio of the two amino acids in the 24 and 17 kda products (data not shown). no cleavage was apparent in the absence of 3clproh6 (data not shown). cleavage at the predicted q/s 7 site was tested by mutation of the cleavage dipeptide to an uncleavable form. failure to cleave at the mutated q/s 7 site should result in the extension of the n-terminus of the 24 kda product by 6 kda resulting in the generation of a new cleavage product of approximately 30 kda. comparison of the cleaved wild-type 24 kda product (lane 2) and the product from the mutated polyprotein fragment (lane 3) revealed a decrease in the mobility of the more intensively labelled product (equating to approximately 30 kda), consistent with failure to cleave at the mutated q/s 7 site. polyprotein fragments constituting the remaining areas of the orf1 region were synthesised and similarly analysed for cleavage by purified 3clproh6. the results of all of these trans assay analyses are summarised in table 1 . we found that all so far identified target dipeptides are utilised by the 3clproh6 enzyme in vitro. it was at q/s 7 and q/n for the largest product of 24 kda and at q/s 8 and q/s 9 for the smaller of 17 kda. the identities of these two products were noted that under conditions expected to result in complete cleavage, not all target sites were digested to completion. these tended to be in orf1b. only three of the predicted sites fail to be cleaved, namely q/g 2 , q/g 5 and q/g 6 and no novel target sites were detected. the first of the uncleaved sites (q/g 2 ) lies upstream of both the 3clp and mp1 domains while the latter pair fall within the mp2 region. either these sites are not utilised by 3clpro or the conformation of the target fragments bearing them in our assays do not facilitate their recognition by the proteinase. however in the case of the q/g 6 -bearing gst fusion, cleavage consistent with use of the thrombin site within the fusion junction region was observed (data not shown), which suggests that no gross conformational abnormality exists that might preclude proteolysis. we have used bacterial expression of viral protein, comprising the ibv orf1a polyprotein fragment that gives rise to 3clpro via autocatalytic processing in vitro (tibbles et al., 1996) , to obtain significant amounts of highly purified active proteinase for trans processing studies. our rationale for modifying the starting pet plasmid containing the 205 sequence was as follows. expression of hydrophobic proteins from picornaviruses with deleterious effects on bacterial cells has been reported (lama and carrasco, 1996) . therefore termination of expression at the ncoi site within mp2 would still allow processing, according to our in vitro data (tibbles et al., 1996) , while at the same time minimising the possible toxicity of the highly hydrophobic mp2 region. the gene 10 sequence was deleted in order to promote the production of a soluble polyprotein fragment, equivalent to that which is able to mature in vitro, and so ensure processing would proceed. evidence suggests that in bacteria folding is post-translational (netzer and hartl, 1997) and so there was potential for the proteinase to become inactive due to the characteristic sequestration of pet fusion proteins into inclusion bodies. in the event this latter measure appeared unnecessary as soluble processed 3clpro was produced by the cells expressing fusion proteins (data not shown). this suggests that folding and processing were completed if not before completion of translation then before the sequestration into inclusion bodies. thus by whatever means the proteinase matures in its usual eukaryotic context seems to be facilitated in the bacterial cell, though not necessarily by analogous interactions. the precise nature of the interaction between mp2 and cellular components (membranes) necessary for maturation of 3clpro and its role in the mechanism of 3clpro processing (maturation or release) remain to be determined. we do not know as yet whether the membrane requirement for maturation of 3clpro also reflects a need for other associated cellular components such as chaperone functions. the fact that processing is able to proceed in an heterologous environment offers a possible means of investigating this further. in order to simplify purification of 3clpro, we introduced a histidine (his 6 ) tag which facilitates separation on a suitable metal chelation matrix. the tag could not be placed at the extreme n or c-termini of the protein as is usual since the cleavage dipeptide and surrounding sequences are crucial for correct processing (ryan and flint, 1997) . we introduced the tag into the nearest region of predicted hydrophilicity to the c-terminal processing site q/s4 so as to minimise interference with the sequences involved in processing and allow interaction of the exposed histidine residues with the purification matrix. the tagged proteinase (3clproh6) appears to be indistinguishable from wild-type and is readily purified to high purity. however, the affinity between modified proteinase and matrix is low and similar to background proteins suggesting that the tag may not be fully accessible. our observation seems to conflict with the interpretation of a mutational analysis of hcv 229e 3clpro by ziebuhr et al., 1997 which concluded that the c-terminus of 3clpro was important to function and intolerant of mutation. the his 6 -tag mutation here (a substitution) was well tolerated and would therefore appear to indicate a so called 'permissive site' in the c-terminus of the protein. we have shown by trans cleavage analysis that processing occurs at the majority of the predicted 3clpro cleavage dipeptides within the ibv orf1 polyprotein and that no further cleavages are apparent. our data reinforce recently reported in vivo studies and also partially overcome a potential shortcoming of these approaches where some products may not be consistently detectable, due to poor reactivity with antisera or short half life for example. this analysis tentatively completes the processing map of the orf1 region with respect to 3clpro. there appear to be no target sites for 3clpro in the n-terminal half of the orf1a polyprotein (covered by the 1a3 clone) so that processing of this region and possibly regions upstream is probably achieved through the plp domain (liu et al., 1995) , alone or in combination with other as yet unidentified proteinase activities. those target dipeptides that were not cleaved in our assays are associated with the hydrophobic domains. cleavage of the mp1 region downstream of 3clpro may be carried out by the plp domain or an alternative activity, although failure for 3clpro to cleave q/g 2 may reflect a shortcoming in the presentation of the targets. the data presented here also suggest that there is no processing between q/s 3 (the c-terminus of the 3clp domain) and q/s 7 so that the mp2 product containing uncleaved q/g 5 and q/g 6 dipeptides would be expected to be approximately 42 kda. notably these three hydrophobic region targets are not represented in either mhv or hcv 229e, although, along with ibv itself, these viruses bear a potential target at a position equivalent to just downstream of the ibv q/g 6 site. these possibilities and the assembly of processed products into polymerase complexes may be addressed using specific antisera raised to amino or carboxyl terminal regions of processing products indicated by the present studies. completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot the coronavirus non-structural proteins coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis characterization of a 105-kda polypeptide encoded in gene 1 of the human coronavirus hcv 229e nucleotide sequence of the human coronavirus 229e rna polymerase locus identification and subcellular localization of a 41 kda, polyprotein 1ab processing product in human coronavirus 229e-infected cells identification of an atpase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229e a simple method for displaying the hydropathic character of a protein screening for membrane-permeabilizing mutants of the poliovirus protein 3ab the complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and rna polymerase a 100-kilodalton polypeptide encoded by open reading frame (orf) 1b of the coronavirus infectious bronchitis virus is processed by orf 1a products 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 identification, expression, and processing of an 87-kda polypeptide encoded by orf 1a of the coronavirus infectious bronchitis virus proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites intracellular and in vitro-translated 27-kda proteins contain the 3c-like proteinase activity of the coronavirus mhv-a59 identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a59 recombination of protein domains facilitated by co-translational folding in eukaryotes 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 virus-encoded proteinases of the picornavirus super-group expression and characterization of a recombinant murine coronavirus 3c-like proteinase single-step purification of polypeptides expressed in escherichia coli as fusions with glutathione s-transferase use of bacteriophage t7 lysozyme to improve an inducible t7 expression system use of t7 rna polymerase to direct expression of cloned genes characterization in vitro of an autocatalytic processing activity associated with the predicted 3c-like proteinase domain of the coronavirus avian infectious bronchitis virus a region of the infectious bronchitis virus 1a polyprotein encoding the 3c-like protease domain is subject to rapid turnover when expressed in rabbit reticulocyte lysate characterization of a human coronavirus (strain 229e) 3c-like proteinase activity biosynthesis, purification, and characterization of the human coronavirus 229e 3c-like proteinase we thank ian brierley for critical reading of the manuscript. this work was supported by the wellcome trust, the biotechnology and biological sciences research council and the ministry of agriculture, fisheries and food, uk. key: cord-266025-bkm486jd authors: tao, ying; tang, kevin; shi, mang; conrardy, christina; li, kenneth s.m.; lau, susanna k.p.; anderson, larry j.; tong, suxiang title: genomic characterization of seven distinct bat coronaviruses in kenya() date: 2012-04-26 journal: virus res doi: 10.1016/j.virusres.2012.04.007 sha: doc_id: 266025 cord_uid: bkm486jd to better understand the genetic diversity and genomic features of 41 coronaviruses (covs) identified from kenya bats in 2006, seven covs as representatives of seven different phylogenetic groups identified from partial polymerase gene sequences, were subjected to extensive genomic sequencing. as a result, 15–16 kb nucleotide sequences encoding complete rna dependent rna polymerase, spike, envelope, membrane, and nucleocapsid proteins plus other open reading frames (orfs) were generated. sequences analysis confirmed that the covs from kenya bats are divergent members of alphacoronavirus and betacoronavirus genera. furthermore, the covs btky22, btky41, and btky43 in alphacoronavirus genus and btky24 in betacoronavirus genus are likely representatives of 4 novel cov species. btky27 and btky33 are members of the established bat cov species in alphacoronavirus genus and btky06 is a member of the established bat cov species in betacoronavirus genus. the genome organization of these seven covs is similar to other known covs from the same groups except for differences in the number of putative orfs following the n gene. the present results confirm a significant diversity of covs circulating in kenya bats. these kenya bat covs are phylogenetically distant from any previously described human and animal covs. however, because of the examples of host switching among covs after relatively minor sequence changes in s1 domain of spike protein, a further surveillance in animal reservoirs and understanding the interface between host susceptibility is critical for predicting and preventing the potential threat of bat covs to public health. coronaviruses (covs) are large, enveloped viruses containing linear, positive-sense, single-stranded rna genomes. their genomes range approximately from 27-to 32-kb in length and contain 7-14 open reading frames (orfs) (woo et al., 2009a) . six major orfs encoding polymerase complex (orf1a and orf1b), spike glycoprotein (s), envelope protein (e), membrane glycoprotein (m), and nucleocapsid protein (n) are present in all covs . in addition, up to seven putative accessory orfs and one orf encoding hemagglutinin-esterase glycoprotein (he) are interspersed between the six major orfs. the numbers and sizes of these accessory orfs differ markedly among covs (woo et al., 2009a) . covs have been identified from a broad range of birds and mammals including humans in which they can cause respiratory, enteric, hepatic and neurologic diseases of varying severity (weiss and navas-martin, 2005) . covs in the subfamily coronavirinae are classified into three genera, alphacoronavirus, betacoronavirus, and gammacoronavirus (former serogroups 1-3) (de groot et al., 2011) . alpha-and beta-coronaviruses have been exclusively isolated from mammals and majority of gamma-coronaviruses from birds. covs of a distinctive lineage were recently detected from birds and pigs (chu et al., 2011; woo et al., 2009b woo et al., , 2012 and have been proposed to belong to a new genus, provisionally named deltacoronavirus (de groot et al., 2011) . the finding that the outbreak of severe acute respiratory syndrome (sars) in early 2003 was caused by a novel cov (sars-cov) has boosted interest in the search for novel covs in humans and animals. at least 30 previously unrecognized distinctive covs from human and various animal reservoirs were reported during recent years, including sars-related covs and covs from all genera in the subfamily coronavirinae which have significantly expanded our understanding of cov diversity and complexity (woo et al., 2009a) . based on available data, bats appear to harbor a great diversity of covs. the frequency and diversity of cov detection in bats, now in multiple continents, suggest that bats are likely a source for cov introduction into other species globally and possibly play an important role in the ecology and evolution of covs. recently we reported the identification of 41 divergent covs in bats from kenya, based on limited orf1b sequences (tong et al., 2009) . these newly discovered bat covs were grouped into 8 different phylogenetic clusters. of these, five clusters belonged to previously identified alphacoronavirus genus, and three clusters belonged to previously identified betacoronavirus genus, including a sars-related cov lineage. in the present study, we expand our sequence data for seven covs, representing 7 of the 8 distinctive clusters we identified in kenya bats during 2006 summer (tong et al., 2009) . the sample representing the eighth cluster of a sars-related cov was a weak positive and had limited specimen amount, therefore further sequencing studies were not included in this analysis. the purpose of our study was to further characterize the genomes and refine the phylogenetic relationships of these seven covs with other covs, based on the orfs 1b, s, e, m, and n. kenya was chosen as a major comparative old world study location in africa as part of the cdc global disease detection program. detailed information on bat capture and sampling is available in the previous publication (tong et al., 2009 ). the protocols for animal capture and use were approved by the cdc animal institutional care and use committee and the ethics and animal care and use committee of the kenya wildlife service (nairobi, kenya). in brief, representative samples at each site were collected from bats of available species, including adult and juvenile of both sexes. after euthanasia, a complete necropsy was performed in compliance with the approved field protocols. samples included blood, various organs (liver, lung, and kidney), rectal and oral swabs. in this study, seven cov-positive rectal swabs were selected as representatives of the seven different phylogenetic groups (tong et al., 2009) (tong et al., 2009) . total nucleic acids (tna) were extracted by using the qiaamp minelute virus spin kit (qiagen, santa clarita, ca) according to the manufacturer's instructions from 200 l of phosphate buffered saline suspension of the rectal swab and homogenized organ tissues (liver, lung, and/or kidney) of each bat except for bats btky33 and btky43 whose organ tissues were not available. the tna was eluted in 80 l depc-treated water and then stored at −80 • c. each cov-positive result on the rectal swab included in this study was repeated from different tna aliquots. the presence of cov rna in organ tissues of these bats was determined using the pan cov rt-pcr assays as described previously (tong et al., 2009) and the sequence specific and/or group specific cov rt-pcr assays (table s1 ). the rt-pcr were performed as described previously (tong et al., 2009) . standard precautions were taken to avoid cross-contamination of samples before and after rna extraction and amplification. purified dna amplicons were sequenced with the rt-pcr primers on an abi prism 3130 automated capillary sequencer using a bigdye terminator v3.1 cycle sequencing kit (applied biosystems, carlsbad, ca). high throughput 454 pyrosequencing on cov rna-positive bat samples was initially attempted, but failed to acquire any cov-associated reads due to lower sensitivity. therefore the rt-pcr-amplicon sequencing by sanger chain-termination method was chosen in this study. each of the seven contiguous sequences was obtained by using 4-6 pairs of semi-nested or nested consensus degenerate group specific primers and 4-7 pairs of semi-nested or nested sequence-specific bridging primers which generated a series of 8-13 overlapping fragments covering 15-16kb genomic sequences at the 3 end (table s1 ). the other half genome sequence containing the orf1a, was not recovered in this analysis due to the limited amount of rectal swab samples. consensus degenerate primers of each group were designed from conserved sequences of known members of the corresponding sequence group or its close group based on codehop strategy (rose et al., 1998) . the 3 end of genome sequence was determined using the 3 race kit (roche, indianapolis, in) according to the manufacturer's instructions. semi-nested or nested primers were used to improve the pcr sensitivity. when nested primers were not available, the pcr product was re-amplified using the same rt-pcr primers. the rt-pcr reactions were performed with superscript iii one-step rt-pcr high fidelity kit (invitrogen, san diego, ca) according to the manufacturer's instructions, and the second round rcr reactions were performed with accuprime taq dna polymerase high fidelity kit (invitrogen, san diego, ca). the rt-pcr products were visualized on 1% agarose gels containing 0.5 g/ml of ethidium bromide, and purified by qiaquick pcr purification kit (qiagen, santa clarita, ca). the rt-pcr amplicons for each sample were first sequenced with the consensus degenerate rt-pcr primers in both directions, and then the remaining internal gaps and 3 end genome were sequenced with sequence-specific bridging primers in both directions as described previously. the genomic sequences (orf1b, s, orf3, e, m, and n) of btky22, btky33, btky27, btky41, btky43, btky06, and btky24 were deposited in ncbi genbank (hq728480-hq728486). sequences were assembled in sequencher (genecodes, ann arbor, mi). each putative orf was predicted using the ncbi orf finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). nglycosylation sites were predicted using netnglyc 1.0 server (http://www.cbs.dtu.dk/services/netnglyc/). blast analyses were performed against ncbi non-redundant protein database (altschul et al., 1990) and against the conserved domain database for protein classification (cdd) (marchler-bauer et al., 2005) to characterize the putative orfs. alignments of the seven kenya bat cov gene sequences with a representative set of 43 other cov sequences, available in the public domain, were performed using the muscle v3.6 (edgar, 2004) . we constructed maximum likelihood trees for each gene alignment (orf1b, s, e, m, and n) in mega software package v5.0 (tamura et al., 2011) with 1000 bootstrap replications. we used general-time-reversible nucleotide (nt) substitution model with 4 categories of gamma distributed rate heterogeneity and a proportion of invariant sites (gtr + ␥4 + i). to identify potential recombination events of the seven kenya bat covs, three methods implemented in recombination detection program rdp version 2 were used, including maxchi (smith, 1992) , chimaera (posada et al., 2002) , and geneconv (padidam et al., 1999) . events detected by all three methods with default parameters were considered as potential recombination events. the aliquots of bat rectal samples for btky27, btky33, btky22, btky41, btky43, btky24, and btky06 were confirmed positive by the pan cov rt-pcr assay, while among tissues (liver, lung, and/or kidney) that were available from bats btky27, btky22, btky41, btky24, and btky06, only the liver from bat btky22 (chaerephon sp.) and the kidney from bat btky24 (eidolon helvum) tested positive by rt-pcr. these data support an infection process rather than transit of ingested infected material through the digestive tract as the source of viral rna in rectal swabs, particularly because these bat species do not feed on vertebrates. negative results for other tissues may be explained by specific pathobiology and a limited tropism to the available tissues. each acquired cov genome sequence covers the complete orf1b, s protein, orf3, e protein, m protein, n protein, other putative orfs after n and the 3 end untranslated region with a poly a tail. the genome organization and size for each of the orfs are shown in fig. 1 and table 1 , respectively. they are similar to other known cov genome organization in the order of 5 -orf1b, s, orf3, e, m, and n-3 , but have a variable number of putative orfs downstream of the n gene. the sizes of these seven genomic sequences from orf1b to the 3 end are between ∼15k and ∼16k and their g + c contents are between 37.6% and 42.6%. btky27 has no evidence of a putative orf downstream of the n gene, but possesses a short untranslated region and poly-a tail similar to bat-cov 1a (chu et al., 2008) . btky22, btky33 and btky43 have one small putative orf (76-161 amino acids (aa)) downstream of the n with no significant homology to previously described cov orfs. btky06 and btky24 have two small putative orfs downstream of the n with sequence similarity to ns7a and ns7b in bat-cov hku9, respectively . btky41 has two small putative orfs downstream of the n, which are overlapped and have no significant sequence homology to the previously described orfs. like most alphacoronaviruses, the btky27, btky33, btky22, btky41, and btky43 viruses share a core sequence 5 -cuaaac-3 or similar putative transcription regulatory sequence (trs) upstream of orfs s, m, n, and orfx and orfy (table 1) (chu et al., 2008; woo et al., 2005) . orf3 and e have putative core trss that sometimes varied from that for the other orfs. the btky06 and btky24 have a core sequence trs 5 -acgaac-3 in the upstream of each orf except e which has a core sequence trs 5 -ucgaac-3 (table 1) . spike proteins are the type i glycosylated membrane proteins, with a putative signal peptide at the n terminal. there are 31, 27, 28, 25, 31, 20, and 19 potential n-glycosylation sites in btky22, btky27, btky33, btky41, btky43, btky24, and btky06, respectively. as shown in fig. 2 , spike proteins of the seven bat covs lack furin protease recognition site, such as rradr-s in murine hepatitis virus (mhv), rrsrg-a in human cov oc43 (hcov oc43), rrsrr-a in bovine cov (bcov) (follis et al., 2006) , and cathepsin l cleavage site (vayt-m) as in sars-cov (bosch et al., 2008) . in spite of lacking conserved cleavage sites, they all consist of two domains, s1 and s2, showing the conserved gxcx motif in s1 around the cleavage site and the conserved nonamer motif iptnfsisi or similar motif in s2. these motifs have been observed in other known covs (follis et al., 2006) . the s1 is responsible for virus binding to the receptor on the target cells and may contain receptor binding domains (rbds) that directly bind to host cellular receptors. for example, the rbds of hcov 229e, tgev, and hcov nl63 in alphacoronavirus are mapped at the c terminus of their s1 domain (bonavia et al., 2003; godet et al., 1994; lin et al., 2008) . the rbds of mhv and sars-cov in betacoronavirus are mapped at n terminus and central region of s1 domain, respectively lin et al., 2008) . alignment of aa sequences of s1 regions from btky22, btky27, btky33, btky41, and btky43 of alphacoronavirus with the corresponding known rbd s1 regions of hcov 229e, tgev, and hcov nl 63 showed 33-41% identity in s1 rbd domains to hcov 229e and 24-29% identity to tgev and hcov nl63 ( fig. s1a -c). btky24 and btky06 from betacoronavirus are quite different in the corresponding rbd s1 regions from sars-cov and mhv (17-19% identity) ( fig. s1d-e) . the dissimilarity of s1 regions of these bat covs to other covs may suggest their different host specificity. we constructed phylogenetic trees using maximum likelihood method based on nt sequences of orf1b, s, e, m and n genes with representative viruses whose corresponding sequences of their genomes were available (fig. 3) . the phylogeny of e gene is not shown due to the short length and limited value for inferring species phylogenies. similar topologies were observed in the phylogenetic trees based on each of 5 orfs (fig. 3) . the analysis revealed that among the seven bat covs, five belonged to alphacoronavirus while the other two belonged to betacoronavirus (fig. 3) . phylogenetic clusterings within alphacoronavirus varied slightly when different genes were analyzed. for example, btky22 and btky43 grouped into one monophyletic clade in orf1b tree while they were grouped differently in the s and n gene trees with generally insignificant bootstrap values (fig. 3) . although recombination was suspected, we found no evidence of recombination in the seven analyzed viruses using maxchi (smith, 1992) , chimaera (posada et al., 2002) , and geneconv (padidam et al., 1999) . since the analyses were based on representatives from each cov species, the results suggest a lack of inter-species recombination in these viruses. one explanation is that the recombination frequency decreases significantly when the sequence divergence is high (kleiboeker et al., 2005; van vugt et al., 2001) . alternatively, the lack of inter-specie recombination is due to rare co-infections as the viruses adapted to different bats species. therefore, the phylogenetic incongruence observed in the gene trees is probably due to low phylogenetic signals, which may be improved by sampling more covs that are related to btky22 and btky43. the pairwise nt comparisons among these seven bat cov gene sequences revealed 67-76% overall nt identity. among the five alphacoronaviruses, three (btky22, btky41 and btky43) were distantly related to other known alphacoronaviruses with only 69-71% overall nt identity and with <90% aa identity in all five conserved domains (nsps 12-16) of orf1b (table 2 ). since we were not able to obtain all the genome portions necessary for definite species classification (de groot et al., 2011), we adopted the separation criteria based on the rdrp group units (rgu) (drexler et al., 2010) . the aa distances in the 816 bp fragment of the rdrp gene from the kenya bat covs described in this study were compared to the aa sequences from their close reference viruses (table s2) . btky22, btky41, and btky43 had >4.8% aa distance in the rdrp fragment (table s2 ). this suggests that they are most likely three distinctive alphacoronvirus species. btky27 and btky33 identified in miniopterus bats were closely related to bat-cov 1a, which was identified from bent-winged miniopterus bat in hong kong (chu et al., 2006) with 85% and 75% overall nt identity and with >90% aa identity in 5/5 and 4/5 conserved domains (nsps 12-16) in orf1b, respectively (table 2 ). btky27 and btky33 had <4.8% aa distance in the 816 bp rdrp to their close reference viruses indicating that they are members of the established bat cov species in alphacoronavirus. as for the two members of betacoronavirus genus identified, one (btky06 identified in rousettus aegyptiacus bat) was likely a member of bat-cov hku9 species identified from rousettus leschenaulti bat in china , sharing 90% overall nt identity and 99% aa identity in 4/5 conserved domains (nsps12-16) in orf1b ( table 2 ). the other (btky24) was distantly related to other known betacoronaviruses with ≤70% overall nt identity and <90% aa identity in all 5 conserved domains (nsps 12-16) from orf1b (table 2) . additionally, based on the rgu criteria, btky24 had >6.3% aa distance in the 816 bp rdrp fragment compared to its closest reference virus indicating that it is most likely a distinctive betacoronavirus. in conclusion, sequence data for the structural and nonstructural orfs in the 3 -end of the genome of seven kenya bat covs confirmed the high diversity and their phylogenetical placement into alphacoronavirus and betacoronavirus genera. the four clusters of kenya bat covs represented by btky22, btky41, btky43, and btky24 respectively, most likely belonged to novel cov species, the two clusters represented by btky27 and btky33 were likely members of bat-cov 1a, and the cluster represented by btky06 was likely a member of bat-cov hku9 species. as noted with other novel covs, the genome organization is similar but differences were found in the number of putative orfs downstream from the orf n. the present results are in line with previous findings of extensive diversity of covs detected in bats and confirm that bat covs mainly belong to the alphacoronavirus and betacoronavirus genera tang et al., 2006; woo et al., 2007 woo et al., , 2009b . consistent with other reports, none of the bat covs characterized in the present study was sufficiently similar to the human sars-cov and other human covs to be suggested their direct progenitors. the examples of host switching among covs after relatively minor sequence changes in s1 domain of spike protein (haijema et al., 2003; kuo et al., 2000; qu et al., 2005) suggest the potential risks for introduction into humans as occurred with sars-cov. therefore characterization of novel covs and understanding species diversity in animals should help understand and respond to emerging zoonotic infections. basic local alignment search tool identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov-229e cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide avian coronavirus in wild aquatic birds genomic characterizations of bat coronaviruses (1a, 1b and hku8) and evidence for co-infections in miniopterus bats coronaviruses in bent-winged bats (miniopterus spp family coronaviridae genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences muscle: multiple sequence alignment with high accuracy and high throughput furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein switching species tropism: an effective way to manipulate the feline coronavirus genome simultaneous detection of north american and european porcine reproductive and respiratory syndrome virus using real-time quantitative reverse transcriptase-pcr retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats complete genome sequence of bat coronavirus hku2 from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome structure of sars coronavirus spike receptor-binding domain complexed with receptor identification of residues in the receptor-binding domain (rbd) of the spike protein of human coronavirus nl63 that are critical for the rbd-ace2 receptor interaction cdd: a conserved domain database for protein classification rdp2: recombination detection and analysis from sequence alignments possible emergence of new geminiviruses by frequent recombination identification of a novel coronavirus in bats recombination in evolutionary genomics identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences analyzing the mosaic structure of genes mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods prevalence and genetic diversity of coronaviruses in bats from china detection of novel sars-like and other coronaviruses in bats from kenya high frequency rna recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia coronavirus diversity, phylogeny and interspecies jumping comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features we thank ivan kuzmin, michael niezgoda, and charles e. rupprecht from division of high consequence pathogens and pathology, cdc, atlanta, ga; robert f. breiman from global disease detection division, cdc-kenya, nairobi, kenya; and bernard agwanda from national museum, kenya wildlife service, nairobi, kenya for excellent technical and logistical assistance and field study. the study was supported in part by the global disease detection program of cdc (atlanta, ga). supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2012.04.007. key: cord-255857-y9wjp0aj authors: yuan, shishan; mickelson, daniel; murtaugh, michael p.; faaberg, kay s. title: erratum to “complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains” date: 2001-11-05 journal: virus res doi: 10.1016/s0168-1702(01)00295-7 sha: doc_id: 255857 cord_uid: y9wjp0aj two full-length porcine reproductive and respiratory syndrome virus (prrsv) genomes, strain vr-2332 and its cell culture passaged descendent respprrs vaccine strain, were compared and analyzed in order to identify possible sites of attenuation. of the 41 nucleotide changes, 12 resulted in conservative changes and 18 produced non-conservative changes. the results suggest that key amino acids in orf1 may contribute to the phenotype of respprrs, which includes increased growth rate on ma-104 cells and decreased virulence in swine. the results provide a genetic basis for future manipulation of a prrsv reverse genetics system. porcine reproductive and respiratory syndrome virus (prrsv) was first recognized as a 'mystery swine disease' in the united states in 1987 (keffaber, 1989 ). since the virus was identified in europe (lelystad virus; wensvoort et al., 1991) and in the usa (vr2332; benfield et al., 1992; collins et al., 1992) , prrsv has become a serious pathogen of swine herds world-wide (rossow et al., 1999) . prrsv, along with lactate dehydrogenase-elevating virus (ldv), equine arteritis virus (eav) and simian hemorrhagic fever virus (shfv), is a member of the family arteriviridae in the order nidovirales (cavanagh et al., 1990; plagemann and moennig, 1992) . prrsv causes respiratory disease in young swine and production of mummified, weakborn or aborted piglets in pregnant sows through an unresolved mechanism. clues to the genetic basis of pathogenicity can be gleaned from comparison of the nucleotide sequence of a virulent parent viral strain with that of the cell-passaged attenuated variant. one such pair is the prototype north american isolate, strain vr-2332, and resp-prrs, its ma-104 cell adapted attenuated descendent. in this article, we report the complete nucleotide sequence of both strains of prrsv and have deduced and characterized their nucleotide and protein differences. several key amino acid changes between parental and vaccine strains were identified using this approach, but many other nucleotide and amino acid changes also occurred in genomic regions of unknown function. the nucleotide sequences were also compared to strain 16244b, which is claimed to be a virulent field revertant of respprrs (allende et al., 2000) . a full comparison of all three prrsv strains shows that the origin of 16244b ('michelle' strain) is unclear. prrsv strains vr-2332 and respprrs have been described in previous reports nelsen et al., 1999; yuan et al., 1999) . strain 16244b (michelle) was described by allende et al. (1999) . for growth curve analysis, ma-104 cells were infected with each prrsv strain at an m.o.i. of 0.1. after 1 h of adsorption, the unattached virus was washed off of the cells and 5 ml of fresh cell medium (emem/ 10% fbs or dmem/2% swine serum) was added. the culture was placed at 37°c, 5% co 2 and 0.5-ml aliquots of virus were removed and replaced with 0.5 ml fresh medium at 4, 8, 12, 16, 24, 36, 48, 60, and 72 h. viral plaque assays of each time point were completed on ma-104 cells with a 5-ml agar overlay (an equal volume of mixture of 2% low gelling point agarose (fmc seaplaque) and 2 ×mem medium). incubation at 37°c, 5% co 2 for 96 -120 h revealed the presence of plaques, which were quantitated for each time point. prrsv was harvested from infected ma-104 cells on day 5 post-infection (p.i.). after pelleting of cellular debris at 12000 rev./min, the supernatants were layered onto a 2 ml 0.5 m sucrose cushion in an sw 27 ultracentrifuge tube and centrifuged at 24000 rev./min for 3 h. the pelleted virions were resuspended in ste buffer (200 ml; 10 mm nacl/10 mm tris ph 7.5/1 mm edta, 4°c) and transferred to a microcentrifuge tube. another 200 ml of ste was used to collect any remaining disrupted virions and the two fractions of viral suspension were pooled and stored at − 20°c. viral rna was isolated using the qiaamp viral rna kit (qiagen) and stored at − 80°c after quantitation by optical density and native rna agarose gel electrophoresis. approximately 1 mg of purified viral rna in 6 ml was added to 2 ml of 2 mm reverse primer /1a-p3668 (5%-ggt-cgttgacaagttggtcatctaccggttt-atcctcgga), incubated at 67°c for 10 min and placed on ice. first strand cdna synthesis was then completed as described previously (yuan et al., 1999) . the reverse transcribed product was purified (microcon 100, amicon) and the cdna was eluted in 40 ml of rnase-free water. multiple adenosine or guanosine residues were added to the 3%-end of purified prrsv cdna ( 1 mg) using a terminal deoxynucleotide transferase procedure described by the manufacturer (tdt; new england biolabs), diluted to 200 ml and stored at − 20°c. first round pcr was completed as described previously using 2 mm qt (5%-ccagt-gagcagagtgacgaggactcgagctca-agcttttttttttttttttt) or qc (5%-cca-gtgagcagagtgacgaggactcgagct-caagcccccccccccccccccc) and 15 mm qo (5%-ccagtgagcagagtg-acg) as the forward primers and 15 mm /1a-p3668 as the reverse primer (frohman, 1994; yuan et al., 1999) . an identical second round of pcr was then completed using 10 ml first round pcr product and qi (5%-gaggactcgagctcaagc) and /1a-p1457 (5%-ccttcggcaggcggggagta-gtgtttgaggtgctcagc; 15 mm each) as the primer pair. automated sequencing reactions were completed with taq dyedeoxy terminator cycle sequencing kit (applied biosystems) using a pe 2400 thermocycler (perkin-elmer) at the univer-sity of minnesota advanced genetic analysis center. comparison of the parental and vaccine genomes and deduced orf amino acid comparison was completed using computer software included in the lasergene package (dnastar inc., madison, wi), wisconsin package version 10.0 and (genetics computer group (gcg), madison, wi). genbank accession numbers used for sequence analysis include the complete vr-2332 (u87392, nelsen et al., 1999) , respprrs (af066183; yuan et al., 1999) and 16244b (af046869) sequences. primer extension experiments had suggested that 20 nucleotides were unaccounted for in the published sequence of strain vr-2332 . in order to determine the unresolved bases, we derived several clones of pcr products generated from different passages of vr-2332 and respprrs (fig. 1) . initially, several standard 5%-race was performed on passage 2 from the original vr-2332 field isolate brain homogenate, passage 31 of strain vr-2332 and passage 4 of respprrs vaccine strain (frohman, 1994) . three different 5%-terminal base patterns were elucidated, with added ambiguity due to the poly t tract added during the 5%-race procedure, immediately preceding an apparent 18 base prrsv sequence gacguauagguguuggc. interestingly, heterogeneity at the 5%-end was detected early during passaging of the original prrsv field isolate, yet no heterogeneity was seen after viral plaque purification to produce strain vrfig. 1 . 5%-terminal nucleotides of strains vr-2332 and respprrs. vr-2332 (vr) or respprrs (r) from different passages were subjected to rapid amplification of cdna ends (race) and products were cloned and sequenced as described in section 2. vr-2332 represents sequence submitted to genbank prior to elucidation of the 5% terminal nucleotides. strain vr-2332-infected brain homogenate was passaged two times on two separate occasions (vr-2a, vr-2b), or plaque purified and passaged 24 (vr-24) or 31 (vr-31) times. respprrs was passaged four (r-4) or 12 (r-12) times. bold letters indicate previously unresolved bases and letter in italics refer to the non-viral nucleotides (t or c) arising from the race procedure. 2332 ( fig. 1 ). further analysis of pcr clones derived from 5%-race experiments using poly (g) addition discriminated the remaining 5% terminal bases of strains vr2332 and respprrs. only one additional base, corresponding to an adenine residue, was identified for strain vr-2332 ( fig. 1) . two additional bases, ta, were detected for the 5% terminal bases of four pcr clones derived from vaccine strain respprrs. the number of subsequent passages shown in fig. 1 (2, 4, 12, 24, 31) indicates the relative stability of the cloned virus strains. these results are discordant with a recent report with respect to the first nucleotide of vr2332 and resp-prrs. oleksiewicz and investigators reported a 5%-terminal u in strain vr-2332 and lack of such in strain respprrs (oleksiewicz et al. 1999) , whereas we observed a 5%-terminal u only in the respprrs vaccine strain. the complete leader sequence of respprrs vaccine strain shares 95.2% identity with another prrsv vaccine strain, primepac prrs (shen et al., 2000) (data not shown). we have determined that the genome size for vr-2332 is 15411 bases (u87392) and that of respprrs, with an extra 5%-terminal u, is 15412 bases (af066183). respprrs leader sequence acquired two additional mutations when passaged for another eight passages at high m.o.i. (ua base 36 and uc at base 63). from the sequence data, we cannot determine exactly when the 5%-terminal thymidine residue of strain respprrs was acquired during passaging. however, the possibility exists that strain respprrs has developed individual genomes of variable nucleotide sequence due to strain evolution over the course of more than 70 passages. the respprrs sequence which we derived from 3-to 7-fold coverage of the genome (af066183) is somewhat different from the sequence of re-spprrs recently reported (allende et al., 2000) . complete genome analysis of the parental strain vr-2332 and its cell-culture adapted descendant, strain respprrs, revealed that 41 nu-cleotides had changed during viral strain attenuation. the changes appeared throughout the genome ( fig. 2a) , except for orf7 and the 3% untranslated region (utr), and no single orf exhibited less than the 99% identity between parental and vaccine strains (fig. 2b) . of the 41 altered nucleotides, 10 resulted in silent mutations such that the encoded protein was not changed. one of these 10 silent mutations resided in the 5%-leader, 8 resided in orf 1, and one silent change was detected in orf3. the rest of the nucleotide changes resulted in amino acid changes in each of the remaining identified orfs. detailed analysis of the nucleotide changes, corresponding amino acid changes and potential coding domains are listed in table 1 . for ease of understanding, nucleotide numbering in table 1 is based on the sequence of vr-2332 (1-15411). the 5%-terminal uracil residue of strain respprrs is numbered as zero, and all site designations are one less than the value of the sequence deposited in genbank. the resolution of the 5% terminal sequence indicated that the 5% ends of both of these prrsv strains coded for a 34 aa peptide. however, no conclusive evidence has been generated to demonstrate the presence of this potential leader protein. the leader sequence of resp-prrs suggested some virus heterogeneity. in one case (table 1 , mutation 2a), u 36 a 36 (vr-2332 respprrs) coded for a conservative change at amino acid 12 (f12y). in the other case (table 1 , mutation 2b), the c 63 u 63 nucleotide change, located in the third base of the 21st codon in this polypeptide sequence, is silent. rna folding predictions (mfold; zucker, 1989) suggested that each of these nucleotide changes resulted in minor alterations in nucleotide-pairing in stems of secondary structure. the 5%-terminal bases, an a residue in strain vr-2332 and an at dinucleotide sequence in vaccine strain respprrs, are predicted to extend 5% from a long stable hairpin located immediately downstream between nucleotides 2 and 45 (data not shown). transversion (3) g c 41 a analysis of complete genome sequences and classification of amino acid changes were completed using gcg computational biology computer programs. predicted domains of viral proteins was based on hopp-woods analysis of peptide structure (jameson and wolf, 1988) were also completed using gcg. predicted orf1 non structural proteins (nsp) were derived from genome comparison of strain vr-2332 with prrsv strain 16244b (allende et al., 1999) and with equine arteritis virus van dinten et al., 1999) . b represent two separate sequence analyses of respprrs leader (fig. 1) . full-genome schematic the nucleotide differences between vr-2332 and respprrs reveals several changes occurred in orf1a, a cluster of changes were seen in the 3%-terminus of orf1b, and discrete changes were seen in all envelope glycoproteins (orfs 2 -5) and in the membrane protein (orf6). when respprrs is similarly compared to strain 16244b, many nucleotide changes are seen throughout the genome. (b) each region of the prrsv genome was analyzed for the number of nucleotide changes and the corresponding orf percent identity between vr-2332 and respprrs. several prrsv orf 1 replicase protein domains have been identified (fig. 3 ; snijder and meulenberg, 1998; allende et al., 1999; . from the analysis of both vr-2332 and respprrs genomes, it was deduced that only four of the 15 orf 1 specific nucleotide changes occurred in these identified domains. one mutation, a c 1181 u 1181 transition, which would alter amino acid 331 of orf 1 from a serine residue in strain vr-2332 to a phenylalanine in the attenuated strain respprrs (s331f), resulted in a nonconservative change located in the putative orf1 cleavage product, nsp2 (den boon et al., 1995; allende et al., 1999 allende et al., , 2000 nelsen et al., 1999) . a second recognizable mutation, a u 10533 c 10533 transition, resulted in a change from tyrosine to histidine within the helicase domain. the final orf1 recognizable mutation was located in the coronavirus-like domain. in this instance, the transversion from g 11329 c 11329 resulted in amino acid 3714 undergoing a non-conservative change from glycine to alanine. a glycine residue at this position, except for strain respprrs, has been shown to be conserved in all nidoviruses analyzed to date . all other amino acid changes located within orf1 are in regions of unknown function. however, the cluster of amino acid mutations located near the carboxyl terminal end suggests that the replicase region was altered during passage to result in a more fit virus for replication in cell culture, as evidenced by the in vitro one-step growth curve comparison shown in fig. 4 . the growth curve comparison appears to be the reverse of virus strain replication in swine (m. roof, unpublished data). prrsv orfs 2 -7 have been shown to code for four glycoproteins (gp2-5), the membrane protein (m) and the nucleocapsid protein (n), respectively. sequence analysis of strains vr-2332 and respprrs indicated that there were 15 nucleotide changes in this region, and all but one of which resulted in amino acid alterations. there were four amino acid mutations within gp2. one amino acid change (aa10, lf) was located within the predicted signal sequence of gp2, but the nucleotide mutation (g 12102 u 12102 ) also altered amino acid 9 from an aspartic acid to a tyrosine of a putative orf2a protein (wu, christopher-hennings, and nelson, unpublished data) . the remaining three gp2 changes (aa 122, a s; aa 128, k r; aa 130, v m) were clustered in an eight amino acid region in the middle of the protein sequence, predicted to be located on the exterior of the virion. the three amino acid fig. 4 . viral growth curves of prrsv strains vr-2332 () and respprrs () reveal that respprrs has enhanced growth kinetics in vitro. results are representative of three separate experiments. the number of plaques were determined in triplicate for two separate viral dilutions and were found to deviate by less than 5% in each individual experiment. vealed that this mutation had reverted back to a vr-2332-like sequence while maintaining all other orf 5 and 6 mutations and the avirulent viral phenotype (data not shown). the other m protein mutation was shown to be the result of two nucleotide changes within the codon for amino acid 121, altering the sequence from an arginine to a glycine (r g). curiously, both carboxyl terminal gp5 and m mutations resulted in a r g change. in 1997, prrsv strain 16244b was isolated from a nebraska herd and has since been shown to be virulent (allende et al., 1999 (allende et al., , 2000 . full-length genome sequencing revealed that this strain was 98% identical to vr-2332 and respprrs (allende et al., 2000) . whether the isolate is a naturally occurring prrsv field variant, or directly related to the original vr-2332 field isolate (isolated in 1989) or the respprrs vaccine strain (released in 1994) is not known. to address this question, a full-length genome comparison of strain resp-prrs to strain 16244b was completed, as it has been suggested that strain 16244b is a field revertant of respprrs (allende et al., 2000) (fig. 2a) . as can be seen in fig. 2a , strain 16244b differs from respprrs at many nucleic acid residues. all three strains were analyzed in detail. we compared identical and non-identical nucleic acid residues between strains vr-2332 and respprrs, strains vr-2332 and 16244b, between respprrs and 16244b, and between all three strains. we observed 15157 instances in which all three viral genomes contained the identical base, 215 instances in which strains vr-2332 and respprrs were identical but different from strain 16244b, 17 cases in which strains vr-2332 and 16244b were identical but different from strain respprrs, and 22 instances in which strains respprrs and 16244b were identical but different from vr-2332. importantly, no instances were observed in which the three strains were all different from one another. if strain 16244b were in fact derived from a field reversion of respprrs, the data would imply that for 237 nucleotide mutations 16244b reverted back to the exact vr-2332 nucleotide sequence and in no modifications that occurred in gp3 (aa 83, ge; aa 116, g s; aa 251, a t) and the two in gp4 (aa 69, c y; aa 135, v a) also are predicted to lie on the exterior of the virion. all of these mutations but one are semi-or non-conservative changes. in the putative viral attachment protein gp5, two amino acids were altered during virus attenuation. one gp5 change occurred within the proposed signal sequence (aa 13, r q) and the other change (aa 151, rg) is predicted to be located in the virion interior . gp5 has been shown to be disulfide linked to the m protein mardassi et al., 1996) and studies with a similar virus have revealed that this disulfide bond is critical to infectivity . while no mutations occurred in the extravirion region of gp5, one mutation did occur within the m protein (aa 16, q e) predicted to be located on the outer surface of the virus. although this m protein mutation was conservative, mutations in this critical region of the prrsv viral attachment heterodimer could be important. however, analysis of the sixth in vivo backpassage of respprrs re-instance would it have mutated to either of the two possible remaining bases. the likelihood of such a probability is exceedingly low. we also considered the possibility that strain 16244b resulted from a viral recombination event between strain respprrs and an unidentified but closely related field isolate. however, no region of considerable length was clearly identified as being derived from strain respprrs, which one would expect if viral recombination had taken place. therefore, the origin of 16244b remains unclear. an attenuated prrsv strain, respprrs, was found to differ in sequence from the parental strain, vr-2332, by only 41 nucleotides. this represents a 0.28% variation during 70 passages in ma-104 cells. this rate of variation is comparable to other rna viruses (steinhauer and holland, 1987) and suggests that prrsv is not susceptible to inherently high spontaneous mutation rates in the absence of immunological and environmental pressures. this may explain the relative stability of strain vr-2332 when passaged alone in cell culture, as this report delineates. however, prrsv has been shown to undergo high frequency recombination in the presence of two or more virus strains (yuan et al., 1999) and appears to evolve rapidly in the presence of biological pressure. attenuation can result from changes in many areas of viral genomes and the 41 nucleotide mutations described include alterations in several key prrsv regions. thus, no definitive site of prrsv attenuation can be identified simply from sequence analysis. twelve of the 41 nucleotide changes in strain respprrs were silent. however, one or more of these silent mutations could change the secondary or tertiary structure of prrsv rna and thus alter the stability of the genome. in other viruses, such as picornaviruses, viral rna folding may play a critical role in host protein association (meerovitch et al., 1989) and in neurovirulence (pilipenko et al., 1989a,b; westrop et al., 1989) . the 5% and 3% ends of viral sequences have been correlated with attenuation in other viruses, such as poliovirus (westrop et al., 1989) , and shown to be important in viral replication and transcription for coronaviruses, another member of the nidovirus order (williams et al., 1999) . only two mutations occurred in the 5%-end of prrsv during attenuation of strain vr-2332 to respprrs, and their relative roles in attenuation must be further investigated. no change occurred in the 3%-end of the prrsv genome. viruses may also acquire attenuated phenotypes as a result of mutations in viral proteases (ni et al., 1995) , protease cleavage sites (tozser et al., 1999) , within the polymerase gene (pelosi et al., 1998; skiadopoulos et al., 1999) , by altering viral proteins to decrease virion stability (bailly et al., 2000) or to interfere with the virus/host interaction (mccright et al., 1999) , as well as by other mechanisms. the comparison of vr-2332 to its vaccine correlate, resp-prrs, revealed that the possibility exists for one or more of these mechanisms to be involved in attenuation. a recent publication suggested that nine specific amino acid changes may be important in attenuation of respprrs (allende et al., 2000) , possibly based on the assumption that prrsv attenuation may be localized by amino acid comparison of a non-parental genome (16244b) with a parental (vr-2332) and its vaccine offspring (respprrs). we have determined that our sequence of the respprrs strain is somewhat different from what has been previously reported (allende et al., 2000) , which brings additional complexity to the derivation of potential prrsv attenuation sites. in addition, the 5%-terminal nucleotides for both vr-2332 and respprrs are markedly different from those reported for strain 16244b. in our view, prrsv attenuation sites may be genotype specific and variable, possibly resulting from a complicated interplay of genomic regions and viral and host specific factors. postulation of attenuation sites can lead to interesting hypothesisbased experiments. however, a reverse genetics system for prrsv must be produced and genetically altered at specific nucleotides in order to discern sites of attenuation. north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype a recombinant human parainfluenza virus type 3 (piv3) in which the nucleocapsid n protein has been replaced by that of bovine piv3 is attenuated in primates characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc 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arteritis virus and simian hemorrhagic fever virus: a new group of positive stranded rna viruses porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterized by marked neurovirulence determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the nsp2 gene with a unique insertion identification of mutations contributing to the temperature-sensitive, cold-adapted, and attenuation phenotypes of the live-attenuated cold-passage 45 (cp45) human parainfluenza virus 3 candidate vaccine the molecular biology of arteriviruses identification of a novel structural protein of arteriviruses rapid evolution of rna viruses effect of serine and tyrosine phosphorylation on retroviral proteinase substrates proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication mystery swine disease in the netherlands: the isolation of lelystad virus genetic basis of attenuation of the sabin type 3 oral poliovirus vaccine a phylogenetically conserved hairpin-type 3% untranslated region pseudoknot functions in coronavirus rna replication recombination between north american strains of porcine reproductive and respiratory syndrome virus on finding all suboptimal foldings of an rna molecule the authors wish to thank chris nelsen and faith klebs for excellent technical expertise. boehringer ingelheim vetmedica, inc., provided financial support for the research. key: cord-270064-hidirfkv authors: tort, fernando l.; castells, matías; cristina, juan title: a comprehensive analysis of genome composition and codon usage patterns of emerging coronaviruses date: 2020-04-12 journal: virus res doi: 10.1016/j.virusres.2020.197976 sha: doc_id: 270064 cord_uid: hidirfkv an outbreak of atypical pneumonia caused by a novel betacoronavirus (βcov), named sars-cov-2 has been declared a public health emergency of international concern by the world health organization. in order to gain insight into the emergence, evolution and adaptation of sars-cov-2 viruses, a comprehensive analysis of genome composition and codon usage of βcov circulating in china was performed. a biased nucleotide composition was found for sars-cov-2 genome. this bias in genomic composition is reflected in its codon and amino acid usage patterns. the overall codon usage in sars-cov-2 is similar among themselves and slightly biased. most of the highly frequent codons are aand u-ending, which strongly suggests that mutational bias is the main force shaping codon usage in this virus. significant differences in relative synonymous codon usage frequencies among sars-cov-2 and human cells were found. these differences are due to codon usage preferences. the family coronaviridae consists of four genera, namely, alphacoronavirus (αcov), betacoronavirus (βcov), gammacoronavirus (γcov) and deltacoronavirus (δcov) (chen et al., 2020) . viruses from this family possess a single stranded, positive-sense rna genome ranging from 26 to 32 kilobases in length (su et al., 2016) . coronaviruses (cov) have been identified in several avian hosts, as well as in mammals, including humans, bats, civets, mice, dogs, cats, cows and camels (clark, 1993; cavanagh, 2007; zhou et al., 2018) . although several cov are pathogenic to humans, most of them are associated with mild clinical symptoms (su et al., 2016) . nevertheless, two notable exceptions have been described: severe acute respiratory syndrome (sars) coronavirus (sars-cov), a novel βcov that emerged in southern china in 2002 (peiris et al., 2004) and middle east respiratory syndrome (mers) coronavirus (mers-cov), which was first detected in saudi arabia in 2012 (zaki et al., 2012) . before the sars epidemic, bats were not known to be hosts for covs. in the last 15 years, bats have been found to be hosts of more than 30 covs. interactions among various bat species themselves, batanimal and bat-human interactions, such as the presence of live bats in wildlife wet markets in china, have been proved to be important for interspecies transmission of covs (wong et al., 2019) . in fact, both sars-cov and mers-cov likely originated in bats, and genetically diverse covs that are related to these viruses were discovered in bats worldwide (cui et al., 2019) . previous studies in different bat species from china permitted the identification of at least 41 new βcov, all of them were rhinolophus spp. bat-cov (li et al., 2017) . at this moment, in wuhan, the capital city of hubei province of the people's republic of china, an outbreak of atypical pneumonia caused by a novel coronavirus (sars-cov-2) is currently underway. the outbreak appears to have started from a zoonotic transmission at a market in wuhan where animals and meat were sold (chan et al., j o u r n a l p r e -p r o o f 2020a). as february 26th, 2020, there have been 77,780 cases of sars-cov-2 confirmed in china, including 2,666 deaths (who, 2020) . in addition, sars-cov-2 has been reported in 33 countries outside china, with 2,459 cases confirmed and 34 deaths (who, 2020) . very recent studies revealed that sars-cov-2 can be considered a new human-infecting βcov (lu et al., 2020) . the world health organization declare this 2019-ncov outbreak as a public health emergency of international concern on january 30 th , 2020 (who, 2020b) and the disease caused by this specific virus species have recently been designated as (who, 2020) . in this sense, the coronavirus study group of the international committee on taxonomy of viruses (ictv), formally recognizes this virus as a relative to severe acute respiratory syndrome sars-covs and designates it as severe acute respiratory syndrome coronavirus 2: sars-cov-2 (gorbalenya et al., 2020) . in order to gain insight into the emergence, evolution, adaptation and spread of the sars-cov-2 viruses, a comprehensive analysis of genome composition and codon usage of βcov circulating in china was performed. available and comparable complete genome sequences of 81 βcov strains isolated in china, including sars-cov-2 as well as βcov isolated from different hosts, were obtained from genbank database (available at: http://www.ncbi.nlm.nih.gov). for accession number, strain name, host and date of isolation, see supplementary material table 1 . for each strain orfs1a+orf1b+s+e+n+m were concatenated. sequences were aligned using the macse program (ranwez et al., 2011) . macse algorithm is a useful tool for accommodating sequencing errors and other biological deviations from j o u r n a l p r e -p r o o f the coding frame (ranwez et al., 2011) . the alignment is available upon request. the dataset comprised a total of 725,433 codons. codon usage, amino acid usage, dinucleotide frequencies, base composition, the relative synonymous codon usage (rscu) (sharp and lee, 1986) , total gc content, gc content in the third position of the codon (gc3s) and effective numbers of codons (enc) were calculated using the program codonw (written by john peden) as implemented in the galaxy server version 1.4.4 (afgan et al., 2018) . the relationship between compositional variables and samples was obtained using multivariate statistical analyses. principal component analysis (pca) is a type of multivariate analysis that allows a dimensionality reduction. singular value decomposition (svd) method was used to calculate the principal components (pc). unit variance was used as scaling method. by the same approach, heatmaps were also constructed, which is a data matrix for visualizing values in the dataset by the use of a color gradient. this gives a good overview of the largest and smallest values in the matrix. rows and/or columns of the matrix are clustered so that sets of rows or columns rather than individual ones can be interpreted. pca and heatmaps analysis were done using the clustvis program (metsalu et al., 2015) . the rscu values of human cells were obtained from kazusa database (available at: http://www.kazusa.or.jp/codon/). to study codon usage preferences in sars-cov-2 in relation to the codon usage of human cells, we employed the codon adaptation index (cai) (sharp & li, 1987) . cai was calculated using the approach of puigbo et al. (2008a) (available at: http://genomes.urv.es/caical) for human cells. this method allows to compare a given codon usage (in our case, sars-cov-2) to a predefined reference set (human). a j o u r n a l p r e -p r o o f statistically significant difference among cai values was addressed applying a wilcoxom & mann-whitney test (wessa, 2012) . in order to discern if statistically significant differences in the cai values arise from codon preferences, we used e-cai (puigbo et al., 2008b) to calculate the expected value of cai (ecai) at the 95 % confident interval. a kolmogorov-smirnov test for the expected cai was also performed (puigbo et al., 2008b) . in order to study the genetic composition of sars-cov-2 emerging in china, 81 orfs sequences from βcov strains isolated in china (including sars-cov-2 and sars-cov from humans and βcov isolated from bats, civets and ferrets) were aligned and the nucleotide frequencies at third codon position, total gc content, gc content at the third codon position, enc and dinucleotide frequencies were established for all strains orfs and pca was performed. the results of these studies are shown in figure 1 . heatmap analysis on genomic composition of all βcov enrolled in these studies revealed a distinct genome composition of sars-cov-2 strains isolated in china, in relation to all other strains isolated from humans as well as bats, civets or ferrets (see to study if these differences in genomic composition can be observed at codon and amino acid usage, these variables were established for all βcov strains enrolled in these studies and their relations were observed by heatmap analysis. the results of these studies are shown in figure 2 . significant differences in codon and amino acid usage was observed among sars-cov-2 strains and all other βcov strains included in these studies (see fig. 2 ). among β-cov strains isolated from bats, a significant variation was also observed. linkage analysis suggests a closer relation among sars-cov-2 and βcov isolated from bats, and a more distant relation with sars-cov and other β-cov isolated from civet and ferret enrolled in these analyses (fig. 2) . to gain insight into the genomic composition of sars-cov-2 strains, the nucleotide frequencies found for this virus where compared to the nucleotide frequencies found for other human covs. the results of these studies are shown in fig.3 . some general characteristics of covs were observed, since the u-frequencies is significantly above average, while the c-frequencies are below the expected frequencies. in the case of purines, a is preferred over g (fig. 3) . as it can be seen in fig. 3 , most variation occurs in the c/u and not the a/g section. moreover, the results of these studies also revealed the presence of species-specific trends, since the c/u ratio differs profoundly per coronavirus type (see fig.3 ). comparison of sars-cov-2 with the other two recent zoonotic transmission to the human population (sars and mers-covs) reveals that sars-cov-2 is quite extreme with a c-count of 18.4 % (fig. 3) . on the other hand, sars-cov-2 has the highest a-count among covs enrolled in these studies (29.9 %, mean a-count of 27.6 ± 1.1 for all human covs enrolled in these studies) (see fig. 3 ). to study the extent of codon usage bias in sars-cov-2, the enc's values were calculated for all sars-cov-2 strains enrolled in these studies. a mean value of 48.54 ± 2.34 was obtained. due to the fact that all values obtained were >40, the results of these studies suggest that the overall codon usage among sars-cov-2 is similar among themselves and slightly biased. mean enc values of 49.11 ± 0.02, 49.66 ± 0.52 and 49.21 ± 0.05 were found for sars and βcov isolated from bats and civets, respectively. enc quantifies how far a codon usage departs from equal usage of j o u r n a l p r e -p r o o f synonymous codons and is a measure of codon usage biases in genomes that ranges from 20 (maximal bias) to 61 (unbiased) (wright, 1990) . although βcov enc values are roughly similar, in the case of sars-cov-2 strains we observed a range of enc values from 45.18 to 50.09. since codon usage by its very nature is multivariate, it is necessary to analyze the data using different and complementary approaches. an enc-gc3s plot (enc plotted against gc content at the third codon position) can be used as a method that quantifies how far the codon usage of a gene departs from equal usage of synonymous codons (wrigth, 1990) . if gc3s is the only determinant factor shaping the codon usage pattern, the values of enc would fall on a continuous curve, which represents random codon usage (jiang et al., 2007) . if g+c compositional constraint influences the codon usage, then the gc3s and enc correlated spots would lie on or below the expected curve (tsai et al., 2007) . when the enc-gc3s plot was constructed with values obtained for all 81 βcov strains enrolled in this analysis (including sars-cov-2 strains), all spots lie below the expected curve, indicating that g+c compositional constraints may play a role in all βcovs codon usage (see fig. 4 ). in order to compare the codon usage preferences of sars-cov-2 with those of human cells, the rscu values of the codons in sars-cov-2 orfs were calculated and compared with those of human. the results of these studies are shown in table 1 . , and ggu (gly). as it can be seen, most of the highly preferred codons are a and u-ending, while most of the highly underrepresented codons are c and g-endind codons, particularly cg containing codons. these results strongly suggest that mutational bias is a main force shaping codon usage in sars-cov-2 (see table 1 ). in fact, when the occurrences of dinucleotides are established, they are not random and no dinucleotide is present at the expected frequencies (see supplementary material table 2 (table 1) . highly underrepresented codons acg (thr), gcg (ala) and ggg (gly) were observed in all three viruses in relation to human cells (table 1) . on the other hand, significant differences in frequencies of two glutamine codons (gag and gaa) were observed among sars-cov-2 and sars and mers-covs (table 1) covs (1.04 and 1.05, respectively). these results suggest that although a general codon usage pattern among human βcov can be found, each virus may evolve to a unique codon usage in its adaptation to the host'cells. in order to compare the codon usage preferences of sars-cov-2 with those of humans, cai values for all triplets were calculated, using human codon usage as reference set. the results of these studies are shown in table 2. cai index ranges from 0 to 1, being 1 if the frequency of codon usage by sars-cov-2 equals the frequency of usage of the reference set. a mean value of 0.710 was obtained for sars-cov-2 genes in relation to human (see table 2 ). to evaluate if the differences were statistically significant, we performed a wilcoxon & mann-whitney test. the results of this tests revealed that the differences in cai values were statistically significant (t = 256, p-value < 0.001). to discern if the statistically significant differences in cai values arise from codon preferences (puigbo et al., 2008a) , the expected cai (e-cai) values were calculated for sars-cov-2 sequences in relation to human codon usage reference set. the e-cai algorithm (puigbo et al., 2008b) generated 500 random sequences with the same nucleotide and amino acid composition as the sequences of interest (in this case sars-cov-2 sequences). then, we calculated the cai values for all of them, and applied a kolmogorov-smirnov test for the e-cai of these random sequences in order to show whether the generated sequences follow a normal distribution. the results of these studies revealed an e-cai value of 0.719 (p < 0.05). kolmogorov-smirnov test revealed a normal distribution of the generated sequences (kolmogorov-smirnov test of e-cai value of 0.028, which is below critical value of 0.061). taking all these results together, our studies revealed that the cai values for sars-cov-2 genes are different from the cai values obtained from human cells. again, these results suggest that these differences are related to codon usage preferences. on january 30 th 2020, the world health organization declared the current sars-cov-2 outbreak a public health emergency of international concern (who, 2020). to gain insight into the biology and evolution of emerging sars-cov-2, a comprehensive analysis of genome composition, codon and amino acid usage of βcov strains isolated in china from humans, bats, civets and ferret hosts was performed, including sars-cov-2 strains recently isolated from current outbreak. the results of these studies revealed that sars-cov-2 strains enrolled in these analyses have a distinct genome composition in relation to other βcov strains isolated from human (sars-cov), bats, civets and ferrets (see fig. 1 ). this is in agreement with very recent studies revealing that sars-cov-2 is sufficiently divergent from sars-cov to be considered a new human-infecting βcov (lu et al., 2020; zhu et al., 2020) . this distinct genomic composition is also reflected in its codon and amino acid usage patterns (see fig. 2 ). moreover, correlation distances and average linkage suggests a closer relation among sars-cov-2 and bats βcov isolated in china and a more distant relation to sars-cov or sars-like βcov isolated from civets or ferrets (see fig. 2 ). this is in agreement with recent results suggesting that bats might be the original host of this virus, an animal sold at the seafood market in wuhan (lu et al., 2020; chan et al., 2020b) . this speaks of the importance of bats as a reservoir of potential emerging cov. moreover, significant numbers of new βcov have been discovered in chinese bats species, particularly rhinolophus affinis (li et al., 2017) . interestingly, a clear degree of variation in codon and amino acid usage was observed among βcov isolated j o u r n a l p r e -p r o o f from bats included in these studies (see fig. 2 ). nevertheless, bats might represent an intermediate host facilitating the emergence of the sars-cov-2 in humans (wong et al., 2019) . more studies will be needed to address this important issue. in these studies, a biased nucleotide composition was found for sars-cov-2 genome (fig. 3) . this bias can also have a major influence on derived parameters, as previously demonstrated for other covs (berkhout and van hemert, 2015) . this is in agreement with the results of this work, since the nucleotide composition of sars-cov-2 has a strong influence in the codons that are used by this virus for the translation of its rna genome. previous studies on codon usage in rna viruses have shown that mutational pressure is the major factor in shaping codon usage patterns in comparison with natural selection (jenkins and holmes, 2003; wang et al., 2011) . although mutational pressure is still a major driving force, it is certainly not the only evolutionary force that might be considered in rna viruses. in these studies, a significant a genomic content was found in sars-cov-2 in comparison with other human covs (fig. 3) . previous studies done in human immunodeficiency virus (hiv) found that this virus has an a-rich genome and this property has been proposed to help the virus to avoid recognition by the innate immune system (vabret et al.,2012) . this could provide a strong selective pressure on retroviruses as well as many others rna viruses, including covs (kindler and thiel, 2014; van hemert et al., 2014) . moreover, previous studies done in hiv suggest that an rna genome with a-rich domains may provide a molecular signature that is recognized during virus replication (van hemert et al., 2013) . in the context of cov, a-rich regions, like the transcription regulation sequence (trs), which is involved in transcription of sub-genomics rnas have been established (pyrc et al., 2004) . this suggest that nucleotide bias may serve distinct biological function in sars-cov-2 as j o u r n a l p r e -p r o o f well as in other covs and is in direct relation to the characteristic codon usage of these viruses (berkhout and van hemert, 2015) . a mean enc value of 48.54 ± 2.34 was obtained for sars-cov-2 strains enrolled in these studies, suggesting that the overall codon usage among sars-cov-2 is similar among themselves and slightly biased. this is in agreement with mean enc values obtained for other cov, like sars-cov (enc = 48.99) (gu et al., 2004) ; bovine coronavirus (bcov) (enc = 43.78) (castells et al., 2017) ; mers-cov (enc = 55.50) (alnazawi et al., 2017) or avian cov (enc = 51.33) (nsp2; brandao, 2013) . enc-gc3s plot of the values obtained for sars-cov-2 revealed that all spots cluster below the expected curve, suggesting that g+c compositional constraints play a role in sars-cov-2 (see fig. 3 ). in these studies, significant differences in rscu frequencies among sars-cov-2 and human cells were found (table 1) . a strong bias toward a and u ending codons was found. this in agreement with very recent studies revealing a significant predominance of a and u at third codon positions in cov genomes (sheikh et al., 2020; wang et al., 2018; kandeel et al., 2020) . these results also suggest that these differences are related to codon usage preferences. previous studies have shown that both cytosine deamination and selection of cpg-suppressed clones are the major factors that shape codon bias in cov genomes (woo et al., 2007) . mers-cov codon usage revealed a bias among hydrophobic amino acids, being ccg (pro) and guu (val) the least and most frequently used codons (chen et al., 2017) . the same results were found in this work for sars-cov-2 orfs, since ccg y guu resulted to be the least and most frequently hydrophobic codons (11 and 352 times, respectively). regarding hydrophilic amino acids, the least and most frequently used codons in mers-cov were cgg (arg) and gau (asp), respectively (chen et al., j o u r n a l p r e -p r o o f 2017). these codons were also the least and most frequently hydrophilic codons used in sars-cov-2 orf's (11 and 310 times, respectively). similarly, cpg and upu dinucleotide frequencies resulted to be the lowest and highest frequencies found in mers-cov (chen et al., 2017) . the same results were obtained in these studies on sars-cov-2, since cpg and upu were the lowest and highest dinucleotide frequencies found (0.22 and 1.96, respectively) (see supplementary material table 2 ). again, these analyses revealed that genomic composition affects the codon usage pattern in both, mers-cov and sars-cov-2 viruses. the results of these studies revealed that sars-cov-2 strains enrolled in these analyses have a distinct genome composition in relation to other βcov strains. this distinct genomic composition is also reflected in its codon and amino acid usage patterns. most of the highly frequent codons are a-and u-ending, which strongly suggests that 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the biased nucleotide composition of hiv-1 triggers type i interferon response and correlates with subtype d increased pathogenicity on the nucleotide compositionand structure of retroviral rna genomes the a-nucleotide preference of hiv-1 in the context of its structured rna genome free statistics software, office for research development and education, version 1.1.23-r7 analysis of codon usage in bovine viral diarrhea virus global epidemiology of bat coronaviruses world health organization (2020) coronavirus disease (covid-19) outbreak. situation report -36 statement on the second meeting of the international health regulations (2005) emergency committee regarding the outbreak of novel coronavirus (2019-ncov) nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study the "effective number of codons" used in a gene preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak a pneumonia outbreak associated with a new coronavirus of probable bat origin fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin a novel coronavirus from patients with pneumonia in china a rscu, relative synonymous codon usage cod, codons; hc, human cells; cov-2, sars-cov-2 highly increased codons in sars-cov-2 with respect to human (∆≥0.30) are shown underlined and in codon adaptation of sars-cov-2 genes in relation to human codon usage, displayed as cai a values cai-hs; codon adaptation index in relation to h. sapiens reference codon usage set. in all cases, mean ± standard deviation values are shown. na we acknowledge drs. pilar moreno, gonzalo moratorio and rodney colina for critical reading of this work. key: cord-262760-mf1pn587 authors: weber, stefanie; ramirez, christina; doerfler, walter title: signal hotspot mutations in sars-cov-2 genomes evolve as the virus spreads and actively replicates in different parts of the world date: 2020-09-24 journal: virus res doi: 10.1016/j.virusres.2020.198170 sha: doc_id: 262760 cord_uid: mf1pn587 severe acute respiratory syndrome coronavirus-2 (sars-cov-2) was first identified in wuhan, china late in 2019. nine months later (sept. 18, 2020), the virus has infected > 30 million people world-wide and caused > 944.000 (3.15 %) fatalities in 220 countries and territories. research on the genetics of the sars-cov-2 genome, its mutants and their penetrance can aid future defense strategies. by analyzing sequence data deposited between december 2019 and end of may 2020, we have compared nucleotide sequences of 570 sars-cov-2 genomes from china, europe, the us, and india to the sequence of the wuhan isolate. during world-wide spreading among human populations, at least 10 distinct hotspot mutations had been selected and found in up to > 80 % of viral genomes. many of these mutations led to amino acid exchanges in replication-relevant viral proteins. mutations in the sars-cov-2 genome would also impinge upon the secondary structure of the viral rna molecule and its repertoire of interactions with essential cellular and viral proteins. the increasing frequency of sars-cov-2 mutation hotspots might select for dangerous viral pathogens. alternatively, in a 29.900 nucleotide-genome, there might be a limit to the number of mutable and selectable sites which, when exhausted, could prove disadvantageous to viral survival. the speed, at which novel sars-cov-2 mutants are selected and dispersed around the world, could pose problems for the development of vaccines and therapeutics. the coronavirus disease 19 pandemic has presented unusual challenges to genetic and virological analyses [fauci et al. 2020; na zhu et al. 2020; qun et al. 2020 ]. one of the major scientific problems confronted with by the sars-cov-2 pandemic lies in our limited understanding of the interactions between the viral and the human host genomes and the latter's defense mechanisms against this pathogen. the frequency of new mutations in viral genomes depends on a multitude of viral and host factors which determine mutant selection. nucleic acid sequence and the presence of repair mechanisms in the viral genome, the secondary and tertiary structures of the viral genome; the intensity of viral replication, the host's genetically determined defense mechanisms, environmental factors, like ambient temperature, uv radiation, among many unidentified factors, all contribute to the stability or instability of viral genomes. sars-cov-2, like sarscov-1 (2003 /2004 ), and mers-cov (2012 , which were also responsible for human severe acute lung diseases, belongs to the group of beta-coronaviruses. human beta-coronaviruses oc43 or hku1 cause less severe seasonal upper respiratory tract infections. coronaviruses carry plus-strand rna genomes of between 26,000 and 32,000 nucleotides in length, the largest genomes among rna viruses [helmy et al. 2020; coronavirus disease pandemic, 2020] . during viral replication, a virus-encoded exonuclease and additional non-structural proteins form a replication complex with the viral rna-dependent-rna polymerase (rdrp) to generate new virion-packaged genomes [hartenian et al. 2020; subissi et al. 2014; wang et al. 2020 ]. this complex functions in proof-reading and corrects copying errors by the viral rdrp [subissi et al. 2014] . for sars-cov-2, this proof reading mechanism is still under study. the debate that during sars-cov-2 rna replication the generation of mutants remains low in comparison to other plus-strand rna viruses, has not been resolved. however, for our understanding of this epidemic, it will be more relevant to research how efficiently sars-cov-2 rna mutants are selected and become dominant in the course of the covid-19 pandemic. mutations affecting the secondary and tertiary structures of sars-cov-2 rna might be the most relevant ones when considering selection for viral survival among human populations. there are unique aspects of sars-cov-2 genetics and mutagenesis in that the virus managed to jump very recently from mammalian to human hosts and thereafter expanded with unprecedented speed among a world population of almost 8 billion who live under vastly different geographic and socioeconomic conditions. the growing distance from that critical animal-to-human transition point and the chance for rapid propagation under an ensemble of environmental factors and human genetic backgrounds has enabled sars-cov-2 genomes to select for replication-efficient mutations. the plasticity of the sars-cov-2 genome has enabled us to study the frequency of occurrence, the types of nucleotide exchanges and the selection of hotspot mutations in 570 sars-cov-2 genomes from isolates that were collected on different continents between december 2019 and the end of may 2020. [ncbi: https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/]. most observed mutations were point mutations and were found once or a few times among a large number of isolates and may not be significant. there arose, however, strongly selected mutations which show predominant representation and increase in frequency as sars-cov-2 explosively replicates in human populations with different geographic, socioeconomic, climatic, and genetic backgrounds. the 570 sars-cov-2 genome sequences, which were analyzed for sequence variations, were randomly selected from the ncbi databank "sars-cov-2 (severe acute respiratory j o u r n a l p r e -p r o o f syndrome coronavirus-2) sequences"(https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2seqs/). the nucleotide sequences included in the comparisons shown in tables s1 to s6 had been deposited in the data bank as follows: table s1 -china: december 23, 2019 to march 18, 2020 table s2 -europe: january 01 to may 30, 2020 table s3 -germany: february to march 23, 2020 table s4 -usa i: february 29 to april 26, 2020 table s5 -usa ii: june 12 to july 07, 2020 table s6 -india: january 27 to may 27, 2020 nucleotide sequences from china, europe, the us and india were compared to the reference genome of the sars-cov-2 isolate form wuhan-hu-1, ncbi reference sequence: nc_045512.2. the program vector nti advance™ 11 (invitrogen™), tool align x was used for the alignment of sequences from the us and germany. nucleotide sequences of isolates from china, india, and europe were analyzed with the program snapgene (gsl biotech) by using the algorithm muscle (multiple sequence comparison by log-expectation). amino acid sequences were also analyzed with the program snapgene. dna sequence analyses of reverse-transcripts of an rna genome will have to be considered with oversight. errors could have been introduced at several steps, e.g., by preferred reading mistakes of the reverse transcriptase due to specific sequence or structural properties of sars-cov-2 rna. we have tried to overcome this obvious complication by analyzing a large number of genomes. more specifically, the absence of the distinct hotspot mutations in the majority of sequences from samples isolated in china, convincingly argues against the possibility of technical problems during the generation of sars-cov-2 nucleotide sequences. in some of the statistical investigations on the data presented, notably comparisons of mutation frequencies between sample collections usa-i and usa-ii, the permutation test as well as the kolmogorov-smirnov test were applied. all sequence alignments performed in our laboratory have been stored and are available to inspection on request. the alignments are also accessible via "google drive" [https://drive.google.com/drive/folders/1fuoqjv_cd_gceaiwh3iiq6gwwsjfoqoi?usp=sha ring]. we have investigated whether and to what extent mutants of the sars-cov-2 rna sequence of 29,903 nucleotides are selected as the virus spreads around the world. and predominate in human populations with different geographic, societal, and genetic backgrounds at the time of beginning our analyses, about 2.500 nucleotide sequences of sars-cov-2 had been published of which 570 were randomly selected and compared to the reference sequence of the wuhan isolate from late 2019 (ncbi reference sequence: nc_045512.2). figure 1 was reproduced here as an example of part of the sars-cov-2 nucleotide sequence at around position 28800 and displays 38 viral isolates from different geographic regions (lines 2-38). this sequence was aligned for comparison with the sequence of the wuhan sars-cov-2 isolate (line 1). in 12 of 37 selected sequences, the ggg sequence of the wuhan sequence (in blue) was mutated to aac (in white). additionally, in sequence positions 28854 and 28863 a few c → t point mutations were apparent. as in this example, all mutations described in this report were identified by inspection and comparison of individual sequences to the wuhan reference. the results of all sequence comparisons were presented as supplemental materials in tables s1 to s6 and summarized in table 1 . we hypothesize that signal hotspot mutations, in particular those identically noted in different populations, have functional significance and have been selected for advantages during active viral replication. the possible impact of these mutations on heightened pathogenicity will require further study. according to the best information available, the world-wide spread of sars-cov-2 originated from wuhan, china, officially in late 2019 [fauci et al. 2020; helmy et al. 2020; na zhu et al. 2020; qun et al. 2020 ]. in table s1 (supplemental materials), the comparison of nucleotide sequences from 99 sars-cov-2 isolates from china with the wuhan standard sequence revealed a total of 228 deviations from the wuhan reference. most notably, in sequence positions 8782 and 28144, the point mutations cc to tc and ta to ca, respectively, were observed in 29 isolates out of the 99 sequences examined. single cytidine (c) to thymine (t) transitions, which were often seen only 1/99, occasionally 2 to 5 times in the 99 sequences, were noted in many different sequence locations. the frequent cytidine-to-uracil (c → t) transitions, as demonstrated here, might be caused by one of the cellular apobec (apolipoprotein b mrna editing enzyme) cytidine deaminases attempting to restrict viral propagation [digiorgio et al. 2020 ]. the high frequency of c to t transitions and its evolutionary implications had been pointed out earlier by simmonds (2020) . among these c to t transitions, 4 originated from cg dinucleotides in sequence positions 204, 9967, 25156, and 29095. such mutations can possibly be attributed to the oxidative deamination of a presumptive 5-methyl-c nucleotide (5-mc) to uracil (u). it is unknown whether 5-mc occurs in sars-cov-2 rna. single nucleotide mutations, other than c to t transitions, which also occurred only in 1/99 isolates or were present 2 to 7 times were also documented among the 99 isolates. the functional meaning of these very rare sequence alterations can be considered questionable, unless the frequency of sequence alterations in a certain spot expands with time and/or at certain geographic locations. some of the hotspot mutations discovered in isolates from locations other than china, like in positions 3037, 14408, 28854, and 28881, were only seen each in 2/99 isolates from china (see table 1 ). thus, the frequency of mutations in these locations strongly increased when the virus massively replicated in regions outside china (see below). this was also true for the presumably important ggg → aac mutation in position 28881 which was found expanded particularly in isolates from europe (see below and discussion section). table s2 presents analyses of 99 nucleotide sequences in sars-cov-2 isolates from european countries (czech republic, finland, france, greece, italy, netherlands, poland, serbia, spain, and sweden). compared to the wuhan reference sequence, five signal hotspot mutations were (5/99), 9477 (5/99), 11083 (8/99), 14805 (8/99), 20268 (9/99), 25563 (7/99), 26144 (7/99), 28144 (5/99) and others, mutations were also found, though much less frequently. the mutation in position 28881 comprised three nucleotides, ggg to aac in 35/98 of the analyzed sequences. this mutation affected the open reading frame for the nucleocapsid phosphoprotein n, altered the original sequence agg gga to aaa cga (see table 2 ), and led to a change in amino acid sequence positions 50 and 51 from arg-gly (rg) to lys-arg (kr) and thus juxtaposed two very polar basic amino acids. possibly due to the strictly implemented lockdown early in the pandemic and by good luck, this country has so-far been spared excessive fatalities, although more recently (september 2020) there has been an increasing number of covid-19 cases. it is solely for this reason that search results from 62 different isolates from germany table s3 have been listed separately from samples from other european countries. some of the previously identified mutation hotspots were confirmed at nucleotide numbers 1059 (21/62), 3037 (41/62), 14408 (39/62), 25563 (21/62), and 28881 (9/62). there were two additional hotspot mutations in positions 1440 (gc → ac, 15/62) and 2891 (gc → ac, 15/62). in positions 241 and 23403, the high mutation rates in samples from europe (table s2) were not observed in the samples from germany. in this context, it will be interesting to compare the results from the published munich study [böhmer et al. 2020] , in which sequences of 14 isolates from patients were described. they had contracted covid-19 in january 2020, and their sars-cov-2 infection could reportedly be tracked to a single conference attendee from china in the munich area. in the munich patient cohort, only sequence positions 241 (14/14), 3037 (14/14), and 23403 (14/14) showed high mutation frequencies, whereas others failed to be represented (data included in synopsis table 1 ). moreover, position 6446 presented with a mutation frequency of 10/14 which did not register in samples from china or any other country. in the samples from china chosen for our analyses (table s1), the high frequency mutations from the munich study were altogether absent or (at nucleotide 3037) present at a low frequency of 2/99. notably, the mutation in position 28881 was absent from the munich list, although it was found at 2/99 in the samples from china (table s1 ). of course, there might have been mutations in chinese sars-cov-2 isolates not represented in the 99 sequences we analyzed (table s1 ). the data on the analyses of 112 isolates from the us confirmed the steady rise in mutation frequencies as sars-cov-2 spread to different parts of the world (table s4 ). these samples from the us were collected before the more recent (june/july 2020) recurrence of covid-19 in several parts of the us ( table s6) . some of the mutations annotated in tables s1 (china) and s2 (europe) were found even more frequently in this us cohort, like in sars-cov the usa, unfortunately, has become the most severely sars-cov-2-hit country in the world with 6.54 million covid-19 cases and has tallied about 194,000 deaths (census september 18). we therefore analyzed an additional 97 sars-cov-2 rna sequences from the most severely affected states arizona (az), california (ca), florida (fl) and texas (tx) (tables 1 and s5) . the inspected sequences (table s5 ) had been deposited between june 12 and july 07, 2020. the sequences discussed here had not been included in table s4 . in table 1 , the usa-i and usa-ii analyses were juxtaposed and demonstrate that the previously identified hotspot mutations were still represented at about the same frequencies as shown in table s4 . the mutations at intermediate frequencies (12/97) in positions 17747, 17858, and 18060 registered at frequencies between 8/112 and 9/112 at slightly lower frequencies in table s4 . we tested the hypothesis of differential distribution of mutation frequencies in the sequences from time point 1 (usa-i) and time point 2 (usa-ii) using a permutation test as well as kolmogorov-smirnov test and failed to reject the null hypothesis of equivalent distributions, p > 0.6, suggesting that there is no significant difference in hotspot mutation frequencies between the two different time periods of sequence analyses from the us. in table s6 , mutation analyses in nucleotide sequences of isolates from india have been summarized. some of the hotspot mutations documented in isolates from the us were found in rna samples from india as well, partly at increased frequencies, at sequence in the samples studied here, the sars-cov-2 sequences from india showed the largest number (7) of hotspot mutations, which also had the highest occurrence (up to 82/99, i.e. 83%) of mutation at a given nucleotide position ( table s6) . as sars-cov-2 infections find susceptible populations around the world, and as the rate of viral replication shoots up in these populations, the number of new hotspots of mutation and the frequencies of mutations in individual hotspots were found increased. permitting the virus to replicate with growing efficiency might render its genome better adapted and increasingly dangerous to human health. alternatively, the possibility has to be considered that the lack of j o u r n a l p r e -p r o o f deleterious effects on the host and its tolerance might have favored the selection of mutants. moreover, repeated bottlenecks in the replication of viruses have been shown in the case of vesicular stomatitis virus to reduce its fitness (so called muller's ratchet) [duarte et al. 1992] . lastly, accumulation of mutants during the world-wide spread of sars-cov-2 might eventually decrease its virulence. hence, the consequences of an increase in the number of viral mutations on its pathogenicity will be very difficult to predict. eventually, a possible decrease in virulence might turn out to set a limit to sars-cov-2's pandemic potential. so far, we have inspected the available seven sars-cov-2 sequences from russia and found mutation hotspots identical to the ones predominant in europe (data not shown) as yet, not enough sars-cov-2 rna sequences have been deposited to allow a meaningful analysis. table 1 juxtaposes all hotspot mutations in isolates from different geographic regions. the majority of de novo mutation hotspots arose after sars-cov-2 had been transmitted to regions outside china and been allowed active replication in different environments (tables s1 to s6). the mutations in positions 8782 and 28144 with frequencies 29/99 in sequences from china (table s1) were found outside china only in the us i and ii samples, though with moderate frequencies (15/97 or 15/112) and at even lower frequencies in the indian samples (7/99) ( table 1) . hence most of the world-wide mutation hotspots described here (tables s1 to s6) must have originated and been selected in the course of massive replication of sars-cov-2 in its world-wide expansion. a challenging aspect arose from the identification of sars-cov-2 mutants in 14 covid-19 patients from the munich, germany area in january 2020 [böhmer et al. 2020 ]. this report explained that a manufacturer of automobile parts in the vicinity of munich was visited by a collaborator from china in january 2020. after the visitor had returned to china, she fell ill with covid-19, and her contacts in the munich area also came down with the disease. a comparison of the occurrence and frequencies of the mutants in positions 241, 3037 and 23403 in all 14 patients in the munich report [böhmer et al. 2020 ] to those in all other parts of the world (table 1) , does not render the chinese traveler the most plausible source for sars-cov-2 in the munich area. these very mutations, however, are frequent hotspots in europe, germany, the us (i and ii) and india. of course, it will have to be investigated whether the hotspot mutations apparent in the isolates from the munich cohort might have been present, j o u r n a l p r e -p r o o f though infrequently, among chinese isolates, in particular in the munich visitor (patient 0) from china. mutations in sequence positions 3037 -14408 -23403 or 25563 occurred at low frequency or were absent in samples from china, but expanded to levels up to 75/111 (68%) and 81/99 (82%) in the us and in india, respectively ( table 1) . increases in the number of hotspot mutations and the heightened percentage of sequences altered in a given hotspot in samples from europe, and even more so from the us and india, suggested that these increases were tied to the intense replication of sars-cov-2 in these countries upon its spreading from china in the course of just a few months. it will be interesting to investigate whether sars-cov-2 spreading to and massively replicating in constantly new, immunologically naïve populations furthers the selection of viral mutants, in particular of those with heightened pathogenicity. the comparison of the number and frequency of hotspot mutations between samples from the usa i (34 states) ( table s4 ) and usa ii (az, ca, fl, tx only) cohorts (table s5 ) revealed no differences ( table 1 , see above), as corroborated by statistical analyses (see above). in an earlier report [pachetti et al. 2020] , the authors analyzed 220 genomic sequences derived from patients infected by sars-cov-2 between december 2019 and mid-march 2020 and found eight mutations of sars-cov-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881 . these authors report mutations in positions 2891, 3036, 14408, 23403 and 28881 to have been observed mainly in europe, those at positions 17746, 17857 and 18060 in north america. their results are interesting in that the mutations in sequence positions 1397, 17746, 17857, and 18060 did not show up in our analyses, whereas mutations in positions 2891, 14408, 23403, and 28881 were found in our collection as well ( table 1) . since their sequence selection anteceded ours by several months, it is conceivable that the non-common mutations might have been counter-selected as the pandemic spread throughout the world. of course, the less likely possibility existed that by chance their group and ours worked on non-overlapping sequences in the data bank. furthermore, in a preprint deposit [laamarti et al. 2020 ], the authors analyzed 3,067 sars-cov-2 genomes isolated from 59 countries during the first three months of the pandemic and found 716 site mutations distributed in six genes of the sars-cov-2 genome. these mutations belonged to certain genotypes which appeared to be specific to certain geographic regions. table 2 summarizes the amino acid exchanges due to sequence alterations in hotspot mutations identified in this study (table 1) . mutations in sequence positions 241, 3037, 8782, 18877, 22444, 25563 , and 26735 did not cause amino acid exchanges and were not included in table 2 . aside from the possible functional and structural alterations in proteins altered by either codon-neutral or codon-affecting mutations, any mutation in sars-cov-2 rna can impinge upon the structure of the single-stranded viral rna molecule itself with consequences for important viral rna-protein interactions. in sequence position 28881, the mutation agg gga → aaa cga changes the amino acid sequence of the nucleoprotein n in positions 50 and 51 from arg-gly (rg) to lys-arg (kr) ( table 2) . the frequency of this mutation was found in isolates from different countries to be 2/99 (china), 35/99 (europe), 9/62 (germany), 3/112 (us i), 6/97 (us ii) and 2/99 (india). the mutant amino acid sequence in nucleocapsid protein n between positions 29 to 35 (kkprqkr) and 49 to 54 (rkrpeq) ( table 2 ) raises questions about the possible generation of a nuclear localization signal [kalderon et al. 1984 ] and strong dna-binding motifs in protein n. for sars-cov-2 very little is known about activities in the nuclei of infected cells. one publication reports cell cycle-dependent nucleolar, not nuclear, localization of protein n, although for non-sars coronaviruses [cawood et al. 2007 ]. it is presently unknown whether a mutated n protein might fundamentally alter the biology and pathogenicity of this sars-cov-2 mutant. it is also interesting to ponder how the wuhan sequence in position 28881 ggg was altered to aac. could rna-rna recombination have played a role? several of the mutants affect virus-encoded proteins with functions in the replication of sars-cov-2, like non-structural proteins (nsp 2, nsp 3) and the helicase. the important surface glycoprotein and the nucleocapsid phosphoprotein were also affected by codon changes. evidence has been adduced that the d614g mutation of the sars-cov-2 spike protein bestows advantages on viral replication both in patients and in cell culture (korber et al. 2020) . in a preprint deposit [isabel et al. 2020 ], a sars-cov-2 this d614g spike protein mutation has also been described. among the 19 mutants listed in table 1 , 17 were transitions, 2 transversions, 12 led to amino acid changes in the proteins they were coding for, 7 were synonymous mutations. of the transitions, 12 were c to t (u) mutations. so far, we have not detected any insertions or deletions in the sequences analyzed. a deletion in the sars-cov-2 genome with evolutionary implications has been recently described [su et al. 2020 ]. it will now be important to correlate the identified hotspot mutations with the course and outcome of individual infections in humans. this demanding problem has not yet been tackled. hopefully, the results of our study will provide a platform for those in sars-cov-2 research who take care of patients with sars-cov-2 infections. sars-cov-2 has the ability to mutate and, in its course of world-wide dissemination, to select for distinct signal hotspot mutations depending on high rates of genome replication and complex environmental and genetic conditions in newly invaded territories of the world. during its intercontinental journey, the exposure of sars-cov-2 to the 21 st century's repertoire of medical resources may have been an additional selective force. the impact of an increase in hotspot sars-cov-2 mutations on immunogenesis and the prospects for vaccine development [jackson et al. 2020 ] might be experienced and will have to be examined in the future. s.w. carried out all work involving sequence selection and formal analyses, was involved in the conceptualization of the project and in the analysis and interpretation of data. c.r. analyzed data for their statistical significance. w.d. initiated the project, was involved in the conceptualization of the project and in the analysis and interpretation of data and wrote the manuscript. yes, i will be a co-author on the paper. tables s1 to s6 -presentation of all data in detail. in these tables all alterations of sars-cov-2 rna/dna reverse transcript nucleotide sequences in comparison to the wuhan sequence have been compiled. sequence positions between 1 to 29,903, nucleotide alteration, and frequency of mutations in individual positions were listed. table s1 -china (99 sequences analyzed -guangzho, hongkong, beijing, wuhan, fuyang, hangzhou, shanghai, yunnan). table s2 -europe (99 sequences analyzed -czech republic, finland, france, greece, italy, netherlands, poland, serbia, spain, and sweden). table s3 -germany (62 sequences analyzed düsseldorf, -heinsberg). table s4 investigation of a covid-19 outbreak in germany resulting from a single travel-associated primary case: a case series cell cycle dependent nucleolar localization of the coronavirus nucleocapsid protein covid-19) pandemic. geneva: world health organization evidence for host-dependent rna editing in the transcriptome of sars-cov-2 rapid fitness losses in mammalian rna virus clones due to muller's ratchet covid-19 -navigating the uncharted the molecular virology of coronaviruses the covid-19 pandemic: a comprehensive review of taxonomy, genetics, epidemiology, diagnosis, treatment, and control evolutionary and structural analyses of sars-cov-2 d614g spike protein mutation now documented worldwide an mrna vaccine against sars-cov-2 -preliminary report a short amino acid sequence able to specify nuclear location tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus large scale genomic analysis of 3067 sars-cov-2 genomes reveal a clonal geo-distribution and a rich genetic variations of hotspot mutations a novel coronavirus from patients with pneumonia in china emerging sars-cov-2 mutation hot spots include a novel rnadependent-rna polymerase variant early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia rampant c→u hypermutation in the genomes of sars-cov-2 and other coronaviruses: causes and consequences for their short-and long-term evolutionary trajectories discovery and genomic characterization of a 382-nucleotide deletion on orf7b and orf8 during the early evolution of sars-cov-2 one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities structural basis for rna replication by the sars-cov-2 polymerase we are grateful to esteban domingo, universidad autónoma de madrid for alerting us to the duarte et al. 1992 the authors declare no competing interests. key: cord-285580-gq7400tq authors: pieretti, joana c.; rubilar, olga; weller, richard b.; tortella, gonzalo r.; seabra, amedea b. title: nitric oxide (no) and nanoparticles – potential small tools for the war against covid-19 and other human coronavirus infections date: 2020-10-18 journal: virus res doi: 10.1016/j.virusres.2020.198202 sha: doc_id: 285580 cord_uid: gq7400tq the endogenous free radical nitric oxide (no) plays a pivotal role in the immunological system. no has already been reported as a potential candidate for use in the treatment of human coronavirus infections, including covid-19. in fact, inhaled no has been used in clinical settings for its antiviral respiratory action, and in the regulation of blood pressure to avoid clot formation. in this mini-review, we discuss recent progress concerning the antivirus activity of no in clinical, pre-clinical and research settings, and its beneficial effects in the treatment of clinical complications in patients infected with coronaviruses and other respiratory viral diseases, including covid-19. we also highlight promising therapeutic effects of no donors allied to nanomaterials to combat covid-19 and other human coronavirus infections. nanomaterials can be designed to deliver sustained, localized no release directly at the desired application site, enhancing the beneficial effects of no and minimizing the side effects. challenges and perspectives are presented to open new fields of research. control of blood pressure (seabra et al., 2015) , and wound healing (seabra, 2017) , no presents important antimicrobial and antitumoral properties, depending on its concentration (hasan et al., 2017; pieretti et al., 2019) . in 1988, no was related to soft tissue relaxation in the penis, allowing the organ to become engorged. eight years later, viagra came to the u.s. market (gur et al., 2017) . due to its small size, neutral charge, and relative lipophilicity, no diffusion through cell membranes is facilitated even in the absence of channels or receptors (dioguardi, 2011) . no action relie s on certain main mechanisms, such as impairment of pathogen replication, interference in the electron transport chain, promotion of snitrosation reactions in cysteine residues of important pathogen enzymes, and generation of oxygen and nitrogen reactive species (no x ) (liu et al., 2017; seabra et al., 2016) . moreover, no quickly reacts with superoxide (o 2 • -), leading to highly toxic and oxidant peroxynitrite (onoo -) (pacher et al., 2008) . no has been widely studied for combating tumor cells, bacteria, fungi, protozoa, and viruses (rolim et al., 2019; santiago-olivares et al., 2019; schairer et al., 2012) . inhaled treatments of gaseous no have been proposed as an efficient route for administering the gas, but systemic administration is also possible using no donors, which are molecules able to release no in the biological medium, and/or by allying no and no donors with nanomaterials (quinn et al., 2015) . the use of no donors and nanomaterials increases the stability of no and enables targeted, controlled no release at desirable concentrations, which is fundamental for no bioactivity (quinn et al., 2015) . there are several combinations of no and/or no donors with nanoparticles, which can be designed for specific applications (seabra and durán, 2017) . there are important recent review articles based on the potential use of no to combat covid-19 and other human coronavirus infections (adusumilli et al., 2020; j o u r n a l p r e -p r o o f hedenstierna et al., 2020; martel et al., 2020) . to the best of our knowledge, these review articles are mainly focused on the administration of gaseous no (inhaled no), on discussion of the pathogenesis of viral infections, and on inflammatory responses modulated by no in vivo. the present review focuses not only on the use of inhaled no but also on the administration of no donors in combination with nanomaterials. noreleasing nanomaterials have been extensively studied in different biomedical applications, including their antimicrobial effects. due to the versatility of nanomaterials, no donors allied to a nanoplatform might be important application in the fight against covid-19 and other human coronavirus infections, and we hope that this review will open the way to new studies in this area. the antimicrobial properties of no usually rely on antifungal and antibacterial effects, although it is well-known that no not only presents antiviral activity, but also acts to modulate host response (akaike and maeda, 2000) . no has already been reported as a potential candidate for the treatment against coronaviruses and other respiratory viral diseases, such as influenza (arabi et al., 2015; keyaerts et al., 2004; murphy et al., 1998) . figure 1 shows an overview of no antiviral activity previously reported against different viral diseases (akaike and maeda, 2000; åkerström et al., 2005; arabi et al., 2015; lin et al., 1997) . j o u r n a l p r e -p r o o f to date, seven coronavirus strains have been identified: hcov-229e, sars-cov, hcov-oc43, hcov-nl63, hcov-hku1, mers-cov and sars-cov-2 (kiyuka et al., 2018; sanchez-nadales et al., 2020) . of these, no has been used against mers-cov, sars-cov and hcov-nl63. more recently, it has been extensively evaluated against sars-cov-2 (arabi et al., 2015; keyaerts et al., 2004; sanchez-nadales et al., 2020; tavazzi et al., 2020) . few reports demonstrate the use of no against mers-cov (al-dorzi et al., 2016; arabi et al., 2015) and this topic should be further explored. inhaled no treatment was administered in combination with traditional therapies, such as antibiotics, to a hypertense male patient who tested positive for mers-cov (arabi et al., 2015) . treatments allied to inhaled no demonstrated positive results, and the patient was discharged to his home 21 days from j o u r n a l p r e -p r o o f the beginning of no treatment (arabi et al., 2015) . moreover, inhaled no was administered for mers-cov in infected healthcare workers and the results also demonstrated that the patients were successfully healed, evidencing that no might have positive effects in the treatment with low harmful side effects (al-dorzi et al., 2016) . more recently, inhaled no treatment was also employed in a 22-year old immunocompromised patient who tested positive for both sars-cov-2 and hcov-nl63 (sanchez-nadales et al., 2020). the authors have hypothesized that no might have played a pivotal role in the treatment, related to the loss of alveolar perfusion and the hypoxic pulmonary vasoconstriction effect (sanchez-nadales et al., 2020) . taken together, these reports suggest beneficial effects of administration of no in patients, and further studies are required. in addition to the few publications reporting no trials in the treatment of coronaviruses, this molecule has already been evaluated against other respiratory viral diseases, such as influenza a/h1n1 (avnon et al., 2015; kovačević et al., 2015; murphy et al., 1998) . during the 2009 h1n1 pandemic, no was used in combination with mechanical ventilation in 67% of the patients under experimental trial, particularly patients with thrombotic complications. in general, authors indicated that critically ill patients were administered deep vein thrombosis prophylaxis in combination with other treatments, such as inhaled no (avnon et al., 2015) . interestingly, considering that no is an endogenous signaling molecule, it has also been reported that serum no levels are higher in patients that survived acute respiratory distress syndrome caused by h1n1, in comparison with non survivors, as well as an increase of oral no levels after 21-days of virus incubation; this offers a strong association of no levels with improved clinical outcomes (avnon et al., 2015; murphy et al., 1998) . no can react with important biomolecules controlling physiological and pathophysiological functions. indeed, no j o u r n a l p r e -p r o o f can nitrosate thiol groups on the surface of red blood cells and the beta chain of hemoglobin, preventing hemolysis and oxidative damage (zhang et al., 2015) . previous studies have suggested the potential beneficial effects of no, opening new areas in this research topic. although positive biological effects have been reported for the administration of no donors, further studies are required to better evaluate the levels of inflammatory mediators and the activity of important heme-containing enzymes, such as indoleamine 2,3-dioxygenase (ido), directly involved in the inflammatory responses in respiratory viral infections (anderson and russel, 2020) . during virus infection, a significant increase in the levels of inflammatory mediators can be observed, known as a "cytokine storm", associated with endothelial and platelet activation; this can lead to vascular permeability, thrombocytopenia, shock (observed in severe cases), pulmonary hypertension and pneumonia (belladonna and orabona, 2020). ido and inducible nitric oxide synthase (inos) are expressed upon monocyte activation. it has been reported that no generated by inos inhibited the sars cov replication cycle (åkerström et al., 2005) . interferon-gamma, a molecule with antiviral activity and a regulator of inflammatory responses to pathogens, is a potent inducer of ido and inos (kang et al., 2018) , in addition to other inflammatory factors, such as tumor necrosis factor- (tnf) and interleukins 6 and 1β (turski et al., 2020) . for instance, both these enzymes were highly expressed in monocytes of patients infected with dengue virus (denv-4), compared to healthy controls, during an epidemic in campo grande (ms, brazil) (fialho et al., 2017) . inflammatory processes and cytokines stimulate inos, which in turn enhance no production against virus infection. ido, induced by inflammation or immune responses, participates in tryptophan catabolism and can be induced by ifn- in monocytes, displaying immunosuppressive and antimicrobial j o u r n a l p r e -p r o o f activities (mellor and munn, 2004; hara et al., 2008) . ido enzymatic activity helps to control pulmonary inflammation and hypertension, allowing immunoregulation in covid-19 patients (belladonna and orabona, 2020) . it should be noted that ido activity can be either suppressed by no or induced by nos inhibition (hara et al., 2008) . ido is a cytosolic immune-modulatory enzyme containing heme iron for which no has a high affinity, thus endogenous no and no donors can inactivate this enzyme (thomas et al., 1994) . some papers reported that blockage of ido activity might enhance antiviral ifn-i/ii (interferon type i/ii) innate and adaptive t-cell responses in japanese encephalitis (kim et al., 2016) and respiratory viral infections (van wissen et al., 2002) . as ido and no have been implicated in t cell immune tolerance (ye et al., 2017) , the effects of no donors on ido and on cytokine production should be further investigated. no inhaled treatment against covid has already been proposed in 2020 in two different countries, china and united states of america gianni et al., 2020a; lei et al., 2020) . the motivation comes from the historical use of no-based treatment against sars-cov, and also against other respiratory diseases caused by viruses. after the sars-cov outbreak in 2002, three different studies demonstrated the potential of no compounds in the inhibition of sars-cov replication (åkerström et al., 2005; åkerström et al., 2009; keyaerts et al., 2004) . in a study that overviewed therapeutic strategies available in 2008 for sars-cov treatment, no was highlighted as a potential drug already available and used on infected humans, while no donors were reported as promising drugs undergoing pre-clinical trials (wong and yuen, j o u r n a l p r e -p r o o f 2008). in vitro studies performed with the no donor s-nitroso-n-acetylpenicillamine (snap) indicated that no was able to inhibit the replication cycle of sars-cov in a concentration-dependent mechanism. moreover, the same experiment showed that viral protein and rna synthesis were also hindered (åkerström et al., 2009) . years later, indepth studies of the same research group into the mechanism of no against sars-cov evidenced that the intermediate peroxynitrite was not responsible for the replication cycle inhibition; the authors demonstrated that the antivirus mechanism relied on two major points: (i) interference in the fusion of s protein and its receptor due to reduced palmitoylation of the s protein, and (ii) a reduction in rna production, probably due to an effect on cysteine proteases (åkerström et al., 2005) . a similar pattern was reported by keyaerts and coworkers, evidencing an ic 50 of snap against sars-cov of 222.3 ± 83.7 µm, and no activity for the non-nitrosated molecule, confirming that the effect relie s on no release (keyaerts et al., 2004) . moreover, no produced from inducible no synthase (inos) has also demonstrated the ability to inhibit the replication cycle of sars-cov (åkerström et al., 2009 ). thus, the protocols published to date against sars-cov-2 rely on the efficient results previously demonstrated. three different strategies were proposed to comprehend the treatment under different conditions, using a randomized experiment evolving 2 centers: boston and xi'an gianni et al., 2020a; lei et al., 2020) . firstly, no application in severely ill patients is proposed in two different conditions: (i) high doses of gaseous no, up to 180 ppm, during short periods of time (20-30 min), and (ii) low doses of gaseous no, up to 80 ppm, during 48 h-treatment j o u r n a l p r e -p r o o f lei et al., 2020) . interestingly, a second protocol proposed inhaled no to prevent covid-19 in healthcare providers who are in direct contact with patients infected with sars-cov-2 (gianni et al., 2020a) . the individuals concerned will receive a maximum of 160 ppm of inhaled no for a short period of time during 14 days. it is expected that the number of healthcare providers contaminated will decrease from 15% to 5% (gianni et al., 2020a) . the three strategies proposed have different parameters for the treatment period, concentration and people involved; they promise to increase knowledge about the use of no against covid-19 in both treatment and prevention. although a promising strategy, the inhalation of gaseous no is not the only possible treatment and/or prevention against covid-19 or future viral diseases. interestingly, the same authors that first proposed the use of inhaled no developed a system for high dose no inhalation, minimizing no concentration oscillations and the inhalation of no 2 , which might have a promising application in this field (gianni et al., 2020b) (fig. 2) . (ferrari et al., 2020) . the dose evaluated was 20 ppm for 30 minutes, in patients already treated with invasive mechanical ventilation (ferrari et al., 2020) . although no acts by improving arterial oxygenation, the same pattern was not observed in all patients, especially those with severe hypoxemia. this highlights the fact that even though no presents promising use in the treatment of patients with covid-19, further studies must be performed to understand which patients might benefit (ferrari et al., 2020) . interestingly, a similar trial employing inhaled no at a concentration of 25 ppm led to complementary results (tavazzi et al., 2020) . the treatment did not improve oxygenation in patients with refractory hypoxemia, although promising results were confirmed for patients with right ventricular dysfunction (tavazzi et al., 2020) . a different pattern was observed in spontaneously breathing patients. parikh and coworkers evaluated the potential of inhaled no treatment in breathing patients diagnosed with covid-19 (parikh et al., 2020) . thirty-nine patients with different characteristics, with or without pre-existent diseases, received a dosage of 30 ppm of no for 2 days. of all the patients treated with inhaled no, 53.9% did not require mechanical ventilation and the spo 2 /fio 2 ratio also improved in those patients (parikh et al., 2020) . this suggests that no might not only prevent hypoxic respiratory failure, but also contribute an antiviral mechanism (parikh et al., 2020) . a higher dose of no (200 ppm/30 minutes) was evaluated in pregnant women diagnosed with covid-19 (safaee fakhr et al., 2020) . in this trial, six pregnant women received 39 treatments with inhaled no after hospitalization with covid-19 with hypoxic respiratory failure. no treatment in high dosages was shown to be well tolerated, and evidenced an improvement in the cardiopulmonary system (safaee fakhr et al., 2020) . all six pregnant patients subjected to no treatment were discharged from hospital, and half of them had already delivered healthy neonates (safaee fakhr et al., 2020) . a singlepatient trial evaluated home treatment with inhaled no, differing from previous reports j o u r n a l p r e -p r o o f (zamanian et al., 2020) . low doses of 20 ppm were administered 12-14 h per day for 11 days with a nasal canula, remotely monitored (zamanian et al., 2020) . positive results were observed, opening venues for further trials employing inhaled no at home as an alternative to hospital treatments (zamanian et al., 2020) . in the war against covid-19, no may not only have a potent antiviral activity but also improve ventilation perfusion in the lungs. in other words, no demonstrates potential for the treatment of patients infected with covid-19 both in severe and nonsevere conditions, improving oxygenation and antiviral mechanisms, and preventing aggravation of the disease (ferrari et al., 2020; parikh et al., 2020) . patients with coronaviruses are susceptible to acute respiratory distress syndrome, which can lead to pulmonary fibrosis (chen et al., 2004) . inhalation of no gas enhanced arterial oxygenation in patients with acute respiratory distress syndrome (chen et al., 2004) . indeed, no gas is approved by us food and drug administration (fda) for neonates in the treatment of pulmonary hypertension, and recently it has been authorized for use as rescue treatment in patients with hypoxic covid-19 symptoms (gebistorf et al., 2016) . moreover, no is synthesized endogenously in the blood vessels, regulating blood pressure and thus avoiding clot formation and destroying potential toxins (ignarro, 1999) . even though no has demonstrated important results in the treatment of covid-19, some authors emphasize that further studies are necessary to better understand which patients will benefit most from inhaled no treatment. a similar pattern has been previously demonstrated in adults and children with acute respiratory distress syndrome, in which treatment with inhaled no led to a transient improvement in oxygenation, but did not alter the mortality rate in critically ill patients (gebistorf et al., 2016) . moreover, j o u r n a l p r e -p r o o f it was observed that no administration might impair the kidney function of the patients treated (gebistorf et al., 2016) . the data presented in this review are fundamental for revealing promising parameters for further trials employing no treatment against covid-19 and/or other respiratory diseases, in order to confirm no potential, target patients and safe dosages. it should be noted that no is a free radical, and its stability is improved by the use of no donors; these fall into different classes depending on the molecule, e.g. organic nitrates, s-nitrosothiols, metal complexes and n-diazeniumdiolates (wang et al., 2002) . besides inhaled no directed for pulmonary treatments, fda has already approved the use of a few no donors, such as nitropress (opasich et al., 2009) . more recently, it has been demonstrated that the combination of no donors with nanoplatforms has been an important approach, which can even improve the antimicrobial properties of no (douglass et al., 2019) . recent works have demonstrated the antimicrobial activity of no donors allied to metal or metal/oxide nanoparticles, such as copper (cunps), silver (agnps), and polymeric nanoparticles. a composite based on snap and cunps enabled a tunable release of no from snap, through catalyzation with cunps (pant et al., 2017) . no release was adjusted through the concentration of cunps, which promoted a no flux 6 times higher at the concentration of 5% when compared to free snap after 3 and 24 h evaluation. the combination of no and nanoparticles promoted enhanced antibacterial activity against both staphylococcus aureus (s. aureus) and pseudomonas aeruginosa (p. aeruginosa). the growth of these strains treated only with snap were 3.5 ± 1.3 × 10 4 and 1.8 ± 1.3 × 10 4 respectively, while the values deceased j o u r n a l p r e -p r o o f significantly when combined with 3% of cunps, reaching 8.9 ± 1.8 × 10 2 and 2.4 ± 1.8 × 10 2 respectively (pant et al., 2017) . a similar pattern was observed for the combination of no and agnps, in which the synergistic effect led to a notable inhibition of escherichia coli (e. coli). interestingly, a recent work reported that silver nanoparticle inhalation (3-7 nm) could be effective in early stage covid treatment (zachar, 2020) . in the same work, the authors indicate that the minimal inhibitory concentration (10 μg ml -1 ) was achievable in the lower airways, but could be an effective initial method for the upper airways to suppress progression of the infection. therefore, its combination with no could be a supplementary therapy for infectious respiratory diseases that should be addressed in future studies. on the other hand, the encapsulation of no donors in polymeric nanoparticles is a promising alternative. the encapsulation of agnps and s-nitroso-mercaptosuccinic acid (s-nitroso-msa) in alginate nanoparticles promoted sustained release of no and a potent antibacterial effect against s. aureus, e. coli and staphylococcus mutans with low cytotoxicity to non-tumoral cells (urzedo et al., 2020) . the effectiveness of this strategy is not limited to antibacterial applications: the encapsulation of no donors in chitosan nanoparticles demonstrated important results against leishmaniasis, a parasitic disease (cabral et al., 2019) . these nanoparticles efficiently inactivated leishmania (l.) amazonensis promastigotes while no toxic effects were observed in macrophages. more studies are required to better investigate how endogenous no (produced by nos) and exogenous no, from a donor, might be related. it has been demonstrated that both sources of no (endogenous and exogenous) can have antiviral activity (åkerström et al., 2005 ). an important advantage of nanotechnology is the ability to design a nanocarrier for sustained, localized drug release (in this case, no release) for pulmonary application (cavalcanti and nogueira, 2020; lammers et al., 2020) . in other j o u r n a l p r e -p r o o f words, nanomaterials can be prepared for pulmonary delivery of no/no donors, depending on nanoparticle size, chemical structure, chemical surface and charge. versatile nanocarriers can be designed to deliver therapeutic amounts of no direct to the desired application site, i.e. the site of the viral infection. chitosan nanoparticles have been explored for pulmonary delivery of active drugs due to their biocompatibility and biodegradability, and their mucoadhesive and non-immunogenic properties (islam and ferro 2016; cavalcanti and nogueira, 2020; tatlow et al., 2020) . thus, important effects that have already been observed for no against sars-cov; these could be improved by combination with nanomaterials, promoting sustained, localized release of no in therapeutic concentrations (keyaerts et al., 2004) . no allied to nanomaterials might thus find important applications against coronaviruses, including in the treatment of covid-19. although several drugs have been evaluated against covid-19, further studies are still required to better elucidate their efficacy, tolerability and side effects (magro, 2020) . in this war against covid-19, the use of no/no donors might find important medical application. there is still little progress on the antiviral effect of no, especially against coronaviruses. the three protocols proposed against covid should significantly contribute to this area, and possibly evidence a promising alternative treatment for the current pandemic. the antiviral effects of no should be further explored. interestingly, it has been reported that ambient ultraviolet a radiation (uva) exposure is associated with lower covid-19 specific mortality, independent of vitamin d (cherrie et al., 2020). uva triggers generation of no from no stores in the skin; this j o u r n a l p r e -p r o o f is released into the blood circulation, increasing the bioavailability of no and reducing blood pressure (weller et al., 2020) . finally, we would like to encourage investment in promising advances in no allied with nanomaterials; this treatment has already shown important results in the area of nanomedicine, but is still undeveloped in the field of viral infections, that are of huge importance not only during the sars-cov-2 pandemic, but also for the treatment of future viral diseases that may arise. definitely, no is bad news for covid-19. the authors declare no competing interest. harnessing nitric oxide for preventing, limiting and treating the severe pulmonary consequences of covid-19 nitric oxide and virus infection dual effect of nitric oxide on sars-cov replication: viral rna production and palmitoulation of the s protein are affected nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus the critical care response to a hospital 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vasoreactive idiopathic pulmonary arterial hypertension and covid-19 infection hemoglobin βcys93 is essential for cardiovascular function and integrated response to hypoxia 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 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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-259044-mubjm22l authors: weng, jing-ru; lin, chen-sheng; lai, hsueh-chou; lin, yu-ping; wang, ching-ying; tsai, yu-chi; wu, kun-chang; huang, su-hua; lin, cheng-wen title: antiviral activity of sambucus formosananakai ethanol extract and related phenolic acid constituents against human coronavirus nl63 date: 2019-09-24 journal: virus res doi: 10.1016/j.virusres.2019.197767 sha: doc_id: 259044 cord_uid: mubjm22l human coronavirus nl63 (hcov-nl63), one of the main circulating hcovs worldwide, causes respiratory tract illnesses like runny nose, cough, bronchiolitis and pneumonia. recently, a severe respiratory illness outbreak of hcov-nl63 has been reported in a long-term care facility. sambucus formosananakai, a species of elderberry, is a traditional medicinal herb with anti-inflammatory and antiviral potential. the study investigated the antiviral activity of sambucus formosananakai stem ethanol extract and some phenolic acid constituents against hcov-nl63. the extract was less cytotoxic and concentration-dependently increased anti-hcov-nl63 activities, including cytopathicity, sub-g1 fraction, virus yield (ic50 = 1.17 μg/ml), plaque formation (ic50 = 4.67 μg/ml) and virus attachment (ic50 = 15.75 μg/ml). among the phenolic acid constituents in sambucus formosananakai extract, caffeic acid, chlorogenic acid and gallic acid sustained the anti-hcov-nl63 activity that was ranked in the following order of virus yield reduction: caffeic acid (ic(50) = 3.54 μm) > chlorogenic acid (ic(50) = 43.45 μm) > coumaric acid (ic(50) = 71.48 μm). caffeic acid significantly inhibited the replication of hcov-nl63 in a cell-type independent manner, and specifically blocked virus attachment (ic(50) = 8.1 μm). therefore, the results revealed that sambucus formosana nakai stem ethanol extract displayed the strong anti-hcov-nl63 potential; caffeic acid could be the vital component with anti-hcov-nl63 activity. the finding could be helpful for developing antivirals against hcov-nl63. human coronavirus (hcov) nl63, the member of the genus alphacoronavirus in the coronaviridae family, is one of common human coronaviruses (li and lin, 2013; huang et al., 2017) . hcov-nl63 genome is a positive-strand rna with near 27.5 kb nucleotides containing 5′ untranslated regions (utr), orf1a/b, spike(s), orf3, envelope(e), membrane(m), nucleoprotein (n), and 3′utr (geng et al., 2012) . orf1a/b encodes overlapping replicase 1a and 1ab via the −1 ribosomal frameshift at the nucleotide 12,424. the papain-like (plpro) and 3c-like (3clpro) proteases embedded within replicase 1a and 1ab process cis-and trans-cleavage activity to divide replicase 1a and 1ab into nonstructural proteins (nsps) that modulate in viral rna replication. among common human coronaviruses like hcov-229e, hcov-hku1, and hcov-oc43, hcov-nl63 is one of main circulating hcovs in the fall and winter worldwide, causing mild upper respiratory tract illnesses like runny nose, cough and sore throat in young children, young adults and elderly (cui et al., 2011; dijkman et al., 2012) . interestingly, hcov-nl63 has been frequently detected than other hcovs, influenza viruses, and rhinovirus in the specimens from the young adults with acute respiratory infection (cough and body aches or chills or fever/feverishness) (davis et al., 2018) . importantly, hcov-nl63 is also associated with lower respiratory tract illnesses, such as pneumonia and bronchiolitis in young children and elderly (huang https://doi.org/10.1016 (huang https://doi.org/10. /j.virusres.2019 received 20 june 2019; received in revised form 20 september 2019; accepted 23 september 2019 et al., 2017) . hcov-nl63 infection is the high prevalence (8.4%) in hospitalized patients with pneumonia in winter. recently, a severe respiratory illness outbreak in a long-term care facility in louisiana has been reported to be associated with the hcov-nl63 infection in winter 2017 (hand et al., 2018) . among 20 cases aged from 66 to 96, 6 patients with pneumonia have to be hospitalized and 3 patients are dead. moreover, screening children with acute undifferentiated febrile illness in rural haiti indicates that hcov-nl63 is identified in the blood samples from four patients aged from 3 to 10 years who have no respiratory symptom, but two cases have headache and the others exhibit influenza virus causing abdominal symptoms (beau de rochars et al., 2017) . therefore, hcov-nl63 is the notable pathogen as the etiology of mild and severe respiratory diseases, even acute undifferentiated febrile illness. sambucus formosananakai, also known by sambucus chinensis lindl and sambucus javanica, is a species of elderberry, belongs to family adoxaceae, and grows in subtropical and tropical asian areas, including taiwan, china, japan, cambodia, india etc. (lin and tome, 1988; yang and chiu, 1998; hong et al., 2013) . sambucus formosananakai is a traditional medicinal herb in taiwan for reducing inflammation, enhancing circulation, and treating rheumatoid and low back pain, neuritis, dermatitis, and infection diseases (yang and chiu, 1998) . the chemical components of s.ambucus formosananakai include sambuculin a, oleanolic acid, α-amyrin and β-amyrin, β-sitosterol, ursolic acid, pomolic acid, lupeol palmitate, glycyrrhetic acid, phenolic acid constituents, and flavonoids (chen et al., 2001 (chen et al., , 2019 liao et al., 2006; lin and tome, 1988; yang and lin, 2004; zhang et al., 2010) . in addition, phenolic acid constituents of s.ambucus formosananakai, including caffeic acid, caffeotannic acid, chlorogenic acid, coumaric acid, ferulic acid, and gallic acid, have been identified as the members of the most important active components with anti-inflammatory, anti-tumor and anti-hepatotoxic activities (chen et al., 2001; liao et al., 2006; yang and lin, 2004; zhang et al., 2010) . active components of sambucus formosananakai are similar to those of other sambucus species, including sambucus nigra l. and sambucus lanceolata, which process antioxidant, antiradical, antiviral, antimicrobial, and anti-inflammatory activities (barak et al., 2001; krawitz et al., 2011; mandrone et al., 2014; pinto et al., 2017; pliszka, 2017; porter and bode, 2017; turek and cisowski, 2007) . especially, the extract of sambucus nigra l. exerts the antiviral activity against influenza a and b viruses, human immunodeficiency virus, and the herpes simplex virus type 1 (krawitz et al., 2011; serkedjieva et al., 1990; zakay-rones et al., 1995; amoros et al., 1992; mahmood et al., 1993; roschek et al., 2009) . sambucus nigraphenolic acids like caffeic acid show the highly inhibitory effect on the in vitro replication of influenza a virus (porter and bode, 2017; utsunomiya et al., 2014) . since sambucus formosananakai contains such antiviral active components in the extract of sambucus nigra l., the antiviral activity of sambucus formosananakai is worthy to further investigate. the study explored the anti-hcov-nl63 activity of sambucus formosananakai stem ethanol extract and some markers of its phenolic acid constituents, like caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, gallic acid. the study indicated the inhibitory activity of sambucus formosananakai extract and its phenolic acid constituents on hcov-nl63 induced cytopathic effect, virus yield, and the early stage of hcov-nl63 replication in concentration-dependent and cell-type independent manners. hcov-nl63 provided by dr. lia van der hoek (academic medical center, the netherlands) was amplified in llc-mk2 cells, as described in our prior study . llc-mk2 cells were cultured in the minimum essential medium (mem) containing 2 mm l-glutamine, 50 μg/ml penicillin, 50 μg/ml streptomycin, and 5% fetal bovine serum (fbs) at 37 ℃ in a 5％ co 2 incubator. llc-mk2 cells were further used to perform antiviral assays and examine antiviral mechanism. human airway calu-3 cells were cultured in mem supplemented 10% fbs and antibiotics mentioned above, maintained at the 80-90% confluent, and then also used to confirm the antiviral activity of indicated phenolic acid constituents. dried stems of sambucus formosana nakai (supplemental fig. 1a) were purchased from the medicinal herb pharmacy in taichung, and identified as described in flora of taiwan (yang and chiu, 1998) . dried stem slices (supplemental fig. 1b) were soaked in 95% ethanol with the sonication for 2 h; the stem ethanol extract was filtered by whatman no. 1 paper, then lyophilized in an iwaki fdr-50 p freeze dryer, as described in our previous study (wang et al., 2012) . the powder of sambucus formosana nakai stem ethanol extract was kept in the sterile bottle; the stock solution of 10 mg/ml in dimethyl sulfoxide was prepared and stored in −20°c freezer and the test solutions of the stem extract were diluted by the media. the phenolic acid constituents such caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, and gallic acid, were purchased from sigma-aldrich company. llc-mk2 cells (3 × 10 4 cells/well) were cultured in the 96-well plates overnight, quintuplicate treated with sambucus formosana nakai stem ethanol extract or its phenolic acid constituents (caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, and gallic acid) for 2 days, and then incubated with 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) for additional 4 h. after the cell lysis by isopropanol, yielding absorbance (od 570-630 ) at the wavelength of 570 nm and 630 nm was assessed using a micro-elisa reader. cell viability (%) and cytotoxic concentration showing 50% toxic effect (cc 50 ) were subsequently determined, as previously described (wang et al., 2012) . llc-mk2 cells (3 × 10 5 cells/well) were cultured in the 6-well plates overnight, and then infected by 3 × 10 4 pfu (plaque formation unit) hcov-nl63, representing as a moi (multiplicity of infection) of 0.1 to significantly induce viral cytopathic effect (cpe) in llc-mk2. the cells were simultaneously added with hcov nl63, and treated with sambucus formosana nakai stem ethanol extract at the concentrations of 0, 1, 10, and 50 μg/ml or the phenolic acid constituents at the concentrations of 0, 10, 50, and 100 μm, respectively. after the 36-h incubation at 37 ℃ and 5％ co 2 , hcov nl63-induced cytopathic effect (cpe) with cell swelling, rounding, vacuoles, and eventual detachment was photographed using microscope, in which vacuoles in cpe from hcov nl63-infected llc-mk2 cells were more predominant at 37°c (lednicky et al., 2013) . in the cell cycle assay, the cells were harvested 36 h post infection, stained with propidium iodide, and then examined using flow cytometry, as described in our prior study . to determine the production of progeny virions (virus yield assay), the cultured media were collected for determined the virus titers using plaque assay. llc-mk2 cell monolayer in the well of 6-well plates was added with 100 μl of serial 10-fold dilutions of above cultured media. after a 1-h incubation, the cell monolayer was overlaid by the medium containing 0.75% agarose (3 ml per well) for 2-day incubation at 37°c in a co 2 incubator to allow the plaque formation. finally, the cell monolayer was stained with the solution of naphthol blue-black dye and plaque number calculated. fifty percent (50%) inhibitory concentration (ic 50 ) for virus yield reduction by the stem ethanol extract and the phenolic acid constituents was determined. in the plaque formation inhibition assays, llc-mk2 cell monolayer cultured in 6-well plates were infected with 200 pfu of hcov-nl63 that were the maximum number of plaques counted in the well of 6-well plates. after the addition with hcov nl63, the cells were immediately treated with the ethanol extract at the concentrations of 0, 1, 5, and 10 μg/ml, caffeic acid and chlorogenic acid at the concentrations of 0, 10, 50, and 100 μm, respectively. after a 1-h incubation at 37°c in a 5% co 2 incubator, the mixtures were removed from the wells; the cell monolayer was cultured with the medium containing 0.75% agarose and performed by the plaque assay mentioned above. in the virucidal assay, hcov-nl63 (2 × 10 6 pfu) was added into in the eppendorf tube and directly treated with the ethanol extract, caffeic acid, or chlorogenic acid for 1 h at 37°c. for minimizing the antiviral effect of indicated agents in the cells, 100 μl (near 200 pfu hcov-nl63) of the 10000-fold dilution from the mixtures of virus and the extract or phenolic acids was added to the mk2 cell monolayer in the 6-well plate to determining the residual viral infectivity using the plaque assay described above. virucidal activity was calculated based on the comparison of the residual infectivity of hcov-nl63 in the treated groups with the un-treated group. in the hcov-nl63 attachment assay, llc-mk2 cell monolayer in 6-well plate was placed at 4°c for 1 h, infected with hcov-nl63 (200 pfu), and then immediately added with the ethanol extract, caffeic acid or chlorogenic acid. after an additional 1-h incubation at 4°c, the mixture of virus and the extract or phenolic acids was removed from the well; the cell monolayer was overlaid with mem medium containing0.75% agarose at 37°c in a 5% co 2 incubator, and then executed by plaque assay, as mentioned above. the attachment inhibition was analyzed according to the ratio of the plaque number in treated groups to the un-treated group. to measure the inhibitory activity of caffeic acid on the replication of hcov-nl63 in human airway epithelial cells, calu-3 cells (1 × 10 5 cells/well) were cultured in the 6-well plates overnight, and then infected by 5 × 10 3 pfu hcov-nl63 (moi = 0.05). after the addition of hcov nl63, the cells were simultaneously treated with caffeic acid at the concentrations of 0, 10, and 50 μm. after the 36-h incubation at 32 ℃ in a 5％ co 2 incubator, swelling, rounding, and eventual de-attachment in hcov-nl63-induced cpe were more predominant at 32°c (lednicky et al., 2013) , and then the images were recorded by microscope. subsequently, the cells were fixed with 4% paraformaldehyde solution in pbs for 30 min, incubated with the quench buffer (50 mm nh 4 ci) for 15 min, permeabilized and blocked using the cell perforation and blocking solution containing 1% albumin bovine (affymetrix) plus triton x-100 (thermofisher) for 4 h at 4°c, and then reacted with hcov-nl63-immunized sera in 1% bsa (1:2000) overnight at 4°c and secondary antibody alexa fluor anti-mouse igg in 1% bsa (1:3000) for 1 h at 4°c (thermofisher). after staining with 4′,6diamidino-2-phenylindole (dapi, thermofisher) 20 min at room temperature, the images of mock, infected, and infected/treated cells were photographed using fluorescent microscopy. the infectivity was represented as the ratio of red fluorescent signals (hcov-nl63-positive cells) to blue fluorescent signals (total nucleuses) was calculated by image j. the p value was calculated based anova using spss program and student t-test with the data from three independent experiments. when the p value was less than 0.05, the result of the assay was statistically significant. sambucus formosana nakai stem ethanol extract had a low cytotoxicity with a cc 50 value of 180.62 μg/ml to llc-mk2 cells (supplemental fig. 2, table 1 ). subsequently, anti-hcov-nl63 ability of the stem ethanol extract was assessed with cytopathicity, cell cycle and virus yield assays (figs. 1 and 2) . sambucus formosana nakai extract concentration-dependently reduced cytopathicity in hcov-nl63 infected cells (fig. 1a) , in which vacuolation in infected cells at 37°c appeared more predominantly, as described in the prior report (lednicky et al., 2013) . the extract also significantly decreased the sub-g1 fraction in infected cells (fig. 1b) . in addition, sambucus for-mosananakai extract inhibited the in vitro production of progeny hcov-nl63 by concentration-dependent manners. the plaque assay indicated that the ic 50 value of sambucus formosananakai extract on the virus yield was 1.17 μg/ml (fig. 2) . remarkably, the results demonstrated that sambucus formosananakai stem ethanol extract served the significantly antiviral activity against hcov-nl63. to discover the inhibitory action of sambucus formosananakai extract on stages of hcov-nl63 replication, plaque formation, virucidal activity, and virus attachment assays were performed using the plaque assays (fig. 3, table 1 ). sambucus formosananakai extract meaningfully inhibited the plaque formation with an ic 50 value of 4.67 μg/ml (fig. 3a) . in the virucidal assay, sambucus formosananakai extract at 1, 5, and 10 μg/ml had a slight virucidal activity with lower than 50% inhibition to interfere with the hcov-nl63 particle infectivity compared to the mock control (fig. 3b ). in the virus attachment assay, sambucus formosananakai extract concentration-dependently reduced the hcov-nl63 attachment onto llc-mk2 cell monolayer in 6-well plates incubated at 4°c, which result demonstrated that the ethanol extract had a influentially inhibitory effect on hcov-nl63 attachment with an ic 50 value of 15.75 μg/ml (fig. 3c) . the results demonstrated that sambucus formosananakai extract specifically suppressed the viral plaque formation and virus attachment during hcov-nl63 replication. the phenolic acid constituents were rich in the extract of sambucus nakai extract were less cytotoxic to llc-mk2 cells, in which cc 50 values of caffeic acid, chlorogenic acid, and gallic acid were greater than 500 μm (supplemental fig. 2 , table 1 ). in the inhibitory assay of hcov-nl63 induced cytopathic effect, caffeic acid, chlorogenic acid, and gallic acid, but not coumaric acid and ferulic acid, diminished the cytopathicity in the infected cells (fig. 4a) . the antiviral activity of the phenolic acid constituents in the in vitro production of hcov-nl63 was ranked in the following order of virus yield reduction: caffeic acid (ic 50 = 3.54 μm) > chlorogenic acid (ic 50 = 43.45 μm) > coumaric acid (ic 50 = 71.48 μm) (fig. 4b , table 1 ). the results verified that the phenolic acid constituents, like caffeic acid, chlorogenic acid, and gallic acid exhibited the prominent antiviral activity against hcov-nl63, as potential anti-hcov-nl63 components in the sambucus for-mosananakai extract. to examine the antiviral mechanism of caffeic acid and chlorogenic acid against hcov-nl63, the assays of plaque formation, virucidal activity and virus attachment were subsequently performed (fig. 5 , table 1 ). caffeic acid had a stronger inhibitory activity on the plaque formation than chlorogenic acid, in which the ic50 values on the plaque formation were 5.4 μm for caffeic acid and 44.38 μm for chlorogenic acid, respectively (fig. 5) . caffeic acid, but not chlorogenic acid, concentration-dependently served the virucidal activity (ic 50 = 91.3 μm) and powerfully reduced hcov-nl63 attachment to fig. 3 . effects of sambucus formosana nakai extract on plaque formation, virucidal activity and virus attachment. mk-2 cell monolayer was infected with hcov-nl63, simultaneously treated with the extract for 1 h, and then covered with the agarose overlay medium. after 3-day incubation at 37°c in a co 2 incubator, plaques were determined after crystal violet staining. the inhibitory activity of the extract on the plaque formation was according to on the ratio of loss plaque number to mock-treated group (a). in the virucidal assay, the extract was mixed with hcov-nl63 (10 6 pfu), then incubated at 37°c for 1 h. the extract/virus mixture was diluted by 1000-fold dilution and examined for the residual infectivity by plaque assay (b). in the attachment assay, hcov-nl63 was mixed with the extract, then immediately added onto mk2 cell monolayer for 1 h at 4°c. after washing, the cell monolayer was overlaid with 0.75% agarose medium for 3 days at 37°c in co 2 incubator. attachment inhibition was determined based on the residual plaques (c). *, p value < 0.05; **, p value < 0.01 compared with mock-treated cells. fig. 4 . inhibitory effects of the phenolic acid constituents on viral cytopathicity and virus yield in hcov-nl63 infected cells. hcov-nl63 at moi of 0.1 was added to llc-mk-2 cell culture and then immediately treated with the phenolic acid constituents. virus-induced cytopathic effect was photographed 36 h postinfection by microscopy (a). supernatant of hcov-nl63-infected/treated cells was harvested 36 hpi; virus yield in supernatant was determined plaque assay. the rate of virus yield reduction was calculated based on the ratio of loss particle number to mock-treated group (b). ca, caffeic acid; ch, chlorogenic acid; co, coumaric acid; fe, ferulic acid; ga, gallic acid. **, p value < 0.01, ***, p value < 0.001 compared with mock-treated infected cells. the cell surface (ic 50 of 8.1 μm), respectively (fig. 5 ). in addition, infectivity inhibition assay with human airway epithelia calu-3 cells was performed when hcov-nl63-infected cells with a moi of 0.05 were treated with 10 and 50 μm caffeic acid after a 36-h incubation at 32°c. microscopic photography indicated that swelling and rounding were observed in hcov-nl63-infected calu-3 cells (fig. 6a, top) , as described in the previous study (lednicky et al., 2013) . caffeic acid at 10 and 50 μm significantly lessening viral cpe (fig. 6a, top) . immunofluorescent staining demonstrated that caffeic acid concentration-dependently reduced hcov-nl63 infectivity determined according to the ratio of hcov-nl63-positive cells to total cells stained with dapi (fig. 6a, middle and bottom) . the inhibitory assay with immunofluorescent staining on hcov-nl63 infectivity indicated that caffeic acid had a potent antiviral activity against the replication of hcov-nl63 in calu-3 cells (ic 50 = 0.2 ± 0.1 μm) (fig. 6b) . the results revealed that caffeic acid displayed the potent anti-hcov-nl63 activity in a cell-type independent manner, specifically inhibiting on the attachment stage of hcov-nl63 replication. this study was the first report on the antiviral efficacy of sambucus formosananakai extract and its related phenolic acid constituents against hcov-nl63. sambucus formosananakai extract exhibited the low cytotoxicity and markedly decreased cytopathic effect and sub-g1 arrest in hcov-nl63-infected cells, in which was associated with the virus yield reduction in a concentration-dependent manner (figs. 1 and 2, table 1 ). moreover, sambucus formosananakai extract showed the potent antiviral activity against hcov-nl63 with the ic50 values in low microg/ml ranges, such ic 50 of 1.17 μg/ml for virus yield, ic 50 of fig. 5 . effects of caffeic acid and chlorogenic acid on plaque formation, virucidal activity and virus attachment. mk-2 cell monolayer was infected with hcov-nl63, simultaneously treated with the caffeic acid or chlorogenic acid for 1 h, and then covered with the agarose overlay medium for 3-day at 37°c in a co 2 incubator. after crystal violet staining, the inhibitory activity of caffeic acid and chlorogenic acid on the plaque formation was according to on the ratio of loss plaque number to mock-treated group (a). in the virucidal assay, the caffeic acid or chlorogenic acid was mixed with hcov-nl63 (10 6 pfu), then incubated at 37°c for 1 h. the 1000-fold dilution of the compound/virus mixture was examined for the residual infectivity by plaque assay (b). in the attachment assay, mk2 cell monolayer was infected with hcov-nl63 (100 pfu), immediately treated with the caffeic acid or chlorogenic acid for 1 h at 4°c, washed, and overlaid with 0.75% agarose medium for 3 days at 37°c in co 2 incubator. attachment inhibition was determined based on the residual plaques (c). ca. caffeic acid; ch, chlorogenic acid. *, p value < 0.05;**, p value < 0.01; ***, p value < 0.001 compared with un-treated infected cells. fig. 6 . inhibition of hcov-nl63 infectivity in human airway epithelial cells by caffeic acid. calu-3 cells were infected with hcov-nl63 at a moi of 0.05 and immediately treated with caffeic acid for 36 h at 32°c. images of virus-induced cpe effect were photographed by a light microscope (a, top). in addition, mock, infected, and infected/treated cells were performed using immunofluorescence staining anti-hcov-nl63 immunized sera and secondary antibody alexa fluor antimouse igg (a, middle); total cells were stained with dapi (a, bottom). infectivity was determined according to the ratio of hcov-nl63-positive cells to total cells (b). **, p value < 0.01; ***, p value < 0.001 compared with un-treated infected cells. 4.67 μg/ml for plaque formation, and ic 50 of 15.75 μg/ml for virus attachment. the results were consistent with the previous reports in that sambucus spp., such sambucus nigra l. served the antiviral properties against influenza a and b viruses, and the herpes simplex virus type1 (krawitz et al., 2011; serkedjieva et al., 1990; zakay-rones et al., 1995) . in addition, sambucus juice was the key recipe for the "virus blocking factor" that processed the in-vitro antiviral activity against many respiratory viral infections, including influenza a h1n1, rhinovirus b subtype 14, respiratory syncytial virus, parainfluenzavirus subtype 3, and adenovirus c subtype 5 (fal et al., 2016) . sambucus nigra l. has been recognized as was a potentially safer alternative to treat upper respiratory symptoms, such common cold and influenza (hawkins et al., 2019) . thus, sambucus formosananakai might be the alternative medicinal herb for caring the respiratory viral infections, especially coronavirus-associated common cold. among six phenolic acid constituents in the sambucus formosananakai extract, caffeic acid had the highest anti-hcov-nl63 potency with the inhibitory effect on the virus yields (ic50 = 3.54 μm), plaque formation (ic50 = 5.40 μm), and virus attachment (ic50 = 8.10 μm) (figs. 4 and 5, table 1 ). caffeic acid has also been demonstrated the antiviral activity against hcov-nl63 in a cell-type independent manner (fig. 6 ). in addition, chlorogenic acid and gallic acid exhibited antiviral activity against hcov-nl63 (figs. 4 and 5, table 1 ). the ic50 value on the inhibition of virus yield was 43.45 μm for chlorogenic acid, and 71.48 μm for gallic acid, respectively. the phenolic acid constituents have been identified as the major components in the sambucus formosananakai extract and sambucus australis by lc-ms/ms analyses (benevides bahiense et al., 2017; zhang et al., 2010) , therefore caffeic acid, chlorogenic acid and gallic acid might be the key components with anti-hcov-nl63 activity in the sambucus formosananakai extract. caffeic acid has been reported to process the antiviral activity against hepatitis b and c viruses, influenza a virus, and herpes simplex virus (wang et al., 2019; shen et al., 2018; tanida et al., 2015; utsunomiya et al., 2014; ikeda et al., 2011; langland et al., 2018) . caffeic acid reduced the production of hepatitis c virus in vitro via the initial stage of viral replication (tanida et al., 2015) . the antiviral mechanism of caffeic acid against hepatitis c virus was also associated with the activation of p62-mediated keap1/nrf2 signaling pathway for the ho-1-dependent production of interferon α, which significantly suppressed the replication of hepatitis c virus (shen et al., 2018) . caffeic acid markedly decreased the virus yield and cytopathic effect in influenza a virus-infected cells, particular during the period within 3 h post-infection, suggesting that caffeic acid works at the early stages of influenza a virus infection (utsunomiya et al., 2014) . in addition, caffeic acid had no virucidal effect, but significantly affected the production of progeny herpes simplex virus and the binding attachment to the heparan sulfate proteoglycans on cell surface (ikeda et al., 2011; langland et al., 2018) . interestingly, hcov-nl63 has been demonstrated to utilize the heparan sulfate proteoglycans as the co-receptor for attachment to target cells (milewska et al., 2014) . caffeic acid has also been identified to exhibit a high inhibitory effect on the enzymatic activity of angiotensin converting enzyme (ace) (chiou et al., 2017) ; docking studies revealed that caffeic acid also presented the good docking score with the hydrogen bond interactions with the residues in the activity site of ace2 (zozimus divya lobo et al., 2017) . thus, the inhibitory mechanism of caffeic acid on hcov-nl63 attachment and infection to cells might be due to that caffeic acid directly target and interfere the binding interaction of hcov-nl63 with heparan sulfate proteoglycans (co-receptor) and ace2 (receptor) on cell surface. chlorogenic acid and gallic acid have also been demonstrated to suppress the in vitro and in vivo replication of influenza a virus, enterovirus 71 and hepatitis b and c viruses (ding et al., 2017; wang et al., 2009; you et al., 2018; govea-salas et al., 2016) . chlorogenic acid specifically inhibited the neuraminidase activity of influenza a viruses h1n1 and h3n2 that was the crucial mechanism of chlorogenic acid for blocking the release of progeny virions from infected cells (ding et al., 2017) . gallic acid served the antioxidant capacity that linked with down-regulating the genomic rna and proteins expression of hepatitis c virus (govea-salas et al., 2016) . therefore, the phenolic acid constituents of sambucus formosana nakai extract, like caffeic acid, chlorogenic acid and gallic acid, play the key antiviral components against hcov-nl63 and the other viruses. the study demonstrated the potent anti-hcov-nl-63 activity of sambucus formosananakai extract through the significant reduction of virus yield, plaque formation and virus attachment. caffeic acid, chlorogenic acid and gallic acid were reported as the phenolic acid constituents in the sambucus formosananakai extract, exhibiting the antiviral capacity with reducing the production of progeny hcov-nl63 particles in vitro. moreover, caffeic acid plays the important component with antiviral activity, as suggested to influence the binding of hcov-nl63 to the co-receptors (such heparan sulfate proteoglycans) and receptor (ace2). like sambucus nigra l., sambucus 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was supported by china medical university under the featured areas research center program within the framework of the higher education sprout project by the ministry of education, taiwan (chm106-6-2 and cmrc-chm-2). this project was also funded by grants from the ministry of science and technology, taiwan (most107-2923-b-039-001-my3, most105-2320-b-039-053-my3), china medical university (cmu106-bc-1, cmu106-asia-06, cmu107-asia-12, and cmu107-s-14). supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10. 1016/j.virusres.2019.197767. key: cord-291962-rp172ugk authors: jing, huiyuan; song, tao; cao, sufang; sun, yanting; wang, jinhe; dong, wang; zhang, yan; ding, zhen; wang, ting; xing, zhao; bao, wenqi title: nucleotide-binding oligomerization domain-like receptor x1 restricts porcine reproductive and respiratory syndrome virus-2 replication by interacting with viral nsp9 date: 2019-07-15 journal: virus res doi: 10.1016/j.virusres.2019.05.011 sha: doc_id: 291962 cord_uid: rp172ugk porcine reproductive and respiratory syndrome virus (prrsv) causes one of the most economically important diseases of swine worldwide. current antiviral strategies provide only limited protection. nucleotide-binding oligomerization domain-like receptor (nlr) x1 is unique among nlr proteins in its functions as a pro-viral or antiviral factor to different viral infections. to date, the impact of nlrx1 on prrsv infection remains unclear. in this study, we found that prrsv infection promoted the expression of nlrx1 gene. in turn, ectopic expression of nlrx1 inhibited prrsv replication in marc-145 cells, whereas knockdown of nlrx1 enhanced prrsv propagation in porcine alveolar macrophages (pams). mechanistically, nlrx1 was revealed to impair intracellular viral subgenomic rnas accumulation. finally, mutagenic analyses indicated that the lrr (leucine-rich repeats) domain of nlrx1 interacted with prrsv nonstructural protein 9 (nsp9) rdrp (rna-dependent rna polymerase) domain and was necessary for antiviral activity. thus, our study establishes the role of nlrx1 as a new host restriction factor in prrsv infection. porcine reproductive and respiratory syndrome (prrs) is one of the most economically important viral diseases affecting the swine industry worldwide since it was first reported in 1987 (harding et al., 2017; snijder et al., 2013) . the etiological agent of this devastating disease, prrs virus (prrsv), is a positive-sense single-stranded rna virus that belongs to the family arteriviridae, order nidovirales (loving et al., 2015) . the virus engages with the host cellular protein interaction network during infection, facilitating virus hijacking of the host molecular machinery to fulfill the viral life cycle (ke and yoo, 2017; lunney et al., 2016; rahe and murtaugh, 2017; shi et al., 2015; sun et al., 2012) . based on their genetic and antigenic differences, prrsv strains are classified into 2 distinct genotypes, prrsv-1 (european type) and prrsv-2 (north american type) (han and yoo, 2014; murtaugh et al., 2010) . the ∼15 kb viral genome contains at least 10 open reading frames (orfs), which encode at least 14 nonstructural proteins (nsp) and 8 structural proteins (han and yoo, 2014) . among the nonstructural proteins, nsp9 contains an rna-dependent rna polymerase (rdrp) domain in its c-terminal portion, which is critical for viral rna synthesis, and replication efficiency (fang and snijder, 2010; yang et al., 2015; zhou et al., 2011) . in this regard, recent advances show that amino acids at positions 519, 544, 586 and 592 in nsp9 contribute to enhanced pathogenicity and determine the fatal virulence of the virus zhao et al., 2018) . in addition, two highly conserved t-cell epitopes have been identified in nsp9, which may provide broad cross-protection against diverse prrsv strains (parida et al., 2012; rascon-castelo et al., 2015) . noteworthy, multiple novel antiviral treatments, including nsp9 specific camel single-domain antibodies, and sirna targeting nsp9, as well as vaccine strategy of de-optimization of codon pair bias in nsp9, significantly decreased prrsv replication in vitro and in vivo liu et al., 2015; xie et al., 2014; zheng et al., 2015; zhu et al., 2018) . finally, nsp9 was found to associate with at least 9 prrsv encoded proteins, including nsp1α, nsp1β, nsp3, nsp7α, nsp7β, nsp8, nsp11, nsp12, and nucleocapsid (n) protein, validating the notion of this protein as a core component of the viral replication/transcription complex (rtc) liu et al., 2016; nan et al., 2018) . further exploring the interaction of nsp9 with host cellular proteins and analyzing the biological significance are necessary for fully understanding the replication mechanisms and pathogenesis of prrsv . nucleotide-binding oligomerization domain (nod)-like receptor (nlr) x1 (also known as clr11.3 and nod9), a member of the nlr family proteins, is initially identified as key mediators of immune defense and inflammation (arnoult et al., 2009; coutermarsh-ott et al., 2016; eitas et al., 2014; imbeault et al., 2014; kang et al., 2015; kanneganti, 2010; koblansky et al., 2016; lupfer and kanneganti, 2013; moore et al., 2008; philipson et al., 2015; singh et al., 2015; soares et al., 2014; tattoli et al., 2016; ting et al., 2008; wang et al., 2013) . to date, accumulated evidence indicated that nlrx1 inhibits nf-κb (nuclear factor-kappa b) signaling, inflammasome activation, double-stranded rna (dsrna) activated kinase pkr and type i interferon (ifn) production but potentiates reactive oxygen species (ros) production and autophagy (abdul-sater et al., 2010; allen et al., 2011; feng et al., 2017; guo et al., 2016; hung et al., 2018; kim et al., 2017; lei et al., 2012; moore et al., 2008; o'neill, 2008; qin et al., 2017; stokman et al., 2017; tattoli et al., 2008; theus et al., 2017; xia et al., 2011; yin et al., 2017; zeng et al., 2017) . recently, nlrx1 was also identified to facilitate human immunodeficiency virus 1 (hiv-1) (guo et al., 2016) , herpes simplex virus 1 (hsv-1) (guo et al., 2016) , hepatitis c virus (hcv) (qin et al., 2017) and kaposi's sarcoma-associated herpesvirus (kshv) infection, whereas restricts influenza a virus (iav) (jaworska et al., 2014) , and hepatitis a virus (hav) (feng et al., 2017) replication. these conflicting reports about whether nlrx1 functions as a pro-viral or antiviral factor imply that the role of nlrx1 in viral infection is more complex than previously thought and remains to be clarified. until now, the specific role of nlrx1 in prrsv infection was scarcely known. thus, the aim of the present study is to seek evidence for a potential role of nlrx1 in prrsv infection. hek293 t and pams cells (jing et al., 2017b) were cultured and maintained in rpmi-1640 supplemented with 10% heat-inactivated fetal bovine serum (fbs). hela and marc-145 cells were cultured and maintained in dmem supplemented with 10% heat-inactivated fbs. prrsv strain hn1 (genbank: ay457635.1) is a highly pathogenic prrsv-2 (north american) strain, which was isolated from the lung of pigs suffering from "high fever" syndrome in henan province, china (cao et al., 2015) . the virus was amplified and titered in marc-145 cells. uv-inactivated prrsv was generated by irradiating the virus with short-wave uv light (254 nm) for 1 h. the loss of infectivity was confirmed by the inability of the uv-exposed virus to produce a cytopathic effect on monolayer of marc-145 cells. mouse monoclonal anti-ha, anti-flag, and anti-β-actin antibodies were purchased from abclonal biotechnology (china). anti-flag polyclonal antibody (macgene, china), anti-nlrx1 polyclonal antibody (proteintech, china), were purchased and used according to the manufacturers' recommendations. hrp-conjugated anti-mouse and antirabbit igg light (or heavy) chain specific antibodies (abbkine science, usa) were purchased and used according to the manufacturers' recommendations. a monoclonal antibody directed against prrsv n protein was produced from hybridoma cells derived from sp2/0 myeloma cells and spleen cells of balb/c mice immunized with recombinant n protein (jing et al., 2017b) . the flag or ha epitope tag was amplified by pcr and cloned into the pcaggs-mcs vector to generate the pcaggs-flag or pcaggs-ha plasmid, encoding an n-terminal flag or ha tag (jing et al., 2017b) . expression plasmids for flag-tagged nsp9 (full-length and truncated) were constructed by pcr amplification of the cdna from prrsv-infected marc-145 cells. plasmids encoding full-length and truncated nlrx1 (nm_024618) were constructed by pcr amplification using the specific primers listed in table 1 . all constructs were confirmed by dna sequencing. the cells were transfected with expression plasmids using lipofectamine 2000 (invitrogen) according to the manufacturer's instructions. where necessary, empty control plasmid was added to ensure that each transfection receives the same amount of total dna. for sirna knockdowns, cells plated in 6-well plates and transfected with 30 nm the indicated sirnas twice over a 48-h period by using lipofectamine 2000. sirna sequences used are as follows: si-nlrx1-1 # , 5′-uugucaaucugcugcgcaa-3′; si-nlrx1-2 # , 5′-gugcugggc uugcggaaga-3′; si-nlrx1-3 # , 5′-gcaugucuuccgccgggau-3′; negative control sirna, 5′-uucuccgaacgugucacgutt-3′ (table 2) . prrsv titers were expressed as the tissue culture infectious dose 50 (tcid 50 ) per milliliter using the reed-muench method as previously described (jing et al., 2017a) . briefly, marc-145 cells were seeded in 96-well plates, following, infected with serial 10-fold dilutions of prrsv samples in eight replicates. plates were incubated for 72-96 h before virus titers were calculated. total rna was isolated at the indicated time points using trizol reagent. qpcr was performed using sybr green real time pcr master mix (toyobo biologics, osaka, japan) in a lightcycler 480 (roche table 1 the sequences of primers used for construction of nlrx1, and nsp9 protein mutants. sequence the restriction enzyme sites used for cloning are underlined in italics. the sequences of primers used for real-time pcr. primer names genebank sequence (5'-3') jing, et al. virus research 268 (2019) 18-26 molecular biochemicals). individual transcripts in each sample were assayed three times. the pcr conditions were as follows: initial denaturation for 10 min at 95°c, followed by 40 cycles of 15 s at 95°c, 15 s at 58°c and 40 s at 72°c. the fold change in gene expression relative to normal was calculated using the delta delta cycles to threshold (δδct) method. gene expression in marc-145 cells and pams was normalized to that of gapdh and β-actin, respectively. primers were designed using the primer express software (version 3.0; applied biosystems, carlsbad, ca). absolute quantitative mrna levels were calculated using standard curves as previously described . cells cultured in 60-mm dishes were prepared by adding 120 μl of 2 × lysis buffer a (65 mm tris−hcl [ph 6.8], 4% sodium dodecyl sulfate (sds), 3% dl-dithiothreitol, and 40% glycerol). the cell extracts were boiled for 10 min, and then resolved with 8%-12% sds-page. the separated proteins were electroblotted onto a polyvinylidenedifluoride (pvdf) membrane (millipore, billerica, ma). run for 1-2 h at 100 v on ice. the western blotting was probed with specific antibodies. the expression of β-actin was detected with a mouse monoclonal antibody to demonstrate equal protein sample loading. densitometry quantification of protein bands of interest was performed using imagej software (table 3) . hela cells seeded on microscope coverslips and placed in 24-well dishes were transfected with flag-tagged nsp9 expression plasmid when the cells reached approximately 70-80% confluence. at 28 h after transfection, cells were fixed with 4% paraformaldehyde for 10 min, subsequently permeated with 0.1%triton x-100 for 10 min at room temperature. after three washes with pbs, cells were sealed with pbs containing 5% bovine serum albumin for 1 h, followed by incubation with rabbit polyclonal antibody against nlrx1 and mouse monoclonal antibody against flag tag for 1 h at room temperature separately. successively, cells were treated with fitc-labeled goat anti-mouse and cy3-labeled goat anti-rabbit (invitrogen) antibodies for 1 h, with dapi for 15 min at room temperature. after washing with pbs, fluorescent images were acquired using a confocal laser scanning microscope (olympus fluoview ver.3.1, japan). to investigate the interactions between proteins, hek293 t cells were lysed in immunoprecipitation lysis buffer (ripa). after the lysates were incubated for 1 h at 37°c with rnase and dnase, the lysate proteins were incubated overnight at 4°c with the indicated antibodies. protein a + g agarose beads (30 μl; beyotime) were then added to each immunoprecipitation reaction for another 6 h. the agarose beads were then washed three times and the captured proteins were resolved on 8%-12% sds-page, transferred to pvdf membranes, and analyzed by immunoblotting. the results represent the means and standard deviations from three independent experiments. graphpad prism 5 software (graphpad software, san diego, ca, usa) was used for data analysis using a twotailed unpaired t-test *p < 0.05; **p < 0.01. our work has previously shown that prrsv infection up-regulates the expression of nlrx1 in marc-145 cells, a highly permissive cell line derived from epithelial cells of a monkey kidney (jing et al., 2014) . to ascertain this result in the main target cells of acute prrsv infection, porcine alveolar macrophages (pams) were infected with prrsv at an moi of 0.1. real-time pcr was performed at 12 h, 24 h, and 48 h post infection (hpi) to determine the expression levels of nlrx1. as shown in fig. 1a , the data showed that nlrx1 expression was significantly upregulated at 24 h, and increased at a steady-state level in prrsv-infected pams when compared to non-infected cells (> 10-folds). moreover, uv-inactived prrsv failed to induce nlrx1 mrna expression, indicating that the up-regulation of nlrx1 dependents on viral replication (fig. 1a) . next, pams were infected at different moi with prrsv for 48 h before analysis for the mrna expression of nlrx1. result indicated that prrsv up-regulated nlrx1 in a dose dependent manner (fig. 1b) . aiming to clarify whether prrsv induces nlrx1 expression in protein level, pams were infected at an moi of 0.1 with prrsv or uvinactived prrsv for the designated time. nlrx1 protein levels measured by western blotting reflected the changes in mrna observed, suggesting that endogenous nlrx1 expression was upregulated by prrsv infection (fig. 1c) . in order to investigate whether the upregulation of nlrx1 might have a correlation with prrsv infection, we transfected marc-145 cells with nlrx1 expression plasmid, followed by infection with prrsv. nlrx1 activity on viral infection was monitored by measuring prrsv titers in the culture supernatants collected at 12, 24 and 48 hpi. as shown in fig. 2a , overexpression of nlrx1 consistently reduced viral titers of prrsv at 12-48 hpi. next, marc-145 cells transfected with nlrx1 were infected with different moi of prrsv, a tcid 50 assay was used to determine the effect of nlrx1 on the production of infectious prrsv particles. as expected, tcid 50 data suggested that overexpression of nlrx1 inhibited prrsv proliferation, especially at low infection doses (fig. 2b) . since marc-145 cells are monkey derived though they are prrsv permissible, we wondered whether endogenous nlrx1 had the same effect on prrsv replication in primary pam cells. to this end, three pairs of sirna duplexes against nlrx1 were individually transfected into pams. the knockdown efficiency of these sirna was evaluated by real-time pcr. result indicated that si-nlrx1-2 # achieved the highest efficiency (fig. 3a) . western blot analysis further showed that si-nlrx1-2 # reduced the level of nlrx1 protein by ∼87% compared to the controls cells transfected with nc sirna (fig. 3b) . to investigate the function of endogenous nlrx1 in prrsv replication, pams were treated with nc sirna or si-nlrx1-2 # followed table 3 the sequences of primers used for quantification of total viral rna and sgrnas. sequence (5'-3') jing, et al. virus research 268 (2019) 18-26 by prrsv infection. then viral titers were measured by tcid 50 at 12-48 hpi. we observed that reduced nlrx1 protein expression was correlated with increased prrsv in pams (fig. 3c ). in aggregate, these data suggested that nlrx1 can potentially inhibit replication of prrsv in pams. nlrx1 contains a carboxy-terminal lrr domain, a central nod domain and a unique n-terminal card-related x domain (moore et al., 2008) (fig. 4a ). to better understand how nlrx1 impaired prrsv replication, we mapped the domains of nlrx1 required for this restriction. to this end, we transfected marc-145 cells with plasmids encoding either the full-length nlrx1 or its mutants prior to prrsv infection. progeny virus in culture supernatants was determined by tcid 50 assay. the results showed that constructs lacking the x domain (δx) impaired prrsv replication. whereas constructs lacking the lrr domain (δl) did not. as shown in fig. 4b , construct encoding lrr alone or lrr in combination with nod domain also impaired prrsv replication, suggesting that lrr is the key domain of nlrx1 that impairs prrsv replication. the c-terminal lrr domain of nlrx1 has been shown to bind rna, and participate in regulation of steady-state levels of a subset of mitochondrial rna (feng et al., 2017; hong et al., 2012; singh et al., 2018) . the observations in fig. 4b led to our working hypothesis that nlrx1 modulated viral rna synthesis. to test this, increasing dose of nlrx1 was transfected into marc-145 cells followed by prrsv infection, qpcr was then performed to test the total viral rna levels. result in fig. 4c showed that nlrx1 inhibited the synthesis of viral rnas in a dose dependent manner. furthermore, knockdown of endogenous nlrx1 in pams increased the levels of viral total rna (fig. 4d) . the prrsv genome is a single-strand positive-sense rna flanked by the 5′ and 3′ un-translated regions (utrs). a set of 3′-coterminal subgenomic rnas (sgrnas) are produced during infection (han and yoo, 2014) . thus, we evaluated the role of nlrx1 in viral sgrnas synthesize. these synthesized viral sgrnas were then quantified by qpcr after the infection of the marc-145 cells with prrsv. as shown in fig. 4e , the relative levels of viral grna and sgrnas, including grna, fig. 1 . prrsv infection up-regulates nlrx1 expression in pams. (a) pams were infected at an moi of 0.1 with prrsv or uv-inactived prrsv for the designated time. total rna was extracted, and real-time pcr was performed to determine the relative levels of nlrx1. gene expression was normalized to that of β-actin. significant differences from the uninfected cells are denoted by asterisks (**) for p < 0.01. (b) pams were infected at the indicated moi with prrsv for 48 h. total rna was extracted, and real-time pcr was performed to determine the relative levels of nlrx1. (c) pams were infected at an moi of 0.1 with prrsv or uv inactived prrsv for the designated time. cell lysates were blotted with the indicated antibodies. sgrna2 to 4, apparently declined in nlrx1-overexpressing cells compared to the control, but the relative levels of viral sgrna5-7 had no significant differences. this result suggested the inhibitory role of nlrx1 in the process of synthesizing long sgrnas and genomic rna. nucleocapsid (n)-nsp9 interaction has been shown to be involved in the production of prrsv sgrnas . to explore the mechanism by which nlrx1 suppresses viral sgrnas synthesis, we investigated whether nlrx1 interacts with prrsv nsp9/n protein. to this end, hek293 t cells were transfected with plasmids expressing flag-nsp9/n and ha-nlrx1 and co-immunoprecipitated with flag antibody or ha antibody. even though no co-immunoprecipitation of n protein was detectable with overexpressed nlrx1 in hek293 t cells (data not shown), ha-nlrx1 was co-precipitated with flag-tagged nsp9 and vice versa (fig. 5a) . next, we investigated whether nlrx1 could colocalize with nsp9. hela cells were transfected with plasmids expressing flag-nsp9. confocal data revealed ectopically expressed flag-nsp9 and endogenous nlrx1 partially co-localized with each other in the cytoplasm (fig. 5b) . to identify the binding region within nlrx1 involved in the nsp9-nlrx1 interaction, hek293 t cells were co-transfected with various combinations of ha-tagged full-length or deleted versions of nlrx1 and flag-tagged nsp9. mapping studies by co-ip revealed that nsp9 interacted with the c-terminal lrr domain of nlrx1, but not with its central nod domain or n-terminal x domains (fig. 5c ). prrsv nsp9 contains n-terminal rdrp domain and a c-terminal domain of unknown function zhao et al., 2015) . to further investigate which domain within nsp9 is required for binding with nlrx1, plasmids expressing nsp9 mutants were constructed and their ability to interact with nlrx1 was assessed by co-ip. result showed that wt nsp9 and nsp9c, but not the nsp9n, bound to nlrx1 (fig. 5e) . the results showed that the rdrp domain of nsp9 is responsible for interaction with nlrx1. nlrx1 was the first nlr shown to reduce type-i ifn production and facilitate viruses infection, by binding to mavs (mitochondrial antiviral signaling protein) on mitochondria and sting (stimulator of interferon genes) on endoplasmic reticulum (er), as demonstrated for sindbisvirus, hiv-1, hsv-1 and kshv (guo et al., 2016; ma et al., 2017; moore et al., 2008; qin et al., 2017) . however, all of these functions reported for nlrx1 are not without controversy. for example, nlrx1 prevented iav-induced macrophage apoptosis. its deficiency led to increased pulmonary viral titer, inflammation, and reduced pulmonary function during iav infection (jaworska et al., 2014) . analogously, nlxr1 was required for immediate irf1 (interferon regulatory factor 1)-directed antiviral responses. replication of both hepatitis a virus (hav) and hepatitis c virus (hcv) was enhanced in nlrx1-deficient hepatocytes (feng et al., 2017) . adding to these similarities, we show here that nlrx1 acts to restrict prrsv replication. based on these observations, it is possible that the effect of nlrx1 during viral infection is a doubleedged sword, potentially weakening irf3 mediated responses as well as presenting a hurdle to be overcome by the virus. nlrx1 and iav pb1-f2 interaction is critical for the control of iav replication and inflammation, indicating that interaction between nlrx1 and viral component might be a key factor in determining the outcome of viral infections (jaworska et al., 2014) . our study shows that nlrx1 inhibits prrsv replication by suppression its rna synthesis. this is achieved by the association of prrsv nsp9 with lrr domain of nlrx1. strikingly, c-terminal lrr domain of nlrx1 has been shown to possess a rna binding site (hong et al., 2012) . in this regard, nlrx1 interacts with rhinovirus (rv) rna in polarized airway fig. 3 . enhancement of prrsv replication by nlrx1 knockdown in pams. (a) pams were transfected with a scrambled control sirna (nc) or three different sirna duplexes against nlrx1 for 48 h. total rna was extracted, and real-time pcr was performed to determine the relative levels of nlrx1. β-action level was monitored as an internal control. (b) pams were transfected with nc sirna or sirna 2 # against nlrx1. after 48 h, cell lysates were blotted with anti-nlrx1 or anti-β-actin antibody. (c) pams were transfected with the control sirna or the nlrx1 sirna for 48 h and then infected at an moi of 0.1 with prrsv. virus titers were determined by tcid 50 assay at 12, 24, and 48 h post infection. epithelial cells (unger et al., 2014) . likewise, nlrx1 also binds hav genomic rna to suppress pkr activation (feng et al., 2017) . collectively, these findings support a model in which nlrx1 interacts with various viral factors to achieve an optimal immune state against diverse viruses. future studies will be necessary to assess whether the nsp9 of prrsv-1 interacts with nlrx1. nsp9, the key enzyme encoded by orf1b for rna-templated rna synthesis (rdrp) (fang and snijder, 2010) , is closely related to the replication efficiency and pathogenicity for piglets (li et al., 2014b; music and gagnon, 2010; xu et al., 2018; zhao et al., 2018) . as a consequence, several host proteins have been reported to interact with prrsv nsp9. on the one hand, interaction between nsp9 and cellular annexin a2, retinoblastoma protein (prb), ddx5 positively regulates the replication of prrsv in vitro, demonstrating that prrsv heavily relies on host cellular proteins to complete life cycle (dong et al., 2014; li et al., 2014a; zhao et al., 2015) . on the other hand, recent literature indicated that the interaction of nsp9 with sumo e2 conjugating enzyme ubc9 and cellular protein interleukin-2 enhancer binding factor 2 (ilf2) through its rdrp domain resulted in a significantly decrease of virus titers, indicating that cells utilize host antiviral factors as defense mechanisms to limit prrsv infection wen et al., 2017) . altogether, these results provide insights into the complex interactions of prrsv rdrp with the host proteome. noteworthy, the nsp9 c-terminal fragment consisting of amino acid residues 599-646 was also identified as the n protein binding region , strengthening the concept that the c-terminal rdrp domain is a common protein interacting platform. thus, the c-terminal rdrp domain of nsp9 might be promising drug targets for interfering with prrsv replication. even though our knowledge on the unique discontinuous transcription of arterivirus and coronavirus has rapidly grown over the last years, the molecular mechanisms controlling template switch still remain unclear (di et al., 2018; sola et al., 2015) . in coronavirus, rna helicase ddx1 was reported to benefit the synthesis of longer sgrnas by unwinding the hairpin loop structure of viral grna, implying that rna secondary structure may hinder the balance between shorter and longer sgrnas synthesis (wu et al., 2014) . in line with this, a recent study suggested that prrsv sgrna2-5, but not sgrna6-7, were impaired in helicase dhx9-knockdown cells . in this study, we observed that ectopic expression of nlrx1 had no effects on fig. 4 . nlrx1 suppresses the synthesis of viral subgenomic rnas. (a) schematic representation of full-length nlrx1 and corresponding deletion mutants is shown. protein motifs are indicated. (b) marc-145 cells were transfected with ha-tagged nlrx1, or its deletion mutants. after 24 h, cells were infected at an moi of 0.5 with prrsv for 24 h. virus titers were determined by tcid 50 assay. (c) marc-145 cells were transfected with an increasing amount of nlrx1 expression plasmid or control plasmid and then infected with prrsv (moi = 0.5) for 24 h. prrsv total rna were measured by qpcr. (d) pams were transfected with control sirna or the nlrx1 sirna for 48 h and then infected at an moi of 0.1 with prrsv. viral total rnas were measured by qpcr at 24 h post infection. (e) marc-145 cells were transfected with the empty vector or nlrx1 expression plasmid for 24 h and then infected at an moi of 0.5 with prrsv. the levels of viral rnas were monitored at 24 hpi by qpcr. the viral rna levels in control cells were normalized to a value of 1. the abundance of shorter sgrna5-7. we therefore hypothesized that longer sgrnas transcript could stop upon reaching nlrx1-nsp9-rna complex, thereby switching to produce shorter sgrnas. further investigations will be required to dissect the nlrx1-dependent mechanism for the effective control of continuous and discontinuous rna synthesis. in summary, these above findings indicate that nlrx1 inhibits the replication of prrsv by interacting with viral nsp9 in vitro, providing more knowledge for understanding the mechanisms associated with the replication regulation of prrsv. significant effort is still required to further uncover the potential interaction of nsp9 with host cellular proteins and translate the current understanding of rdrp biology into effective antiviral drug development. none. ha-tagged nlrx1 was cotransfected with flag-tagged nsp9 into hek293 t cells. cell lysates were immunoprecipitated (ip) with anti-ha antibody or anti-flag antibody and then blotted as indicated. (b) hela cells were transfected with flag-tagged nsp9 for 24 h. cells were then fixed and incubated with anti-nlrx1 and anti-flag antibodies. dapi, 4′6diamidino-2-phenylindole. (c) flag-tagged nsp9 was cotransfected with the indicated ha-tagged nlrx1 mutants into hek293 t cells. cell lysates were immunoprecipitated with anti-ha antibody and blotted with the indicated antibodies. (d) the schematics of nsp9 and corresponding truncation constructs. numbers indicate the residues where deletions begin or end. (e) ha-tagged nlrx1 was cotransfected with the indicated flag-tagged nsp9 mutants into hek293 t cells. cell lysates were immunoprecipitated with anti-flag antibody and blotted with the indicated antibodies. h. jing, et al. virus research 268 (2019) 18-26 enhancement of reactive oxygen species production and chlamydial infection by the mitochondrial nod-like family member nlrx1 nlrx1 protein attenuates inflammatory responses to infection by interfering with the rig-i-mavs and traf6-nf-kappab signaling pathways an nterminal addressing sequence targets nlrx1 to the mitochondrial matrix inhibition of highly pathogenic porcine reproductive and respiratory syndrome virus replication by recombinant pseudorabies virusmediated rna interference in piglets structural analysis of porcine reproductive and respiratory syndrome virus non-structural protein 7alpha (nsp7alpha) and identification of its interaction with nsp9 nlrx1 suppresses tumorigenesis and attenuates histiocytic sarcoma through the 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perspectives novel insights into host responses and reproductive pathophysiology of porcine reproductive and respiratory syndrome caused by prrsv-2 structure and functional characterization of the rna-binding element of the nlrx1 innate immune modulator nlrx1 modulates differentially nlrp3 inflammasome activation and nf-kappab signaling during fusobacterium nucleatum infection nlrx1 regulates neuronal cell death nlrx1 prevents mitochondrial induced apoptosis and enhances macrophage antiviral immunity by interacting with influenza virus pb1-f2 protein porcine reproductive and respiratory syndrome virus nsp1alpha inhibits nf-kappab activation by targeting the linear ubiquitin chain assembly complex porcine reproductive and respiratory syndrome virus infection activates nod2-rip2 signal pathway in marc-145 cells dexd/ h-box helicase 36 signaling via myeloid differentiation primary response gene 88 contributes to nf-kappab activation to type 2 porcine reproductive and respiratory syndrome virus infection suppression of nlrx1 in chronic obstructive pulmonary disease central roles of nlrs and inflammasomes in viral infection the viral innate immune antagonism and an alternative vaccine design for prrs virus cholesterol 25-hydroxylase inhibits porcine reproductive and respiratory syndrome virus replication through enzyme activity-dependent and -independent mechanisms fas-associated factor-1 positively regulates type i interferon response to rna virus infection by targeting nlrx1 the innate immune receptor nlrx1 functions as a tumor suppressor by reducing colon tumorigenesis and key tumor-promoting signals the mitochondrial proteins nlrx1 and tufm form a complex that regulates type i interferon and autophagy nlrx1 attenuates apoptosis and inflammatory responses in myocardial ischemia by inhibiting mavs-dependent nlrp3 inflammasome activation the interaction between host annexin a2 and viral nsp9 is beneficial for replication of porcine reproductive and respiratory syndrome virus nsp9 and nsp10 contribute to the fatal virulence of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china an intracellularly expressed nsp9-specific nanobody in marc-145 cells inhibits porcine reproductive and respiratory syndrome virus replication porcine reproductive and respiratory syndrome virus nucleocapsid protein interacts with nsp9 and cellular dhx9 to regulate viral rna synthesis innate and adaptive immunity against porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system the expanding role of nlrs in antiviral immunity nlrx1 negatively modulates type i ifn to facilitate kshv reactivation from latency nlrx1 is a regulator of mitochondrial antiviral immunity the everexpanding diversity of porcine reproductive and respiratory syndrome virus the role of porcine reproductive and respiratory syndrome (prrs) virus structural and non-structural proteins in virus pathogenesis the network of interactions among porcine reproductive and respiratory syndrome virus nonstructural proteins innate immunity: squelching anti-viral signalling with nlrx1 location of t-cell epitopes in nonstructural proteins 9 and 10 of type-ii porcine reproductive and respiratory syndrome virus modeling the regulatory mechanisms by which nlrx1 modulates innate immune responses to helicobacter pylori infection nlrx1 mediates mavs degradation to attenuate hepatitis c virus-induced innate immune response through pcbp2 mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus immunological features of the non-structural proteins of porcine reproductive and respiratory syndrome virus prrsv receptors and their roles in virus infection nlrx1 acts as tumor suppressor by regulating tnf-alpha induced apoptosis and metabolism in cancer cells nlrx1 resides in mitochondrial rna granules and regulates mitochondrial rna processing and bioenergetic adaptation arterivirus molecular biology and pathogenesis the mitochondrial protein nlrx1 controls the balance between extrinsic and intrinsic apoptosis continuous and discontinuous rna synthesis in coronaviruses nlrx1 dampens oxidative stress and apoptosis in tissue injury via control of mitochondrial activity interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus nlrx1 is a mitochondrial nod-like receptor that amplifies nf-kappab and jnk pathways by inducing reactive oxygen species production nlrx1 acts as an epithelial-intrinsic tumor suppressor through the modulation of tnf-mediated proliferation loss of nlrx1 exacerbates neural tissue damage and nf-kappab signaling following brain injury the nlr gene family: a standard nomenclature nod-like receptor x-1 is required for rhinovirus-induced barrier dysfunction in airway epithelial cells interaction of porcine reproductive and respiratory syndrome virus proteins with sumo-conjugating enzyme reveals the sumoylation of nucleocapsid protein the involvement of nlrx1 and nlrp3 in the development of nonalcoholic steatohepatitis in mice interleukin-2 enhancer binding factor 2 interacts with the nsp9 or nsp2 of porcine reproductive and respiratory syndrome virus and exerts negatively regulatory effect on the viral replication nucleocapsid phosphorylation and rna helicase ddx1 recruitment enables coronavirus transition from discontinuous to continuous transcription nlrx1 negatively regulates tlr-induced nf-kappab signaling by targeting traf6 and ikk inhibition of porcine reproductive and respiratory syndrome virus by specific sirna targeting nsp9 gene nonstructural protein 9 residues 586 and 592 are critical sites in determining the replication efficiency and fatal virulence of the chinese highly pathogenic porcine reproductive and respiratory syndrome virus lipid rafts both in cellular membrane and viral envelope are critical for prrsv efficient infection nlrx1 accelerates cisplatin-induced ototoxity in hei-oc1 cells via promoting generation of ros and activation of jnk signaling pathway interactions of traf6 and nlrx1 gene polymorphisms with environmental factors on the susceptibility of type 2 diabetes mellitus vascular complications in a southern han chinese population two residues in nsp9 contribute to the enhanced replication and pathogenicity of highly pathogenic porcine reproductive and respiratory syndrome virus the deadbox rna helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral nsp9 in vitro inhibition of porcine reproductive and respiratory syndrome virus replication in vitro using dnabased short antisense oligonucleotides mutational analysis of the sdd sequence motif of a prrsv rna-dependent rna polymerase in vitro inhibition of porcine reproductive and respiratory syndrome virus replication by short antisense oligonucleotides with locked nucleic acid modification key: cord-275413-e2rhioty authors: rowland, raymond r.r. title: the interaction between prrsv and the late gestation pig fetus date: 2010-09-09 journal: virus res doi: 10.1016/j.virusres.2010.09.001 sha: doc_id: 275413 cord_uid: e2rhioty porcine reproductive and respiratory syndrome virus (prrsv) crosses the placenta during late gestation and productively infects the fetus. virus replication and cytokine responses were measured in tissues of fetuses recovered at 109–112 days of gestation, just prior to parturition. at the time of recovery, gross anatomical abnormalities were evident in both infected and non-infected fetuses from the infected dams. virus isolation and immunohistochemistry identified the thymus as the primary site of virus replication. steady state rt-pcr amplification of inflammatory, th1 and th2 cytokines, showed elevated ifn-γ and tnf-α mrnas in tissues from infected fetuses, which corresponded to elevated cytokine proteins in serum but not amniotic fluid. further evidence for induction of immunity was found in the hyperplastic response of lymph nodes, which included the development of germinal centers occupied cdw75+ b cells. collectively, these data support the notion that the immunocompetent fetus is capable of initiating an antiviral response, which is compartmentalized within the infected fetus. furthermore, fetal pathology may not be a direct result of virus replication in the fetus. porcine reproductive and respiratory syndrome (prrs) is caused by an enveloped positive-stranded rna virus, prrsv, belonging to the family arteriviridae cavanagh, 1997; nelsen et al., 1999; wensvoort et al., 1991) . other members of the arterivirus group include lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv; for review see plagemann, 1996) . the arteriviruses, toroviruses, roniviruses and coronaviruses form a single order, nidovirales. arteriviruses structurally resemble togaviruses, but similar to coronaviruses, replicate via a nested 3 -co-terminal set of subgenomic mrnas that possesses a common leader and a poly-a tail (reviewed in snijder and mulenberg, 1998) . the arteriviruses exhibit several important properties relevant to the study of viral pathogenesis, including cytopathic replication in macrophages, the capacity to establish and maintain an asymptomatic infec-tion, as well as cause severe and fatal disease (plagemann, 1996) . infection of adult pigs with prrsv usually produces a non-fatal disease, characterized by flu-like symptoms, a transient elevation in temperature and inappetance (reviewed in benfield et al., 1999; christianson et al., 1992) . the reproductive form of prrs occurs following the infection of late gestation pregnant gilts or sows. natural infection of the fetus with prrsv is initiated with the infection of gilts and sows at 90 days gestation. after productive replication on the maternal side, the virus crosses the placenta and productively infects the fetus. the mechanism of transplacental infection is unknown, but could be similar to the infected "trojan horse" macrophage, described for ldv (cafruny and bradley, 1996) . since the pig fetus becomes immunocompetent at about 70 days of gestation, prrsv infection occurs in an immune environment containing functional b and t cells. accordingly, virus-induced reproductive failure can present clinically as delayed returns to estrus, as well as abortions, mummified fetuses, stillborn and weak-born pigs christianson et al., 1993; collins et al., 1992; mengeling et al., 1994; rossow et al., 1999; rowland et al., 2003) . surviving neonates can exhibit the severest form of respiratory disease with mortality sometimes reaching 100% within three weeks after birth (feng et al., 2001; rossow et al., 1994; rossow, 1998) . the complex pathology following exposure to prrsv in utero represents a unique form of the disease referred to as congenital prrs (rowland et al., 2003) . the purpose of this study was to characterize the interaction between prrsv and the pig fetus by (1) identifying sites of virus replication, (2) measuring immune and inflammatory cytokines in different compartments, and (3) evaluating the response of lymph nodes. experiments involving animals were approved by the kansas state university iacu committee. pregnant sows, obtained from a closely monitored prrsv-negative herd, were challenged at 90 days gestation with a sixth passage isolate of sd-23983, a typical north american field isolate (rowland et al., 2001) . the methods for the preparation of the prrsv inoculum on marc-145 cells and infection of pigs are described in rowland et al. (2003) . virus was cultivated on marc-145 cells in mem supplemented with antibiotics (pen/step) and 7% fbs. dams, at 90 days gestation were challenged with approximately 10 5 tcid 50 of virus diluted in 5 ml of culture medium. one half of the inoculum was administered by intramuscular injection in the neck. the remaining dose was administered intranasally. mock-infected sows were challenged with medium recovered from marc-145 cells. dams were monitored for clinical signs and blood collected weekly. at between 109 and 112 days of an approximate 114 days gestation period, the dams were euthanized. the uterine horns were immediately removed and the individual fetuses with intact placenta were carefully removed and immediately necropsied. a sample of amniotic fluid was collected prior to removal. the brachial artery of each fetus was severed and blood collected using a disposable syringe and serum stored at −80 • c. maternal, accessory and fetal tissues were collected and stored in formalin for histological staining and immunohistochemistry (ihc), or storage in rnalater (ambion) for rt-pcr of cytokine mrnas. prrsv-specific antibody was measured in sera using the herdcheck ® prrs elisa (idexx) and performed by personnel at kansas state university veterinary diagnostic laboratory. serology results were reported as a sample/positive (s/p) ratio. an s/p ratio greater than 0.39 was considered positive for prrsv antibody. virus isolation (vi) in serum and tissues was performed as described in rowland et al. (2003) . briefly, serum was serially diluted in mem supplemented with pen/step antibiotics and 7% fbs and placed on 96 well plates of confluent marc-145 cells. after three days, plates were fixed in 80% acetone and stained with fitc-sdow-17 anti-nucleocapsid antibody, diluted in pbs with 5% fbs (nelson et al., 1993) . the results were reported as the log 10 of the inverse dilution of the last positive well. virus isolation from tissues was the same except that tissues were weighed and homogenized in hanks balanced salt solution and then centrifuged at 500 × g for 20 min to remove debris. the sequencing of the hypervariable region of orf5 is described in rowland et al. (1999) . total rna was prepared from serum or infected marc-145 cells using an rneasy kit (qiagen) according manufacturer's instructions. for pcr, cdna was prepared using mlv reverse transcriptase (promega) and 4msb as the primer. the sense and antisense primers for the outer amplification were 4msa, 5 -cttcgtcccttcttttcctcgtgg, and 4msb, 5 -ccgctctagagccaacgatagagtctgc, respectively. the product was re-amplified with a nested set of sense and antisense primers, 04a, 5 -accgtgtatgttaccatcacagcc and 04b, acgggaaagatgacaaaactctcc. thirty-two cycles of amplification were performed for each primer pair. the conditions for both amplifications included a 95 • c denaturing step (25 s), a 58 • c annealing step (10 s), and a 74 • c (25 s) polymerization step. the final pcr product, which contained the last 312 nucleotides of orf4, the 10 nucleotide untranslated region (utr), and the first 215 nucleotides of orf5 was sequenced directly by automated dna sequencing. pcr products were cloned into a pcr2.1 ta cloning vector (invitrogen), propagated in escherichia coli and individual plasmids sequenced using m13 forward and reverse primers. sequences were analyzed using gene jockey ii software. tissue samples for rt-pcr were immediately placed in rna-later (ambion) and stored at −80 • c. total rna was extracted from approximately 50 g of tissue using rneasy kit (qiagen) according to manufacturer's instructions. the design of cytokinespecific primers and rt-pcr procedures were performed according to reddy and wilkie (2000) . primer sequences are listed in table 1 . rna was diluted to a final volume of 50 l in nuclease free water. cdna was prepared from10 l of total rna by reverse transcription using molony murine leukemia virus reverse transcriptase (promega) and random hexamers as primers. the amplification of ␤2m mrna was used as an internal control. pcr amplification of cytokine and control cdnas consisted of 35 cycles (45 s at 94 • c, 45 s at 55 • c, and 45 s at 72 • c) and dna products electrophoresed on a 2.0% agarose gel and visualized using ethidium bromide. the identity of the dna products was confirmed by dna sequencing. tissue samples were collected and immediately placed in 10% buffered formalin. paraffin-embedded thin sections were mounted on slides, deparaffinized and stained with hematoxylin for the detection of prrsv antigen, slides were incubated for 30 min with a 1:100 dilution of mab sr-30 anti-nucleocapsid antibody (rural technologies). other antibodies included a polyclonal anti-human cd3 and b cell antibodies, anti-cdw75 and anti-cd79␣. bound antibody was detected with biotinylated goat anti-mouse or anti-rabbit ig followed by avidin-hrpo and dab chromagen (ventana medical). slides were counterstained with hematoxylin. the experiment incorporated four prrsv-infected and twomock-infected dams. all maternal serum samples were vi-negative and seronegative for prrsv prior to infection. between one and two weeks after virus challenge, all infected sows were vi-positive in serum, confirming the presence of an active infection. by the time of necropsy, the concentration of circulating virus in the dams had dipped to below detectable levels by vi. a total of 44 viable fetuses were recovered from the four infected dams (see table 2 for summary). two fetuses were dead and partially autolysed and not subjected to further study. 10 (22%) of the 44 viable fetuses were positive for prrsv by vi. since dams were vi-negative in blood at the time of necropsy, it was concluded that the presence of virus serology results, shown in parentheses, are presented as the s/p ratio. s/p ratios greater than 0.4 were considered positive for prrsv antibody. dead fetuses were partially autolysed and not tested. gross pathology key: *1, partially mummified; *2, non-viable fetus or relatively low quantity of amniotic fluid; *3, necrotic placenta; *4, merconium-and/or blood-stained amniotic fluid; *5, small, underdeveloped fetus. in the fetal circulation was the result of virus replication in the fetus and not from contamination with maternal blood. (the virus isolation technique is not subject to false positives from minute amounts of cross-contamination with viral protein or rna.) the number of infected fetuses in each litter varied from no infected fetuses (dam no. 2) to five of 14 (36%) infected fetuses for dam no. 4. fetuses that were vi-negative in serum were confirmed as prrsv-negative by vi-negative results in placenta, lung, lymph nodes and thymus (data not shown). one fetus, 1-1, was seropositive for prrsv (s/p ratio = 0.94). since there is no maternal transfer of antibody from mother to fetus (tizard, 1996) , it was concluded that this fetus generated an antibody response to prrsv in utero. 7 of the 10 infected fetuses showed some form of gross pathology, including growth retardation (two fetuses) or reduced amounts and/or merconium-stained amniotic fluid (five fetuses). ongoing virus replication in the fetus as the source of these gross pathological changes is questionable, since non-infected fetuses from infected dams exhibited similar changes. for example, fetuses from dam no. 2 (see fig. 1 ) were either merconium stained (two fetuses), non-viable, possessed reduced amniotic fluid levels (four fetuses) or small (one fetus). except for one autolysed fetus, the 23 fetuses from the two control dams showed no evidence of gross pathology (data not shown). rna viruses frequently exist as a heterogeneous population, frequently referred to as a quasispecies. the appearance or disappearance of individual viral sequences within a quasispecies population is often used as evidence to support the existence of positive or negative selection during infection (elena et al., 2000; forns et al., 1999; tsibris et al., 2009) . mutations that appear in the prrsv genome are useful as markers to identify and follow the appearance and disappearance of viruses within the population (allende et al., 2000; rowland et al., 1999) . dna sequencing of orf5 pcr products, amplified directly from the sera of infected dams, table 3 frequency of t at nucleotide position 77 of orf5. consensus nucleotide at position 77 of orf5 t:c ratio in cloned pcr products (percent) t 3/5 (60) fetus 4-09 t 6/7 (86) fetus 4-10 t 3/6 (50) fetus 4-11 c 1/4 (25) fetus 4-12 t 6/6 (100) fetus 4-13 t 5/5 (100) identified one dam, no. 4, which possessed a virus with a mutation within the hypervariable region of orf5. the change detected by sequencing the pcr product was a c to t (u for rna) nucleotide transition at position 77 that resulted in a non-conserved amino acid change from threonine to isoleucine in gp5. the t-77 mutation was not detected after sequencing the orf5 pcr products from the other dams (see table 3 ). pcr products were cloned into a ta plasmid and the individual plasmids sequenced. even though t-77 was detected in the pcr product from dam no. 4, the sequence of individual clones showed that two of the five sequences possessed a c at position 77 mutation, which indicated that viruses with the wild-type orf5 sequence were still present in the population. whole pcr and plasmid-cloned pcr products were sequenced for the five infected fetuses from dam no. 4. the frequency of the t-77 mutation ranged from 25% (fetus 4-11) to 100% (fetuses 4-12 and 4-13). these results indicate that the fetus is capable of selecting for a particular virus population, which either arises in the dam or fetus. therefore, fetal infection is a potential source of prrsv diversity. during acute infection of the postnatal pig, the largest quantity of virus and greatest number of cells supporting virus replication are found in the lung, a consequence of targeting alveolar macrophages. during the later stages of prrsv infection, secondary lymphoid organs, including tonsil and lymph nodes, become sources of virus replication (allende et al., 2000; rossow, 1998; rowland et al., 1999; . virus replication in fetal tissues was assessed using a combination of virus isolation and ihc detection of nucleocapsid antigen in formalin-fixed tissues. as summarized in table 4 , virus was isolated from all tissues from infected fetuses, including placenta, umbilical cord, heart, lung, spleen, lymph nodes and thymus. overall, the thymus contained the largest quantity of virus. nine of ten fetuses yielded measurable amounts of virus with five of the ten fetal thymuses producing titers greater than 3.0. for lung, 6 of 10 fetuses were vi-positive and only one fetal lung yielded a virus titer greater than 2.0. the recovery of virus from a tissue can represent virus in circulating blood. therefore, to determine if cells in the thymus and other tissues were a source of prrsv, tissue thin sections were stained with prrsv anti-nucleocapsid antibody. the ihc staining procedure included two sets of negative controls: tissue thin sections from non-infected fetuses and from infected fetuses stained with only secondary antibody. both controls were negative for staining (data not shown). the results in table 4 showed the largest number of positive tissues for the thymus (8 of 10 positive), followed by spleen (2 of 8 positive) and lymph node (1 of 9 positive). within the thymus, antigen-positive cells were located in both medullar and cortical regions (data not shown). prrsv antigen-positive cells were not detected in lung or tonsil. taken together, the virus titration and ihc results identify the thymus as a principal source of virus replication in the prrsvinfected fetus. in the post-natal pig, prrsv infection results in distinct pathology in the lung, including the appearance of interstitial pneumonia. representative lymph node and lung tissues from non-infected and infected fetuses are shown in fig. 2 . there was no discernable difference between lungs from infected and non-infected fetuses (compare fig. 2 panels c and d) . lymph nodes from prrsv-negative fetuses appeared largely undeveloped and devoid of well-defined germinal centers. in contrast, the lymph nodes from prrsv-infected fetuses appeared much more pronounced and enlarged. at the microscopic level, the increased lymph node volume was associated with increased numbers of cells and the formation of distinct germinal centers (see fig. 2 panels a and b) . the overall appearance is consistent with an antigen-activated lymph node. to determine the source of the increased cell volume, formalin-fixed thin sections of lymph nodes were stained with t cell-specific (cd3) and b cell-specific (cdw75 and cd79␣) antibodies. representative results for mandibular lymph nodes from infected and non-infected fetuses are shown in fig. 3 . the lymph nodes from both infected and non-infected fetuses showed exten sive areas of staining with anti-cd3, indicating the presence of t cells. lymph nodes from non-infected fetuses were negative for cwd75 staining, but possessed some regions that were positive for cd79␣. the b cell receptor for antigen (bcr) signal transduction complex is composed of a heterodimer of ig␣ and ig␤ chains, which are also known as cd79␣ and cd79␤, respectively. cd79␣ staining in the lymph node from non-infected fetuses is consistent with the presence of pro-and pre-b cells (lee et al., 2008) . the principle difference between infected and non-infected fetuses was found in an overall increase in cd79␣+ cells as well as the appearance of cdw75+ cells, which were associated with germinal centers. cdw79 is beta-galactoside alpha-2,6-sialyltransferase, which is up-regulated in activated b cells (erikstein et al., 1992) . the absence of cdw75 staining in non-infected fetuses is consistent with the overall quiescent nature of the non-stimulated fetal immune system. the up-regulation of cdw75 after prrsv infection is consistent with b cell activation and the formation of mature germinal centers in response to infection. it should be noted that infected fetuses showed different degrees of staining, a likely consequence of the different stages of fetal infection; i.e. less staining was the result of early infection. together, the overall morphology and lymphocyte marker expression results indicate that the increased volume in the lymph nodes of infected fetuses is largely the result of increased numbers of mature activated b cells, which occupy germinal centers. immune cytokines are important factors in antiviral immunity and can influence the outcome of pregnancy (arck et al., 1999; basurko et al., 2009 ). the detection of cytokine gene expression in tissues was performed using a steady state rt-pcr procedure. rt-pcr was performed on rna isolated from four fetuses randomly chosen from the two mock-infected dams and four randomly selected prrsv-infected fetuses. the tissues selected for rt-pcr amplification were lung, lymph node and placenta. lung and lymph node represent sites cytokine alterations in the post-natal pig. placenta was selected as an accessory tissue located at the fetal maternal interface. amplification of mrnas included cytokines associated with inflammatory (il-6, il-8), th1 (il-12, ifn-␥, il-2) and th2/regulatory (il-4, il-10) responses. the amplification of ␤2m mrna was included as an internal control. the determination of a cytokine response was based on the presence or absence of a pcr product. il-6, il-8, il-12 and il-10 products were detected in tissues from both control and infected fetuses. because of the qualitative nature of the pcr method, it was not possible to accurately determine quantitative differences between control and infected fetuses; and therefore, these cytokines were not subjected to further study (data not shown). il-4 mrna was not detected in any of the selected tissues for control and infected fetuses. marked differences in expression were observed for tnf-␣ and ifn-␥ mrnas. the results for ifn-␥ and tnf-␣ from lung, mandibular lymph node and placenta are presented in fig. 4 . the results for il-10 are also shown. the internal control mrna, ␤2m, was amplified from all tissues, indicating that the rna was intact. il-10 was amplified from lung and mandibular lymph nodes from two of the four control fetuses and from all infected fetuses. the presence of il-10 was not unexpected, since increased il-10 production is associated with fetal t cell responses (lin et al., 1993; rainsford and reen, 2002) . as shown in fig. 4a , ifn-␥ and tnf-␣ pcr products were not detected in lung, lymph node or placenta from the non-infected fetuses. however, ifn-␥ pcr products were obtained for lung and lymph nodes from infected fetuses. one infected fetus, 4-3, yielded a faint ifn-␥ product for placenta. for infected fetuses, tnf-␣ mrna was amplified from lung, but not lymph node or placenta (fig. 4b) . to determine if cytokine gene expression was the direct result of prrsv infection, rt-pcr for ifn-␥ and tnf-␣ was performed on the same tissues from fetuses of infected dam no. 2, which produced only prrsv vi-negative fetuses (see fig. 1 ). the analysis of mrna expression in lungs and lymph nodes from six fetuses from dam no. fig. 4 . cytokine gene expression in fetal tissues. rt-pcr for cytokine mrnas was performed on lung (l), mandibular lymph node (n) and placenta (p) for control (panel a) and infected (panel b) fetuses as described in the text using the primers in table 2 . amplification of ␤2-microglobulin mrna was used as an internal control. pcr products were electrophoresed on a 1.2% agarose gel and stained with ethidium bromide. 2 showed only the amplification of the ␤2m, but not tnf-␣ or ifn-␥ mrnas (data not shown). similar results were obtained from virusnegative fetuses located immediately adjacent to infected fetuses (data not shown). in order to confirm that tnf-␣ and ifn-␥ were produced during infection, cytokine protein levels were measured in fetal sera and amniotic fluid from infected fetuses and fetuses from mock-infected dams. as shown in fig. 5 , the concentration of tnf-␣ in serum for prrsv-infected fetuses ranged between 20 and 100 pg/ml compared to less than 30 pg/ml for control fetuses. even though the maximum quantities obtained for infected fetuses were near the lower limit of detection for the elisa test, it was clear that tnf-␣ was elevated during infection. detectable concentrations of tnf-␣ (>23 pg/ml) were found in amniotic fluid from only two control and two infected fetuses. compared to fetuses from mockinfected dams, ifn-␥ concentrations were detected in sera, with values ranging from a low of 60 to more than 500 pg/ml. a maximum level of 60 pg/ml was obtained for a single non-infected fetus. ifn-␥ was not detected in amniotic fluid from either the infected or control fetuses. the presence of ifn-␥ and tnf-␣ in serum, but not amniotic fluid, further supports the notion that the ifn-␥ and tnf-␣ responses are primarily restricted to the prrsv-infected fetus and do not extend to the accessory tissue compartments. this study characterizes the unique biology associated with the interaction between prrsv and the late gestation fetus. consistent with the body of published literature, the fetuses from the infected dams obtained in this study exhibited several anatomic pathological features typical of prrsv infection of the fetus, including lesions associated with the accessory organs, such as umbilical cord and amniotic sac (lager and halbur, 1996; mengeling et al., 1996) . one interesting observation from this study was the apparent absence of a correlation between the presence of gross abnormalities and productive fetal infection. for example, several fetuses from dam no. 2 exhibited several types of gross pathology, including death, growth retardation, or merconium/blood stained amniotic fluid. there were also examples of productively infected fetuses that showed no evidence of gross pathology (see fetuses 1-2, 3-12, 4-9, 4-11, 4-13 in fig. 1 ). the mechanism for fetal pathology remains unclear, but the results suggest that the source pathology is likely result of the infection of tissues on the maternal side and damage to maternal tissues or production of maternal factors that affect the fetus. in this study we did not observe lesions in the myometrium or placenta; however, stockhofe-zurwieden et al. (1995) reported prrs virions budding from maternal vascular endothelial cells at the maternal-fetal interface. lager and halbur (1996) reported damage to the myometrium during prrsv infection. virus-associated lesions in the myometrium are observed for horses infected with eav (coignoul and cheville, 1984) . the appar-ent discrepancy between pathology and infection helps to explain the stealthy nature of the prrsv. a significant number of apparently healthy, but infected fetuses, are likely go on to become healthy growing pigs with the capacity to shed virus. rna viruses often exist as a population of closely related sequences, frequently referred to as a quasispecies. the appearance or disappearance of individual gene sequences in the population over the course of infection is used as evidence for changes in fitness that result from selection. prrsv variants with mutations in orf5 typically appear during the serial passage of virus in culture and during infection of pigs allende et al., 2000) . the significance of mutations in orf5 as a source for increased fitness during infection is not completely understood; but is useful for following the fate of individual prrsv subpopulations over the course of a long-term infection . in this study, one dam, no. 4, showed evidence of a mixed prrsv infection as indicated by the presence of two different orf5 sequences, which were distinguished from each other by a nucleotide substitution at position 77 in orf5. the c to t change was observed in approximately 60% of the cloned plasmid products obtained from dam no. 4 (see table 2 ). however, the same frequency was not transferred to the individual fetuses, which included two fetuses that possessed only viruses with the t-77 mutation and a single fetus with a virus population dominated by c at position 77. these results suggest that fetal infection can alter the selection of prrsv variants and may represent a source of prrsv genetic diversity. to identify the targets of virus replication in the fetus, a variety of tissues were assessed for the presence of virus and virus-infected cells. vi, as opposed to more sensitive approaches, such as pcr, was selected as the means for measuring virus, because the relative insensitivity of vi avoids the possibility of false positive pcr results that might result from small amounts of contaminating maternal material. within the group of selected tissues, virus could be isolated from all tissues. however, the most consistent source and largest quantity of virus were obtained from the thymus. collectively, these data identify the thymus as a primary site of virus replication in the prrsv-infected fetus and confirm an earlier observation for prrsv by benson et al. (2002) . the specific cell population in the thymus targeted prrsv replication remains unknown. the natural predisposition of maternal immunity towards th2like responses aids in protecting the fetus by blocking cell-mediated th1-related allorejection responses (arck et al., 1999; entrican, 2002; raghupathy, 2001 ; recently reviewed in challis et al., 2009) . the negative influence of th1 cytokines on fetal development can be demonstrated experimentally in mice, which show that a single injection of il-2 or ifn-␥ into certain strains induces fetal resorption (lala, 1990; chaouat et al., 1990) . tnf-␣, when infected into mice is abortifacient (clark et al., 2004) . th1 cytokines can also have important long-term impacts. for example, newborn mice that survive maternal infection with influenza virus exhibit behav-ior similar to hyperanxiety and autism. the behavioral changes are a consequence of the altered distribution of dopamine and glutamine receptors during fetal brain development. the effect of influenza virus infection on the fetal brain can be mimicked by the administration of poly i:c, an inducer of ifn (shi et al., 2003) . furthermore, the addition of ifn-␥ to hippocampal neuron cultures alters the distribution of glutamate receptors, sufficient to affect synaptic activity between neurons (vikman et al., 2001) . virus infections during pregnancy present an interesting paradox: those cytokines that protect the fetus from viral infection, tend to inhibit fetal development or potentiate rejection of the fetus; while those cytokines that maintain and promote fetal development are associated with the inhibition of antiviral immune responses. because of the potential negative impact of th1 cytokines on fetal outcome, there is a natural predisposition for the fetus to block the induction of th1-associated cytokines. for example, the ifn-␥ response in the developing fetus can be blocked by a combination of factors, including (1) defects in the capacity of fetal dendritic cells to express mhc class ii and synthesize il-12, (2) hypermethylation of the ifn-␥ gene in fetal t cells, (3) the absence of target macrophages, and (4) the presence of an immune environment dominated by th2/regulatory cytokines (goriely et al., 2001; langrish et al., 2002; melvin et al., 1995; marodi et al., 2001; murphy et al., 2009; prescott et al., 1998; white et al., 2002,) . with this in mind, there are examples of viruses, such as epstein-barr virus (ebv), which are capable of stimulating antigen-specific ifn-␥ responses in human umbilical cord blood lymphocytes (ito et al., 1998; wilson and morgan, 2002) . these and other data provide a description of the fetus as capable of initiating a robust antiviral th1 immune response (chaouat et al., 2002; chipeta et al., 2000; murphy et al., 2009) . in this study ifn-␥ and tnf-␣ mrnas were identified in tissues from infected fetuses. rna message was not identified in tissues of fetuses from mock-infected dams or non-infected fetuses from infected dams. this indicates that altered gene expression is the direct result of fetal infection. up-regulated expression was confirmed by the presence of detectable levels of ifn-␥ and tnf-␣ proteins in serum. furthermore, the results indicate that inf-␣ and ifn-␥ cytokines response are compartmentalized within the fetus and do not extend to other compartments, such as amniotic fluid or placenta, thus lessening the probability of allorejection by the dam. from these data, it is apparent that the fetus is capable of initiating a th1-like response; however, the capacity of ifn-␥ and tnf-␣ to control prrsv infection in the fetus is not known. additional evidence for induction of virus-specific immunity was found in the development of germinal centers containing activated (cdw75+) b cells and seroconversion in at least one fetus. pro-inflammatory cytokines, such as tnf-␣ and ifn-␥ can contribute to pulmonary distress through the activation of alveolar macrophages and other cell populations. the increased quantities of ifn-␥ and tnf-␣ found in the lungs of prrsv-infected fetuses may not be sufficient to cause pulmonary damage to the fetal lung, primarily because fetal macrophages have a reduced capacity to respond to inflammatory stimuli. in addition, il-10, up-regulated in response to prrsv infection, is a potent antagonist of ifn-␥ and tnf-␣ activation of macrophages (burchett et al., 1992; marodi et al., 2001; johnsen et al., 2002; thanawongnuwech et al., 2003) . however, within days after birth, adult macrophages, including mature alveolar and intravascular macrophages emerge into an environment already enriched in ifn-␥ and tnf-␣. the outcome is the rapid recruitment, activation, and cytopathic killing of large numbers of virus-permissive macrophages (choi and chae, 2002) . this scenario as a cause of severe interstitial pnuemonia in the prrsv-infected newborn requires further investigation, but has obvious implications in the 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of ifn-gamma positive cells in porcine reproductive and respiratory syndrome virus-infected lung immunity in the fetus and newborn. in: veterinary immunology: an introduction, chapter 19 quantitative deep sequencing reveals dynamic hiv-1 escape and large population shifts during ccr5 antagonist therapy in vivo interferongamma-induced changes in synaptic activity and ampa receptor clustering in hippocampal cultures differential patterns of methylation of the ifn-gamma promoter at cpg and non-cpg sites underlie differences in ifn-gamma gene expression between human neonatal and adult cd45ro-t cells primary immune responses by cord blood cd4(+) t cells and nk cells inhibit epstein-barr virus b-cell transformation in vitro this work was partially supported by the usda national research initiative for competitive grants program grants # 97-35204-5071. key: cord-252048-ftbjsoup authors: mckinley, enid t.; jackwood, mark w.; hilt, deborah a.; kissinger, jessica c.; robertson, jon s.; lemke, cornelia; paterson, andrew h. title: attenuated live vaccine usage affects accurate measures of virus diversity and mutation rates in avian coronavirus infectious bronchitis virus date: 2011-04-22 journal: virus res doi: 10.1016/j.virusres.2011.04.006 sha: doc_id: 252048 cord_uid: ftbjsoup the full-length genomes of 11 infectious bronchitis virus (ibv) field isolates from three different types of the virus; massachusetts (mass), connecticut (conn) and california (cal) isolated over a 41, 25 and 8 year period respectively, were sequenced and analyzed to determine the mutation rates and level of polymorphisms across the genome. positive selection was not detected and mutation rates ranged from 10(−4) to 10(−6) substitutions/site/year for mass and conn ibv types where attenuated live vaccines are routinely used to control the disease. in contrast, for cal type viruses, for which no vaccine exists, positive selection was detected and mutation rates were 10 fold higher ranging from 10(−2) to 10(−3) substitutions/site/year. lower levels of genetic diversity among the mass and conn viruses as well as sequence similarities with vaccine virus genomes suggest that the origin of the mass and all but one of the conn viruses was likely vaccine virus that had been circulating in the field for an unknown but apparently short period of time. the genetic data also identified a recombinant ibv isolate with 7 breakpoints distributed across the entire genome suggesting that viruses within the same serotype can have a high degree of genetic variability outside of the spike gene. these data are important because inaccurate measures of genetic diversity and mutation rates could lead to underestimates of the ability of ibv to change and potentially emerge to cause disease. avian infectious bronchitis virus (ibv) a gamma-coronavirus in the coronaviridae family causes a highly contagious upperrespiratory tract disease of domestic chickens characterized by coughing, sneezing and tracheal rales. serotype-specific attenuated live vaccines are routinely used to control the disease, yet ibv remains one of the most widely reported respiratory diseases of chickens worldwide resulting in significant economic loss due to reduction in weight gains and feed efficiency, and condemnations at the processing plant. the virus can also cause permanent oviduct damage in layers and some strains of the virus cause nephritis, which when severe can result in significant mortality. infectious bronchitis virus is an enveloped, positive-sense single-stranded rna virus with a genome size of approximately 27 kb (holmes, 1991) . the 5 two-thirds of the genome, approximately 21 kb, encodes two large overlapping open reading frames (orfs), 1a and 1ab, which encode 15 non-structural proteins (nsps), nsps 2-16 (ibv does not have nsp 1) including a papainlike protease 2 (the pl1 protease is truncated and non-functional in ibv), a main protease, the rna-dependent rna-polymerase (rdrp) and several other non-structural proteins involved in making up the replication transcription complex. the remaining one-third of the genome encodes four structural proteins; spike (s), envelope (e), membrane (m) and nucleocapsid (n), and a number of nonstructural proteins. the spike glycoprotein of ibv forms projections on the surface of the virion. spike is post-translationally cleaved into s1 and s2 subunits with the s1 subunit forming the outermost portion and s2 forming a stalk like structure that is embed in the viral membrane. the s1 subunit contains hypervariable regions that play a role in attachment to host cell receptors, membrane fusion and entry into the host cell, and it contains conformationally-dependent virus-neutralizing and serotype-specific epitopes (cavanagh et al., 1998; niesters, 1987 it is extremely difficult to control ibv because little or no cross protection occurs between numerous different types of the virus. coronaviruses evolve by a process of natural selection working on mutations and recombination events (domingo and holland, 1997; moya et al., 2004; vijaykrishna et al., 2007) . these factors contribute to diverse subpopulations of the virus that continually emerge to form new and variant ibv types. in the absence of a specific vaccine, the mutation rate for the hypervariable region in the s1 gene of the 793/b type of ibv was reported as 3 × 10 −3 substitutions/site/year (cavanagh et al., 1998) while the mutation rate for the ga98 virus, which emerged as a result of selection pressure driven by vaccine usage, was reported to be 1.5 × 10 −2 substitutions/site/year in the hypervariable region of s1 (lee and jackwood, 2001) . sequence alignments and phylogenetic reconstructions indicate that recombination events play a significant role in coronavirus evolution. recombination in ibv was demonstrated with coinfection of different ibv types in embryonated eggs (kottier et al., 1995a) . sequence analysis of the s1 gene from field isolates of ibv also revealed evidence of multiple recombination events (jia et al., 1995; wang et al., 1993) and that recombination occurred between vaccine viruses (mondal and cardona, 2007) . in a recent study, recombination in ibv, which replaced the spike gene with an unknown sequence, likely from another coronavirus, resulted in the emergence of turkey coronavirus (tcov) a new enteric disease of young turkeys (jackwood et al., 2010) . characterization of ibv is based on analysis of the spike glycoprotein by either serotyping or genotyping methods (cavanagh and gelb, 2008) . however, pathogenicity in ibv is associated with spike as well as genes outside of spike (ammayappan et al., 2009; cavanagh, 2007; hodgson et al., 2004) . because typing is focused on the analysis of spike, there is no clear understanding of whether viruses with similar spike proteins also share high sequence similarities in other regions of the genome especially in regions associated with pathogenicity. this gap in knowledge results from the lack of complete genome sequences from viruses of the same serotype. the objective of this study was to determine the levels of polymorphism across the entire genome of ibv isolates with similar spike genes and to examine the mutation rates for viruses with and without vaccine selection pressure. we sequenced the full-length genome from 11 ibv field isolates of three different serotypes, massachusetts (mass), connecticut (conn) and california (cal) isolated over a 41, 25 and 8-year period respectively. the genome sequences were aligned and phylogenetic analysis was conducted to examine genetic relationships between the viruses. estimates of the rates of mutation were calculated and evidence of recombination was investigated for each of the full-length genome sequences. to our knowledge, this is the first study to identify polymorphisms and determine mutation rates using full-length genome sequences of ib viruses isolated over a multi-year time span. in this study, we sequenced mass viruses isolated in 1965 mass viruses isolated in , 1972 mass viruses isolated in , 1979 mass viruses isolated in and 1985 obtained from dr. jack king (southeast poultry research laboratory, athens, ga) and one isolated in 2006 obtained from dr. pedro villegas (poultry diagnostic and research laboratory, athens, ga). in addition, conn viruses isolated in 1966 , 1972 , 1983 and 1991 obtained from dr. king, and cal viruses isolated in 1995 and 2003 obtained from dr. peter woolcock (california animal health and food safety laboratory system, university of california, fresno, ca) were sequenced. the specific viruses were all isolated from broiler chickens with clinical signs of disease. the viruses were propagated in embryonated eggs as described (gelb and jackwood, 2008) . the strains and embryo passage number are; ibv/mass/65 (passage 1), ibv/mass/72 (passage 7), ibv/mass/79 (passage 2), ibv/mass/85 (passage 15), ibv/mass/06 (passage 3), ibv/conn/66 (passage 3), ibv/conn/72 (passage 7), ibv/conn/83 (passage 15), ibv/conn/91 (passage 1), cal99/ne15172/95 (passage 3), cal/ca557/03 (passage 5). 2.2. viral rna extraction, amplification and genome sequence determination viral rna extraction, reverse transcription and polymerase chain reaction (rt-pcr), library construction and sequencing were conducted as previously described (jackwood et al., 2010) . briefly, the viruses were filtered through a 0.8 m filter then through a 0.22 m filter (millipore, billerica, ma) and viral rna was purified using the high pure rna isolation kit according to the manufacturer's recommendation (roche diagnostic corporation, foster city, ca). the rt-pcr amplifications were performed with the takara rna la pcr kit (takara bio inc., otsu, shiga, japan) using a random primer and an amplification primer in a strand displacement amplification reaction following the manufacture's protocol. the sequence of the random reverse transcription primer was 5 -agcgggggttgtcgaatgtttgannnnn-3 , and the amplification primer sequence was 5 -agcgggggttgtcgaatgtttga-3 . both primers were obtained from integrated dna technologies, inc. (idt, coralville, ia). a master mix for the rt reaction was prepared, which included mgcl (5 mm), 10x rna pcr buffer (1×), dntp mixture (1 mm), rnase inhibitor (1 units/l), reverse transcriptase (.25 units/l), 5 degenerate primer (2.5 m), and rna (5.75 l/reaction) then 10 l per sample was aliquoted in a thermocycler tube. the rt reaction conditions were 10 min at 30 • c for the primer annealing then 1 h at 50 • c for extension followed by a five-minute incubation at 99 • c for inactivation of the enzyme and a 5-min period at 5 • c. a pcr master mix, which included at the final concentrations: mgcl (2.5 mm), 10x la pcr buffer (1×), sterilized distilled water (32.25 l), takara la taq (1.25 u/50 l), and 5 primer (0.2 m) was prepared and 10 l of the rt reaction was added to 40 l of the mix. the amplification reaction consisted of a 94 • c step for 2 min followed by 30 cycles of 94 • c for 30 s, 60 • c for 30 s, and 72 • c for 3 min. ten pcr reactions were combined for each virus and purified using the qiaquick pcr purification kit (qiagen, foster city, ca) then run on a 1% agarose gel. the pcr products were size selected by removing amplicons between 500 and 1500 bp from the gel, followed by purification using the qiaquick (qiagen) gel purification kit. the pcr products were cloned into the pcr-xl-topo vector (invitrogen, life technologies, carlsbad, ca) according to the manufacturers recommendations and transformed into one shot topo electrocompetent e. coli cells (invitrogen) using 30 l of competent cells mixed with 2 l of the ligation reaction. electroporation settings were 20 kv and 200 using a biorad gene pulser (bio-rad, hercules, ca), and the electroporated cells were incubated at 37 • c in 480 l of super optimal broth medium for 1 h on a rotary shaker. the cultures were mixed with 70% glycerol and frozen at −80 • c until plated on q-trays (genetix, boston, ma) containing liquid broth agar cat#3002-032 (mp biomedicals, llc, solon, oh) with 50 g/ml of kanamycin. the q-trays were pre-warmed at 37 • c before the entire culture (approximately 500 l) was spread on the plates and incubated overnight at 37 • c, then robotically picked with a q-bot (genetix, boston, ma). plasmid dna from the libraries of cloned cdna fragments for each virus were isolated using an alkaline lysis method modified for the 96-well format, and incorporating both hydra and tomtek robots (http://www.intl-pag.org/11/abstracts/p2c p116 xi.html). cycle sequencing reactions were performed using the bigdye tm terminator ® cycle sequencing kit version 3.1 (applied biosystems, foster city, ca) and mj research (watertown, ma) thermocyclers. finished reactions were filtered through sephadex filter plates into perkin-elmer microamp optical 96-well plates. a 1/12strength sequencing reaction on an abi 3730 was used to sequence each clone from both the 5 and 3 ends. each viral genome was sequenced to approximately 10× coverage. the accuracy of the sequence is insured by generating sequence reads for both strands. gaps and areas with <2× coverage were identified and specific primers were synthesized (idt) for rt-pcr amplification and sequencing of the ambiguous areas. the rt-pcr was conducted as described above, and the reaction conditions were 42 • c for 60 min, 95 • c for 5 min, then ten 10 cycles of 94 • c for 30 s, 50 • c for 30 s, 68 • c for 90 s, followed by 25 cycles of 94 • c for 30 s, 50 • c for 30 s, 68 • c for 90 s + 5 s/cycle added. the final elongation step was 68 • c for 7 min then the reaction was cooled to 4 • c. the pcr products were directly sequenced in both directions using the amplification primers at a concentration of 15 ng/reaction and the abi prism bigdye terminator v3.0 (applied biosystems, foster city, ca). the amount of cdna added to the reaction ranged from 20 to 30 ng and the sequencing reactions were analyzed on an abi 3730 (applied biosystems). the extreme 5 end of each genome was obtained using the 5 race system for rapid amplification of cdna ends, according to manufactures protocol (invitrogen, life technologies, carlsbad, ca). the primer design was based upon an alignment of ibv sequences. the race primers are designated; ibv 5 race sp1 (5 -cgtatagaaaaacaaagcgtcac-3 ), ibv 5 race sp2 (5 -gtcactgtctattgtatgtctgctc-3 ) and ibv 5 race sp3 (5 -tagccgaccttatgcgagaacg-3 ). the extreme 3 end of each genome was obtained using primers designated; m41 3 end reverse (5 -gctctaactctatactagccta-3 ) and genome 3 f 900 bp (5 -tgacaagatgaatgaggaaggtat-3 ). sequences for some viruses were verified and sequence gaps were filled by amplification using overlapping primer pairs. briefly, the primer design tool located at http://www2.eur.nl/fgg/kgen/primer/overlapping primers.html was used with a template genome sequence of a virus with the same serotype as the virus to be sequenced. thirty-two overlapping primer pairs (available upon request) were synthesized (idt) for each virus and used to amplify the genome. the amplicons were sequenced directly using the amplification primers and the abi prism bigdye terminator v3.0 (applied biosystems) as described above. the sequence reads for each virus were assembled using the seqman program (dnastar, inc. madison, wi) and the orf finder at the national center for biotechnology information (ncbi, http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to predict open reading frames. genome annotation was entered using the annotation editor seqbuilder (dnastar, inc.). the blastn program (http://www.ncbi.nlm.nih.gov/blast/) was used to search genbank (national center for biotechnology information, http://www.ncbi.nlm.nih.gov/) for similar ibv sequences. the megalign program implemented in dnastar was used to align sequences and to calculate percent identity and number of substitutions between selected sequences. whole genome alignments were generated using clustalw and phylogenetic trees were constructed with the neighbor-joining method, minimum evolution method, maximum parsimony method, and upgma with 1000 bootstrap replicates (mega4, http://www.megasoftware.net/index.html (tamura et al., 2007) ). to reconstruct evolutionary trees, maximum likelihood (tree-puzzle, http://www.tree-puzzle.de/) and bayesian analysis (beast 1.4, http://beast.bio.ed.ac.uk/) were conducted. a codon-based test of positive selection (z-test, mega4) was used to analyze the numbers of non-synonymous and synonymous substitutions per site (dn/ds ratio) in the spike glycoprotein. because only 2 cal type viruses were sequenced herein, additional reference sequences for the cal type viruses were included in the codon-based test (z-test) of positive selection; cav/cav56b/91 (af027509), cav/cav9437/95 (af027510), cal99/cal99/99 (ay514485) and ca/ca1737/04 (eu925393). to determine mutation rates, the full length genomes as well as the coding sequences for orf1ab, s1, e, m and n gene from each virus within the same virus type were aligned using clustalx 2.0.8 (larkin et al., 2007) and manually edited in jalview (waterhouse et al., 2009) . the bayesian markov chain monte carlo (mcmc) implemented in beast v1.4.8 (drummond and rambaut, 2007) was used to estimate mutation rates of the aligned sequences and the sampling dates for each isolate were used as calibration points. the mutation rates were analyzed using the general time-reversible (gtr) (rodriguez et al., 1990 ) substitution model with gamma-distributed among site rate variation with four gamma rate categories (yang, 1994 ). the srd06 model was used to partition codon positions and an uncorrelated lognormal relaxed clock model was selected. each mcmc chain was run for 10 million states and sampled at every 1000 states. tracer v1.4.1 (http://tree.bio.ed.ac.uk/software/tracer/) was used to confirm convergence of mcmc chains with 10% of each chain discarded as burn-in. complete genome nucleotide sequences for cal99/ne15172/95, ibv/ck/ca99/99, ca/ca557/03, ibv/conn/66, ibv/conn/72, ibv/conn/83, ibv/conn/91, ibv/mass/65, ibv/mass/72, ibv/mass/79, ibv/mass/85, ibv/mass/06 and the ibv/ck/a2 china strain (used as out-group) were aligned using clustalx 2.0.8 (larkin et al., 2007) . to identify recombinants as well as major and minor parents, the data set was scanned using a recombination detection program (rdp) v2 with implemented algorithms geneconv, bootscan, maxchi, chimera and siscan. similarity plot and bootscan analyses were performed using the simplot program (lole et al., 1999) to further identify recombination events and recombination breakpoints. the window width and step size were set to 1000 bp and 40 bp, respectively. genome sequences generated in this study were submitted to the genbank database and assigned the follow the full-length genome sequence of 11 ibv isolates from the field obtained over a 41 year period (5 mass table 1 ). genomic libraries and rt-pcr fragments from overlapping specific primer pairs were used to assemble genomic sequences with seqman pro and genome annotation was performed using seqbuilder (dnastar, inc., v.8.0.2, madison, wi), to reveal a typical gamma coronavirus gene order for all of the viruses; 5 utr, orf1a/ab, spike glycoprotein, orf3a, orf3b, envelope protein, membrane protein, orf 4b, orf 5a, orf 5b, nucleocapsid protein, orf 6b, 3 utr and a poly(a) tail. the full-length genome sequences were aligned using clustalw and all the phylogenetic trees constructed with the neighbor-joining method, minimum evolution method, maximum parsimony method, and upgma using mega 4.0.2 had similar topography (tamura et al., 2007) . a representative tree with the 11 viral genomes sequenced herein and other ibv full-length genomes available in genbank is presented in fig. 1 . the nucleotide sequence similarities between the genomes of the viruses sequenced herein are from 93.8% to 94.9% for the cal isolates, 99.8% to 99.9% between the conn isolates and 92.4 to 100% between the mass isolates. to determine the relationship between different virus types for sequences in specific areas across the genome, phylogenetic analysis of the nucleotide sequences of the 5 and 3 utrs and the amino acid sequences of individual orfs was conducted (fig. 2) . the spike glycoprotein, which is used to define ibv isolate type, shows individual groupings for conn, mass and cal viruses with the exception of the ibv/mass/06 virus, which is positioned as an offshoot of the conn group. similar groupings were observed for the envelope and nsp 3 (pl2) protein trees, whereas ibv/mass/06 clearly grouped with conn type viruses in the membrane, nucleocapsid, nsp 5 (mpro) and nsp 11/12 (rdrp) protein trees (fig. 2) . a blastp search showed the ibv/mass/06 virus spike glycoprotein to have 98% similarity to h120 (acc. no. acq55230), which is a commonly used mass type vaccine. a blastp search and phylogenetic analysis of the other mass spike glycoprotiens identified mass type attenuated viruses (acc. nos. eu283075, eu283081, eu283082), which grouped together (data not shown). a blastp search and phylogenetic analysis of the conn spike glycoproteins identified conn attenuated viruses (acc. nos. eu283059, eu283061 and eu283062), which also grouped together (data not shown). the phylogenetic analysis of the 5 utr showed individual groupings for conn, mass, and cal viruses, however the ibv/mass/06 virus grouped with the cal viruses (fig. 2) . in the 3 utr, the cal/ca557/03 is an offshoot of the mass group, the ibv/mass/06 virus is an offshoot of the conn group, and cal99/ne15172/95 was clearly distinct from the other viruses. a blast analysis of the cal99/ne15172/95 virus 3 utr showed that it was 99% identical to the de072 strain of ibv (acc. no. af203002), a delaware type virus. although the 3 utrs sequence divergence was as high as 13.5% among the viruses, sequence alignment of the stem-loop (s2m) region in the 3 utr revealed identical sequences among cal99/ne15172/95, ibv/conn/66, ibv/conn/72, ibv/conn/83, and ibv/conn/91, one nucleotide difference (nt. position 9: a to g) in the cal/ca557/03 and ibv/mass/79 viruses, and two nucleotide differences (nt. positions 9: a to g and 26: a to c) in the ibv/mass/65, ibv/mass/72 and ibv/mass/85 viruses while ibv/mass/06 had an major a and a minor g at nt. position 36 (data not shown). the optimal tree with the sum of branch lengths is shown. the tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances. a bootstrap consensus tree was constructed from 1000 replicates (percentage of replicate trees in which associated strains clustered together are presented at nodes). the p-distance scale is presented at the bottom of the figure. the analyses were constructed using mega4 (tamura et al., 2007) . to further examine the relationship of the ibv/mass/06 virus with the other viruses examined in this study as well as selected ibv genomes available in genbank we conducted a recombination detection program (rdp) analysis, which indicated that the ibv/mass/06 isolate was a recombinant with conn and cal viruses as the potential major parents (data not shown). the recombination events in ibv/mass/06 were identified using simplot, which showed that the ibv/mass/06 genome shared a high sequence similarity, 97-100%, with cal and conn isolates in different regions of orf1ab, with mass in the s1 gene and with conn isolates in the remainder of the 3 end of the genome (fig. 3a) . at the extreme 3 end of orf1ab, between nucleotides 15,500 and 19,800, the ibv/mass/06 virus did not show any significant sequence similarity with any of the genomes in this study. a blast analysis of that region showed 99% similarity with ibv strain h120 (acc. no. fj807652). the simplot bootscan analysis revealed a total of seven breakpoints located at approximate nucleotide positions 1530, 5895, 8188, 11,200, 12,266, 19,918 and 21,975 in the ibv/mass/06 virus (fig. 3b) . the sequences located between the breakpoints were designated regions i-viii, respectively. regions one through five were located in nsp2, nsp3, nsp4, nsp9 and nsp11/12 respectively. region vii contained the entire s1 glycoprotein gene, whereas the s2 glycoprotein gene was in region viii. evidence of recombination in the ibv/mass/06 isolate was further confirmed when phylogenetic trees constructed from the regions delineated by the breakpoints showed incongruence (fig. 4) . the ibv/mass/06 isolate grouped with mass type viruses in region vii, which contained the s1 gene. however, ibv/mass/06 grouped with cal type viruses in regions i, iii and v, and with conn type viruses in regions ii, iv and viii. in region vi, ibv/mass/06 did not group with any of the genomes included in this study. as indicated above, a blast search of the ibv/mass/06 virus sequence in that region showed that it was most closely related to the h120 mass type vaccine virus. fig. 2 . infectious bronchitis virus amino acid sequences a through j as indicated in the figure, were aligned with clustalw and the phylogenetic trees were constructed using the neighbor-joining method. the optimal trees with the sum of branch lengths are shown. the trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances (the p-distance scale is presented at the bottom of each tree). bootstrap consensus trees were constructed from 1000 replicates and the percentage of replicate trees in which associated strains clustered together are presented at the nodes. the analyses were conducted using mega4 (tamura et al., 2007) . a codon-based test of positive selection (z-test, mega4), which measures the numbers of non-synonymous and synonymous substitutions per site (dn/ds ratio) showed that positive selection was occurring in the cal viruses and between ibv/conn/66 and other conn viruses but not in mass type viruses (table 1 ). the beast v1.4.1 software, used to estimate the mutation rates for individual coding regions within each virus type, indicated that the cal isolates had mutation rates that were 2-4 orders of magnitude higher than the mass and conn isolates (data not shown). data for the fulllength genomes and the s1 glycoprotein are presented in table 2 . the mutation rate for the orf1ab, s1 and n gene was calculated to be 10 −3 substitutions/site/year and 10 −2 for the e and m genes from the cal viruses, whereas the conn and mass viruses had mutation rates that were 10 fold lower and ranged from 10 −4 to 10 −6 in all of the coding regions except for the e gene in the conn viruses, which did not show any polymorphisms. the majority of emerging infectious diseases are caused by rna viruses. presumably, high rates of mutation and rapid replication drive their evolution. infectious bronchitis virus, like most rna viruses, undergoes a high rate of genetic change that contributes to diverse subpopulations of the virus that continually emerge to form new variants of existing strains as well as completely new types of the virus. new variants and types of ibv are defined by the sequence of the spike glycoprotein and/or by specific antibodies against spike, and for many ibv types, only spike sequences are available. to examine sequence changes not only in spike but also over the entire length of the genome, we sequenced the full-length genomes of 5 mass type, 4 conn type and 2 cal type ibv field viruses that were isolated over a 41, 25 and 8 year period respectively. all of the viruses were obtained from chickens with clinical signs of the disease. alignments of the full-length genome sequences showed that the mass viruses had ≥92.4% identity (≥93.7% if the recombined ibv/mass/06 is not included), the cal viruses had ≥93.8% identity and the conn viruses varied the least, at ≥99.8% identity. examination of the mutation rates for the orf1ab, s1, e, m and n genes showed, in general, that the cal viruses had rates between 10 −4 and 10 −2 substitutions/site/year, whereas the conn and mass viruses had lower mutation rates that ranged from 10 −4 to 10 −6 substitutions/site/year. in addition, the e gene in the conn viruses showed no signs of change. although the egg passage level of all the viruses used in this study was relatively low (≤15 passages) it is possible some genetic changes could have been introduced as a result of propagation in embryonated eggs. however phenotypic change is likely to be minimal since it typically takes 75 or more egg passages to attenuate ibv and in a study that examined genetic changes in the highly variable s1 gene following 10 passages in embryonated eggs, five viruses had no changes, two viruses had only 2 nucleotide changes one virus had only 1 change (mckinley et al., 2008) . it has been shown that vaccine usage can result in faster evolutionary rates in ibv field viruses (lee and jackwood, 2001; wang fig. 4 . the nucleotide sequences a through h as indicated in the figure, were aligned with clustalw and the phylogenetic trees were constructed using the neighbor-joining method (saitou and nei, 1987) . the optimal trees with the sum of branch lengths are shown. the trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances (the p-distance scale is presented at the bottom of each tree). bootstrap consensus trees were constructed from 1000 replicates and the percentage of replicate trees in which associated strains clustered together are presented at the nodes. the analyses were conducted using mega4 (tamura et al., 2007) . mutation and evolutionary rates of the genome and s1 genes for california (cal), connecticut (conn) and massachusetts (mass) isolates. mutation rate (nucleotide changes/year) evolutionary rate (amino acid changes/year) genome a s1 gene b genome a s1 gene b cal 1.0 × 10 −2 9.4 × 10 −4 2.0 × 10 −2 2.4 × 10 −3 conn 1.5 × 10 −4 1.7 × 10 −4 2.6 × 10 −4 3.5 × 10 −4 mass 1.9 × 10 −3 2.4 × 10 −4 3.3 × 10 −3 5.8 × 10 −4 a viruses used in the genome analysis were the cal, conn and mass viruses shown in fig. 1 . b viruses used in the s1 gene analysis are the same viruses shown in table 1 . et al., 1993) . thus, it was surprising that mutation rates for mass and conn type viruses were lower than mutation rates for cal type viruses because live attenuated vaccines have been used against the mass and conn type viruses in poultry for many years, whereas no vaccine exists for the cal type viruses. one possible explanation for the differences in mutation rates is that the mass and conn viruses were possibly re-isolated vaccine viruses that have been circulating in the field for an unknown but relatively short period of time, with the exception of the ibv/conn/66 virus, which did show positive selection when compared to the other conn viruses. in contrast, because no vaccine exists for cal viruses, those viruses represent true field isolates that have been circulating for known periods of time. the possibility that the origin of the mass and conn isolated viruses is vaccine, is supported by lower levels of genetic diversity observed for many of the genes within each virus type (fig. 2) as well as the genetic similarity of the viruses, previously characterized as attenuated viruses. it has been shown that ibv can be shed for long periods of time and that vaccine viruses can persist in the field and revert to cause disease (farsang et al., 2002; jackwood et al., 2009; matthijs et al., 2008; naqi et al., 2003) . thus, it is possible that vaccine viruses are more likely to be isolated from the field because they may be more abundant having displaced field viruses of the same type or because they are more easily isolated since they are adapted to grow in embryonated eggs. because the mass and conn type viruses were isolated from clinically diseased chickens suggests that if they were originally vaccine viruses then they have likely acquired the sequence changes necessary for pathogenicity. further studies would need to be conducted on vaccine viruses and pathogenic strains to gain a better understanding of the molecular changes necessary for coronavirus reversion to pathogenicity. another explanation for lower mutation rates in mass and conn viruses could be that some of the mutations observed are incidental, meaning that they are not deleterious and provide no selective advantage to the virus rather than due to selection pressure. this possibility was suggested in another study where no evolution was detected in the 5 hypervariable region of the s1 gene for a number of 793/b type viruses isolated over a 11 year period (cavanagh et al., 1998) . this however does not explain why polymorphisms were observed for the cal type viruses. recombination contributes to the genetic diversity of coronaviruses and can lead to the emergence of new viruses and outbreaks of new diseases (holmes, 2009; jia et al., 1995; woo et al., 2009) . although the ibv/mass/06 virus is identified as a mass type virus based on characterization of the spike glycoprotein, it was shown herein to be a recombinant virus with conn and cal viruses as major parents. eight regions defined by 7 breakpoints distributed across the entire genome were identified. region vii was similar to other mass type viruses, which was expected because that region includes the entire s1 gene. however, cal like sequences were found in regions i, iii and v, conn sequences were found in regions ii, iv and viii, and region vi was found be similar to the h120 mass type vaccine strain. high frequencies of recombination including recombination events between vaccine and field strains have been reported for ibv (brooks et al., 2004; cavanagh et al., 1992; fang et al., 2005; jackwood et al., 2010; kottier et al., 1995b; lee and jackwood, 2000; yu et al., 2001) . in addition, ibv pathogenicity was shown to be polygenic, involving spike as well as replicase proteins (ammayappan et al., 2009; armesto et al., 2009; cavanagh, 2007; hodgson et al., 2004) . although ibv type is defined by the spike glycoprotein, our data shows that viruses within the same type can have vastly divergent sequences in other regions of their genome including regions that contain genes important for pathogenicity. if recombination occurs outside of the spike gene where a pathogenic field virus acquires attenuated vaccine virus sequences, the outcome would likely be a relatively benign virus regardless of the virus types (spike sequences) involved. furthermore, if a vaccine virus circulating in a flock recombines with a pathogenic virus outside of the spike gene, resulting in the vaccine virus becoming pathogenic, the outcome is still likely to be of little or no consequence since the birds would presumably be immune to the unchanging spike. however, in the event that a pathogenic field virus recombines with either a vaccine virus or another pathogenic virus and acquires a new or unique spike glycoprotein gene, the outcome could be the emergence of a new virus capable of causing disease. this was recently shown for tcov (jackwood et al., 2010) . although new coronaviruses can emerge and cause disease as a result of recombination it appears that the emergence of most variant or new ibv types is due to accumulation of mutations in the spike glycoprotein over time (cavanagh et al., 2005; jackwood et al., 2005 jackwood et al., , 2007 lee and jackwood, 2001; nix et al., 2000) . thus, until pathogenicity genes can be specifically identified, monitoring the spike gene in ibv appears to be the most reliable measure of genetic change leading to the emergence of new viruses capable of causing disease. this is the first study to identify genetic changes and mutation rates using full-length genome sequences of ibvs isolated over many years. our data show that mutation rates are lower and that positive selection was not occurring for ibv types where attenuated live vaccines are used compared to an ibv type were no vaccine exists, suggesting that the virus isolates examined were re-isolated vaccine viruses that have been circulating in the field for a relatively short-period of time. this observation is extremely important because it could lead to incorrect estimates of mutation rates for rna viruses where modified live vaccines are used to control the disease. in this study, we also identified a recombinant ibv isolate with two major parents and no fewer than 7 breakpoints distributed across the entire genome. although recombination among coronaviruses can contribute to genetic diversity of the virus and recombination has been shown to lead to the emergence of new viruses, it appears that monitoring mutations in the spike gene that accumulate over time is currently the best method of identifying potentially new genetic variants and ibv types capable of causing disease. identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strain arkansas dpi the replicase gene of avian coronavirus infectious bronchitis 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chickens the general stochastic model of nucleotide substitution the neighbor-joining method: a new method for reconstructing phylogenetic trees mega4: molecular evolutionary genetics analysis (mega) software version 4.0 evolutionary insights into the ecology of coronaviruses evidence of natural recombination within the s1 gene of infectious bronchitis virus jalview version 2 -a multiple sequence alignment editor and analysis workbench coronavirus diversity, phylogeny and interspecies jumping maximum likelihood phylogenetic estimation from dna sequences with variable rates over sites: approximate methods molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia abbreviations: ibv, infectious bronchitis virus; nsps, nonstructural proteins; orf, open reading frame; pbs, phosphate buffered saline; rdrp, rna-dependent rna-polymerase; rt-pcr, reverse transcriptase-polymerase chain reaction; utr, untranslated region. this research was supported by usda, csrees award number 2007-35600-17786. supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2011.04.006. key: cord-272408-8flsy5o1 authors: chen, rui; fu, jiayu; hu, jingfei; li, cheng; zhao, yujia; qu, huan; wen, xintian; cao, sanjie; wen, yiping; wu, rui; zhao, qin; yan, qigui; huang, yong; ma, xiaoping; han, xinfeng; huang, xiaobo title: identification of the immunodominant neutralizing regions in the spike glycoprotein of porcine deltacoronavirus date: 2020-01-15 journal: virus res doi: 10.1016/j.virusres.2019.197834 sha: doc_id: 272408 cord_uid: 8flsy5o1 porcine deltacoronavirus (pdcov), is an emerging enteropathogenic coronavirus in pigs, that poses a novel threat to swine husbandry worldwide. crucial to halting pdcov transmission and infection is the development of effective therapies and vaccines. the spike (s) protein of coronavirus is the major target of host neutralizing antibodies, however the immunodominant neutralizing region in the s protein of pdcov has not been defined. here, three truncations of the pdcov s protein were generated, the n-terminal domain of the s1 subunit (ntd, amino acids (aa) 50–286), the c-terminal domain of the s1 subunit (ctd, aa 278–616), and s2 subunit (aa 601–1087). the proteins were expressed using an e. coli expression system. polyclonal antisera against the three recombinant proteins were produced in rabbits and mice. all three antisera were able to inhibit pdcov infection in vitro, as determined by virus neutralization assay, fluorescent focus neutralization assay, and plaque-reduction neutralization. the ctd-specific antisera had the most potent pdcov-neutralizing effect, indicating that the ctd region may contain the major neutralizing epitope(s) in the pdcov s protein. based on these findings, ctd may be a promising target for development of an effective vaccine against pdcov infection in pigs. porcine deltacoronavirus (pdcov) was first reported in 2012 in hong kong during an investigation of novel coronaviruses (covs) (woo et al., 2012) . thereafter, pdcov was detected in the united states and isolated from pigs suffering from severe diarrhea in 2014 by hu et al. in ohio, usa (hu et al., 2015; li et al., 2014; wang et al., 2014b) . pathogenesis and virulence were subsequently investigated using gnotobiotic and conventionally raised pigs. pdcov infection is characterized by watery diarrhea and vomiting 1-3 days after infection (hu et al., 2016; ma et al., 2015) ; it has since been detected in swine populations throughout the world, resulting in substantial economic losses (lee and lee, 2014; saeng-chuto et al., 2017; song et al., 2015; suzuki et al., 2018; wang et al., 2014a) . pdcov is an enveloped, positive-sense, single-stranded rna virus belonging to the order nidovirales, family coronaviridae, subfamily coronavirinae, and genus deltacoronavirus. the pdcov genome (∼25.4 kb) consists of eight open reading frames (orfs) and contains six common coronaviral genes in the conserved order: 5' untranslated region (utr)-orf1a-orf1b-s-e-m-n-3'utr (woo et al., 2012) . the 5'orf1a/b comprises two-thirds of the genome and encodes two overlapping viral replicase polyproteins (1a and 1ab), the six following orfs encode four structural proteins and two strain-specific accessory proteins in the order: spike (s), envelope (e), membrane (m), nonstructural protein 6 (ns6), nucleocapsid (n), nonstructural protein 7 (ns7) (lee and lee, 2015) . among cov structural proteins, the s glycoprotein is abundantly produced in infected cells and has multiple functions in viral entry and pathogenesis (li et al., 2017a; zhang, 2016) . the s1 subunit mediates virus binding to cells through its receptor-binding domain (rbd), while the s2 subunit mediates virus-cell membrane fusion. in addition, the s protein is postulated to harbor epitopes that induce neutralizing antibodies zumla et al., 2016) . hain et al. (hain et al., 2016 ) generated a recombinant orf virus (orfv) that expresses the fulllength pedv s protein. an immunization challenge study in pigs showed that intramuscular inoculation with orfv-pedv-s elicited sspecific igg, iga, and a neutralizing antibody response. inoculation with pdcov s protein may therefore be able to inhibit pdcov infection and induce neutralizing antibodiess against pdcov infection. several studies have shown that potent neutralizing antibodies against alpha-or beta-covs target the rbd region of the s protein (du et al., 2013a, b; he et al., 2004a; li et al., 2017a; yoo and deregt, 2001) . the rbd regions in several cov genera have been identified. for example, the c-terminus of the s1 domain is the rbd region of transmissible gastroenteritis virus (tgev) in the genus alphacoronavirus (godet et al., 1994) and in severe acute respiratory syndrome coronavirus (sars-cov) in the genus betacoronavirus (li et al., 2005) . in contrast, the rbd regions of murine hepatitis virus and bovine coronavirus, both in the genus betacoronavirus, are located in the n-terminus of the s1 domain (peng et al., 2011 (peng et al., , 2012 . however, the immunodominant neutralizing region associated with delta-covs, such as pdcov, has not been identified. recent elucidation of pdcov spike protein structures by cryo-electron microscopy reveal that the s1 subunit consists of four individually folded domains, designated a, b, c, and d (xiong et al., 2018) . recently, li et al. demonstrated that the porcine aminopeptidase n, previously known to be a functional receptor for transmissible gastroenteritis virus (tgev), also interacts with the b domain of pdcov s1 subunit and functions as a major cell entry receptor for pdcov. therefore, in this study, we aimed to identify the immunodominant region of pdcov s protein, and its neutralizing epitopes. based on previous structural data and the location of the rbd region in the s protein of pdcov , three truncated s proteins spanning the entire s domain were produced using an e.coli expression system. the constructs were designated ntd (n-terminal domain of the s1 subunit, amino acids (aa) 50-286), ctd (c-terminal domain of the s1 subunit (aa 278-616), and the s2 subunit (aa 601-1087). we purified the recombinant proteins and inoculated rabbits and mice to produce ntd-, ctd-, and s2-specific polyclonal antisera. sera from ntd-, ctd-, and s2-inoculated mice had pdcov neutralization activity after the second boost. all antisera, from mice and rabbits, exhibited anti-pdcov activity in vitro, determined by virus neutralization, fluorescent focus neutralization, and plaque-reduction neutralization assays. among the three antisera, the ctd-specific sera showed the most potent pdcov inhibitory effect, indicating that the ctd region may contain the major neutralizing epitope(s) of the pdcov s protein. all the animal experiments were performed in strict accordance with the guidelines and regulations of the care and use of laboratory animals of the ministry of science and technology of the people's republic of china. all experiment in this study were approved by the institutional animal care and use committee of sichuan agricultual university(iacuc#rw2016-090). swine testis (st) cells (atcc crl-1746) were cultured in dulbecco's modified eagle medium (dmem, gibco, usa) with 10 % fetal bovine serum (pan-biotech, germany) at 37 ℃ in a humidified 5 % co 2 atmosphere. the pdcov strain chn-sc2015 was isolated from a diarrheal piglet in sichuan province (genbank accession no.mk355396). the virus was propagated in st cells and dmem supplemented with 5 μg/ml of trypsin. the titers of chn-sc2015 strain during several passages were up to 10 6 · 64 tcid 50 /ml. horseradish peroxidase (hrp) conjugated goat anti-rabbit igg and fluorescein isothiocyanate (fitc) labeled goat anti-mouse igg were purchased from beijing biosynthesis biotechnology co., ltd. (bioss, beijing, china). hrp conjugated goat anti-pig igg was purchased from abclonal technology co., ltd (abclonal, wuhan, china). an anti-pdcov n-protein monoclonal antibody (mab) and pig anti-pdcov chn-sc2015 polyclonal serum were prepared and stored in our laboratory the s gene of pdcov consists of three overlapping regions, the nand c-terminal domains of the s1 subunit (ntd, aa 50-286 and ctd, aa 278-616), and the s2 domain (aa 601-1087) (fig. 1a ). viral rna was extracted from infected cell supernatants using trizol reagent (sangon biotech, china). the rna was reverse transcribed using a transcription first strand cdna synthesis kit (takara) according to the manufacturer's instructions. gene-specific primers were designed using primer 5.0 software based on the published chn-sc2015 sequence ( table 1) . the three s gene segments were amplified by rt-pcr then each was locations and lengths of the three antigenic s fragments. the s gene was divided into overlapping sections, the n-terminal domain of subunit s1 (ntd, aa 50-286), the c-terminal domain of s1 (ctd, aa 278-616), and s2 (aa 601-1087). the segments were amplified by rt-pcr and then cloned into a pet32a (+) expression vector. transetta (de3) cells harboring pet32a-ntd, pet32a-ctd, or pet32a-s2 were induced by iptg, and cells were collected 4 h after induction. the proteins were purified and diluted to the same concentration (750 μg/ ml). (b) sds-page was performed to analyze protein expression. (c) expression products were specifically recognized on a western blot using polyclonal pig antisera against pdcov. (d) band intensities on the western blot were analyzed using imagej. experiments were performed three times. in each experiment, band intensity values were compared and the intensity of ntd was defined as 100 %. r. chen, et al. virus research 276 (2020) 197834 cloned into a pet32a (+) expression vector, the resulting plasmids are henceforth referred to as pet32a-ntd, pet32a-ctd, and pet32a-s2. the recombinant plasmids were separately transformed into transetta (de3) cells, protein expression was induced with 0.8 mm iptg for 3-6 h. protein expression was analyzed by sds-page. the recombinant proteins were purified as previous described (luo et al., 2017) . the concentration of the purified proteins was determined using an enhanced bca protein assay kit (beyotime, china). equal amounts of purified proteins were separated on a 10 % sds-page gel, then transferred to a polyvinylidene fluoride membrane (pvdf, bio-rad). the membrane was blocked with 2 % bovine albumin v (bsa, solarbio, china) in pbst (pbs with 0.1 % polysorbate-20), after 2 h the membrane was incubated with pig anti-pdcov serum (dilution) for 8 h at 4°c. the membrane was then washed four times with pbst, and incubated with hrp conjugated goat anti-pig igg (1:5000 in pbst) for 1 h at room temperature. the membrane was washed again four times and the proteins were visualized using enhanced chemiluminescence reagents (ecl; bio-rad, usa). rabbits were inoculated with the purified recombinant proteins (ntd, ctd, or s2) for production of polyclonal antibodies, as previously described with some modifications (luo et al., 2017) . briefly, rabbits were inoculated with 1 mg/rabbit of recombinant protein in montanide™ gel 01 pr adjuvant (seppic, france), then boosted three times with the same immunogen and adjuvant at 2-week intervals. sera was taken from pre-immunized rabbits and then 10 days after each inoculation, sera were heat-inactivated for 1 h at 56°c and stored at −20°c. female balb/c mice (6-8-weeks-old) were purchased from chengdu dossy experimental animal co., ltd (chengdu, china). mice were divided into 4 groups of 6 mice/group. three of the groups were subcutaneously injected with 50 μg/mouse of purified protein (ntd, ctd, or s2). the last group was the negative control; each mouse was injected with the same volume of sterile pbs. all animals were boosted twice at two-week intervals. serum samples were collected each week until 6 weeks after the first immunization. all sera were heat-inactivated for 1 h at 56°c and stored at −20°c. mouse and rabbit serum titers were assessed by elisa as previously described with some modifications (quan et al., 2018) . briefly, 96-well elisa plates were pre-coated with 1 ug/well of purified pdcov overnight at 4°c. wells were blocked with 5 % non-fat milk for 2 h at 37°c, then 100 μl of serially diluted sera was aliquoted into each well and plates were incubated for 1 h at 37°c. wells were washed four times then incubated with hrp-conjugated anti-mouse igg or hrp-conjugated anti-rabbit igg (1:5000 in pbst) for 1 h at 37°c. bound antibody was visualized by addition of tmb substrate (3,39,5,59-tetramethylbenzidine) (invitrogen) for 15 min, the reactions were stopped with 1 n h 2 so4. absorbance at 450 nm was read using microplate absorbance reader (bio-rad, usa). a standard micro-neutralization [21] assay was used to quantify the neutralizing activity of the polyclonal serum from each rabbit and mouse group. briefly, serial dilutions of sera, rabbit or mouse anti-ntd, ctd or s2, were mixed with pdcov (200 tcid 50 in 50 μl) in dmem. the mixtures were incubated for 1 h at 37°c to allow for formation of virus-antibody complexes. st cells grown in 96-well plates were washed 2 times with dmem supplemented with 5 μg/ml of trypsin, then the virus-antibody mixtures were added to each well. control sera were treated the same way. after 1 h incubation at 37°c, unabsorbed virus was removed and cells were overlaid with dmem supplemented with 2.5 μg/ml of trypsin. after 72 h at 37°c, the cells incubated with virus and preimmune sera exhibited obvious cpe, the cells incubated with no virus remained healthy. neutralizing antibody titers were determined by the method of reed and muench (reed and muench, 1938; tumpey et al., 2005) . results are expressed as the average of triplicates ± standard deviation. the binding of mouse anti -ntd, -ctd, and -s2 antibodies to pdcov-infected st cells was measured by flow cytometry. briefly, 2 × 10 6 st cells were infected with pdcov (moi 0.1) for 24 h, washed twice with pbs and fixed with 4 % formaldehyde in pbs, followed by membrane permeabilization with tritonx-100 for 10 min at 4°c. the cells were again washed twice with pbs, then incubated with 1 ml of a 1:50 dilution of anti -ntd, -ctd, or -s2 mouse sera for 1 h at 4°c. cells were washed then incubated with fitc-goat anti-mouse igg for 40 min at 4°c, cells were washed again then analyzed by flow cytometry. naive mouse antiserum was used as negative control. the flow cytometry data were analyzed using flowjo software. the neutralizing activity of the rabbit and mouse anti -ntd, -ctd, and -s2 polyclonal antisera was assessed 4 weeks after the initial inoculation by a ffn assay, as previously described (okda et al., 2015) . the heat-inactivated serum samples were 2-fold serially diluted in dmem and 200 tcid 50 in 50 μl of pdcov stock was added to each dilution and incubated for 1 h at 37°c. the virus/serum mixtures were then added to confluent monolayers of st cells, which had been washed twice with dmem supplemented with 5 μg/ml trypsin, and incubated for 1.5 h at 37°c. the overlays were removed and the cells were then incubated for 72 h in dmem with 2.5 μg/ml of trypsin to allow for replication of non-neutralized virus. indirect immunofluorescence staining of allowed visualization of pdcov-infected cells. endpoint neutralization titers were determined as the highest serum dilution resulting in a 90 % or greater reduction in fluorescent foci relative to pbs-immunized antisera controls. plaque-reduction neutralization tests (prnt) were performed to quantify the titer of pdcov-neutralizing antibodies in the sera of pdcov-challenged and unchallenged mice as previously described (matrosovich et al., 2006) . heat-inactivated sera were two-fold serially diluted from 1:40 to 1:320 in 100 μl of dmem, to each dilution 50 pfu of pdcov in 100 μl of dmem was added, the serum/virus mixtures were then incubated for 1 h at 37°c. after incubation, 200 μl of each mixture was aliquoted into wells of 6-well plates containing a confluent monolayer of st cells, plates were incubated for 1.5 h at 37°c with r. chen, et al. virus research 276 (2020) 197834 periodic rocking. after incubation the inoculum was removed and cells were overlaid with 1 × dmem containing 0.6 % avicel rc-661(fmc biopolymer). the plates continued incubating at 37°c for 3 days, when plaques were easily observed. the monolayers were stained with 1 % crystal violet in 20 % methanol for 30 min at rt, then washed with water. the percent inhibition was calculated as follows: % plaque reduction = (plaque count in virus only sample -plaque count in serum/ virus sample) / (plaque count virus only sample) × 100. the prnt is defined as the reciprocal of the antibody dilution required to reduce the number of plaques by 50 % relative to the control wells all experimental data were analyzed using graphpad prism version 7.0 and expressed as mean ± sd. the differences among the three groups were analyzed using two-way anova. statistical significance is indicated by* p value < 0.05, ** p value < 0.01, *** p value < 0.001, ****p value < 0.0001. sds-page analysis showed that the ntd (aa 50-286), ctd (aa 278-616), and s2 (aa 601-1087) fusion proteins were efficiently expressed. the proteins were purified using affinity chromatography, and their concentrations were adjusted to 0.75 mg/ml with pbs (fig. 1b) . after separation using sds-page and transfer for western blot, the proteins were specifically recognized by pig anti-pdcov polyclonal antisera (fig. 1c) . based on band density, the reaction of ctd with the polyclonal antisera was more intense than with the ntd or s2 proteins (fig. 1d) , indicating that the ctd region may be a stronger antigenic site. sera from rabbits inoculated with ntd, ctd, and s2 were tested for neutralizing antibodies against pdcov by elisa, virus neutralization (vn), and fluorescent focus neutralization (ffn) assays. as shown in fig. 2 , all rabbit antisera, anti -ntd, -ctd and -s2, effectively neutralized pdcov in vitro. neutralizing antibody titers were 1:88 ± 10, 1:212 ± 11, and 1:125 ± 9.0, respectively. pre-immune serum exhibited no significant neutralizing effect. the ffn assay (fig. 3) shows that the endpoint neutralizing titer (1:256) of the rabbit polyclonal serum vs. ctd was higher than that for the serum vs. ntd or s2 (1:32 and 1:64, respectively). these results demonstrate that the ntd, ctd, and s2 proteins induced potent anti-pdcov neutralizing antibody responses in the immunized animals. to evaluate the immunogenicity of ntd, ctd, and s2, mice were inoculated by subcutaneous injection with identical concentrations (50 μg/mouse) of the three proteins (fig. 4a ). sera were collected every week after the primary inoculation and pdcov-specific antibodies were detected using elisa. the results showed that each protein induced pdcov-igg antibodies after the first inoculation, and all mice showed a significantly enhanced immune response three weeks after the secondary boost. ctd induced the highest level of igg antibody (fig. 4b) . the pdcov neutralizing-activity of the mouse sera was assessed using a virus neutralization (vn) assay. pdcov neutralization was detected 2 weeks after mice were initially inoculated and the neutralizing titers increased significantly thereafter (fig. 4c) . sera from the pbs inoculated control group had no significant neutralizing activity. after 4 weeks, each sera effectively neutralized pdcov infection in vitro; antibody titers were 1:218 ± 38, 1:545 ± 81, and 1: 291 ± 68, respectively (fig. 4c ). more importantly, neutralizing antibody titers induced by ctd were significantly higher than those induced by ntd or s2 at weeks 4 and 6 ( fig. 4c) , indicating that ctd contains the main neutralizing epitope in the pdcov s protein. pdcov infected st cells were reacted with ntd-, ctd-, and s2specific mouse antisera, collected at week 4 after the initial inoculation, then with fitc-anti mouse igg. antibody binding to pdcov was evaluated by flow cytometry. as shown in fig. 5 , pdcov-specific fluorescence signal was generated by each sera, but the percentage of pdcovinfected cells bound by the ctd-antisera (85.9 %), was considerably higher than ntd (60.9 %) or s2 (73.8 %) antisera. neutralizing activities of the mouse polyclonal antisera were also tested by fluorescent focus neutralization (ffn) assay. as shown in fig. 6 , the mouse antisera differed in the extent of neutralization of pdcov infection. based on relative amount of infected st cells, the endpoint neutralizing titer (1:320) of the ctd serum was higher than the ntd or s2 sera (both 1:160). the results suggest that the ctd region may contain the major neutralizing epitope(s) of the pdcov s protein. and evaluation of neutralizing activity for sera of rabbit vaccinated with ntd, ctd and s2, respectively. sera from 10 days post-last vaccination were used for the detection. (a) elisa of anti-pdcov serum igg levels, data are presented as mean a450 ± sd. (b) pdcov-neutralizing antibody titers, pre-immune serum was used for a negative control. **p = 0.0031, *p = 0.0378, ****p < 0.0001 for ntd compared to pre-immune serum, s2 and ctd, respectively. ***p = 0.0005 for s2 compared to ctd. the experiment was repeated three times. statistical differences are indicated by * p value < 0.05, ** p value < 0.01, *** p value < 0.001, **** p value < 0.0001. r. chen, et al. virus research 276 (2020) 197834 to further demonstrate neutralization activity, a plaque reduction neutralization test (prnt) was used to evaluate the mouse sera collected at week 4 after the initial inoculation. the prnt titer (1:320) of the anti-ctd sera was higher than the titers obtained for anti-ntd (1:80) and -s2 sera (1:160) (fig. 7) . of the three protein domains, the ctd may play a more important role in the viral infection process. week four, *p = 0.0398, **p = 0.0058, ****p < 0.0001 for pbs compared to ntd, ctd and s2, respectively. **p = 0.0016 for ntd compared to ctd, and *p = 0.0105 for s2 compared to ctd. at week six, both ***p = 0.0002 for pbs compared to ntd and s2, and ****p < 0.0001 compared to ctd. **p = 0.0028 for ntd compared to ctd, and **p = 0.0033 for s2 compared to ctd. statistically significant differences are indicated by* p value < 0.05, ** p value < 0.01, *** p value < 0.001, **** p value < 0.0001. r. chen, et al. virus research 276 (2020) 197834 several studies have shown that the s glycoprotein of covs is primarily involved in virus attachment and cellular entry (gallagher and buchmeier, 2001) . the s protein of covs contains two subunits, s1 and s2. subunit s1 is located at the n-terminus and binds to cellular receptor(s), whereas s2 is involved in virus-cell membrane fusion (eckert and kim, 2001) . given its critical functions in cellular entry, s protein is a high-priority target for vaccine development. s protein-specific serum or secretory neutralizing antibodies may play a key role in protection against coronavirus infection (okda et al., 2017; song et al., 2016) . several neutralizing epitopes have been identified in the full-length pedv s protein, with the majority of epitope(s) mapping to a collagenase equivalent domain (coe) (okda et al., 2017) . vaccine development has therefore been focused exclusively on the coe epitope. an oral vaccine (ppg-coe-dcpep/l393), a recombinant lactobacillus casei that expresses dc-targeting peptide fused with a coe protein, was developed by hou et al. (hou et al., 2018) . pdcov, a newly emerged and lethal swine enteric coronavirus, poses a worldwide threat to the pig industry. to date, the immune neutralizing region or epitope of pdcov remains unknown. using approaches similar to those used to study pedv, we successfully identified the immune dominant region(s) of pdcov s protein. these regions are potentially useful as targets for vaccine or therapeutic agents to control pdcov infection in pigs. in order to identify the immune dominant region of pdcov s protein, we expressed three truncated fragments covering residues 50-1087 of s but excluding the signal peptide, guided by a previous study that used cryo-electron microscopy to analyze the structure of the pdcov s protein (xiong et al., 2018) . the three recombinant fragments contained the ntd (aa 50-286), ctd (aa 278-616), and s2 (aa 601-1087) regions of the s protein. the purified fragments were specifically recognized on a western blot probed using polyclonal pig antisera against pdcov. the bands migrated as expected for each protein (ntd, 46 kda; (b) serum from mice inoculated with pbs was used as negative control and hyperimmune pig anti-pdcov serum was used as positive control. (c) neutralizing activity of the s-specific antisera was quantified by counting virus-infected cells after immunofluorescent staining. percent infection based on the images in panels a and b, the fitc-positive cells in the control serum was set as 100 %. the total number cells and the pdcov + cells were counted using three or more pictures for each dilution of sera. r. chen, et al. virus research 276 (2020) 197834 ctd, 58 kda; s2, 66 kda) (fig. 1c) . interestingly, ctd bound pdcov at higher a level than did ntd or s2, suggesting that the ctd region may have better antigenicity and contain more or stronger epitopes. purified recombinant ntd, ctd and s2 proteins were injected into rabbits to produce specific antisera. all antisera were able to inhibit pdcov infection in vitro, as determined by vn and ffn assays. amongst the three antisera, ctd-specific serum was the most effective (figs. 2 and 3). however, since the neutralizing antibody in these experiments was obtained from a single time point after immunization, the result is not conclusive. to verify the relative efficacies of the antisera, female balb/c mice were inoculated with purified protein and the corresponding antibodies was evaluated several times after immunization. the inoculated mice showed a significant increase in anti-pdcov antibody titer after receiving the second immunization (fig. 4b ). as shown in fig. 4c , anti-ctd antiserum consistently showed the highest neutralizing antibody titers by vn assay across time points. at week 4, mice inoculated with ctd showed a significant increase in neutralizing antibody titers to 1:545 ( ± 81.7). similarly, anti-ctd antiserum collected at week 4 also had a markedly higher neutralizing titer (1:320) in ffn and prnt assays (figs. 6 and 7) . these results suggest that pdcov ctd has an important role in the viral infection process. neutralizing antibody titers in mice inoculated with the rbd regions of mers-cov and sars-cov reached 1:1000 in vn assays after the second immunization (he et al., 2005; tai et al., 2017) . the three recombinant pdcov proteins we tested generated somewhat lower neutralizing antibody responses, perhaps because the truncated region of the s protein contains non-neutralizing epitopes that compete with the neutralizing epitopes in eliciting antibody responses (du et al., 2013a ). more precise localization and identification of specific immune dominant epitope(s) of ctd will be needed to test this hypothesis. the s glycoprotein of covs plays a critical role in mediating viral entry and inducing neutralizing antibody responses in infected individuals spaan et al., 1988; zumla et al., 2016) . previous work demonstrated that an anti-pedv-s2 mab recognizing the linear epitope gprlqpy at the carboxy-terminal of the s2 subunit exhibited neutralizing activity against pedv (cruz et al., 2008) . similarly, a neutralizing mab that specifically recognizes the s1-ntd of mers-cov s protein has been identified (chen et al., 2017) . in our study, we also found that regions s2 and s1-ntd can induce neutralizing antibody, consistent with previous reports (chen et al., 2017; cruz et al., 2008) . in addition, all covs use the s1-ctd or s1-ntd regions to interact with cellular receptors or co-receptors for viral attachment. the main functional rbd regions of most α-covs and some β-covs, including sars-cov, mers-cov, and hcov-hku1, are located in the s1-ctd (chen et al., 2017; deng et al., 2016) . in contrast, the rbds of human cov oc43, mhv, and avian γ-cov are located in the s1-ntd region. importantly, the s-rbd region in most covs contains critical neutralizing sites that induce potent neutralizing antibodies (du et al., 2013a; he et al., 2004b; yoo and deregt, 2001) . using ten anti-pedv-s1 mabs, li et al. (li et al., 2017a) showed that six non-overlapping neutralizing and non-neutralizing epitopes exist in the s1 subunit of the pedv spike protein, and that most of the neutralizing antibodies target the receptor binding domain. the antibodies induced by ntd, ctd, and s2 in our study differed in neutralization capacity against pdcov in vitro (figs. 4-6). the superior inhibitory and neutralizing capacity of ctd antiserum suggests that this region may contain the main rbd region or neutralizing epitopes in the pdcov s protein. recently, three research groups demonstrated that papn is a major cell entry receptor for pdcov, and its catalytic domain can interact with s1-ctd wang et al., 2018; zhu et al., 2018) . in these studies, apn-knockout ipi-2i cells and st cells were still susceptible to pdcov infection. by analyzing the three-dimensional structures of pdcov s1-ctd, tgev s1-ctd, and papn, zhu et al. found that the shorter β1-β2 and β3-β4 turns in pdcov s1-ctd may cause insufficient contact with papn compared with tgev s1-ctd. these data indicate that papn may be a major entry receptor for pdcov, but other unknown pdcov receptors also exist. li et al. (li et al., 2017b) found that mers-cov uses both protein and sialoglycanbased receptor determinants, with binding to dipeptidyl peptidase 4 (dpp4) required for entry and cell surface sialic acids (sias) serving as attachment factors, respectively. data also show that dpp4 interacts with the ctd region of mers-cov s1 subunit , and the sia binding site lies within s1-ntd of the mers-cov (li et al., 2017b) . in our current study, we showed that the ntd of pdcov s1 subunit can induce neutralizing antibody responses. importantly, the sugar-binding capability of pdcov s1-ntd to mucin has been demonstrated using an enzyme-linked immunosorbent assay (shang et al., 2017) . therefore, we speculate that one sugar receptor may interact with the ntd region in the s1 subunit and influence the entry of pdcov into the cell, just as sia is used as an additional receptor for mers-cov cell entry. to our knowledge, this is the first report describing the neutralizing regions within the pdcov s protein. we found that three regions in pdcov s protein, including ntd (aa 50-286), ctd (aa 278-616), and s2 (aa 601-1087), can induce neutralizing antibody responses, and the specific polyclonal mouse and rabbit sera efficiently inhibit pdcov entry and infection in st cells. because receptor binding domains on covs are often targets for potent neutralizing antibodies, we speculate that the ctd and ntd regions may bind to protein and sugar receptors, respectively. our results provide a foundation for the study of pdcov receptors and their receptor binding domains. ctd may be more immunogenic than the ntd and s2 regions and may contain the major neutralizing epitope(s) in the pdcov s protein. therefore, ctd potentially offers an attractive target for the development of vaccines to prevent pdcov infection and combat future pdcov-disease pandemics. fig. 7. (a) plaque reduction neutralization activity of s-specific mouse polyclonal antisera. sera collected at week 4 after the primary inoculation were used. approximately 50 pfu of pdcov were used to infect st cells in a 12-well plate with or without pdcov sspecific polyclonal antisera. pdcov alone and pbs were used as the virus and blank controls, respectively. the images correspond to the neutralizing percentages presented in panel b. r. chen, et al. virus research 276 (2020) 197834 the authors declare that we have no commercial or associative interests that represent a conflict of interest in connection with the work submitted. a novel neutralizing monoclonal antibody targeting the nterminal domain of the mers-cov spike protein the gprlqpy motif located at the carboxyterminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus 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alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections a single amino acid change within antigenic domain ii of the spike protein of bovine coronavirus confers resistance to virus neutralization porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, prevalence and genetic evolution contribution of porcine aminopeptidase n to porcine deltacoronavirus infection coronaviruses -drug discovery and therapeutic options this study was supported by the national key research and development program of china (award 2016yfd0500700). we thank dr. kwonil jung from the ohio state university for critical reading and editing of the manuscript. key: cord-267228-g2tf1jz6 authors: huang, ke-yan; yang, gui-lian; jin, yu-bei; liu, jing; chen, hong-liang; wang, peng-bo; jiang, yan-long; shi, chun-wei; huang, hai-bin; wang, jian-zhong; wang, guan; kang, yuan-huan; yang, wen-tao; wang, chun-feng title: construction and immunogenicity analysis of lactobacillus plantarum expressing a porcine epidemic diarrhea virus s gene fused to a dc-targeting peptide date: 2018-03-02 journal: virus res doi: 10.1016/j.virusres.2017.12.011 sha: doc_id: 267228 cord_uid: g2tf1jz6 porcine epidemic diarrhea virus (pedv) is one of the most important causative pathogens of swine diarrhea, which is widely prevalent throughout the world and is responsible for significant economic losses in the commercial pig industry, both domestic and abroad. the spike (s) protein in the pedv capsid structure can carry the major b lymphocyte epitope, which induces production of neutralizing antibodies and provides immunoprotective effects. moreover, the conserved region encoded by the s gene can be considered a target for establishing a new diagnostic method and is a new candidate for vaccine design. in this study, use of anchorin pgsa' allowed the fusion protein of s-dcpep to express on the surface of recombinant lactobacillus plantarum (nc8-psip409-pgsa'-s-dcpep) nc8 strain. mice were immunized by lavage administration of the recombinant nc8-psip409-pgsa'-s-dcpep, which was observed to induce dc activation and high production of siga and igg antibodies in experimental animals, while also eliciting production of significantly more iga(+)b220(+) b cells. more importantly, secretion of cytokines ifn-γ, il-4 and il-17 in mice that were vaccinated with nc8-psip409-pgsa'-s-dcpep was remarkably increased. the results of our study suggest that nc8-psip409-pgsa'-s-dcpep potently triggers cellular and humoral immune responses. the obtained experimental results can provide a theoretical basis that lays the foundation for production of a novel oral vaccine against ped. porcine epidemic diarrhea (ped) is a highly contagious disease with typical symptoms in piglets that include severe diarrhea, vomiting and intestinal dehydration (ko et al., 2017) . the morbidity and mortality rates of suckling piglets caused by pedv are more than 50%, and it is the primary cause of death in piglets and weight loss in fattening pigs (choi et al., 2009) . this viral disease was first reported in britain in 1971 and then became an epidemic in other european countries (yu et al., 2017) . in 1976, pedv was detected for the first time in china, and since october 2010, pedv outbreaks worldwide have become more frequent (song et al., 2015) . for instance, ped broke out in minnesota and iowa in the united states in 2013, resulting in huge losses to the pig industry (diep et al., 2017; lee, 2015) . as a coronavirus, pedv has a typical structure that consists of four structural proteins, including the nucleocapsid protein (n), a membrane protein (m), a glycosylated spike protein (s) and a envelope protein (e) (kocherhans et al., 2001) . the pedv spike protein-encoding gene is an immune dominant gene that encodes a protein with a very dense hydrophobic region of amino acids that harbors multiple glycosylation sites and has a high antigenic index . several studies have reported that the s protein is a structural surface protein of pedv, containing both the receptor binding domain, which mediates invasion of the virus into the host cell, and the antigenic epitope, which mediates production of the neutralizing virus antibody . thus, the s-glycoprotein plays a crucial role in viral infection, pathogenicity and adsorption. it has been shown that the s-glycoprotein is immunogenic and has the potential to be a good vaccine candidate (fan et al., 2015) . lactic acid bacteria (lab) are diverse species that are widely distributed in nature and produce large amounts of lactic acid by fermenting carbohydrates (riaz rajoka et al., 2017) . lab, which are generally recognized as safe bacteria, colonize the intestines of humans or animals and have potentially beneficial effects (landete et al., 2015) . the study of lab has many advantages, including that they are easy to culture, convenient to manipulate and highly safe, which is why lab can be regarded as a heterologous antigen protein delivery system (wanker et al., 1995) . l. plantarum has been manipulated to deliver a number of viral antigens in previous studies (li et al., 2017b; yang et al., 2017c) . the l. plantarum strain nc8 was isolated from ensilages and has a better antireversion characteristic compared with lab isolated from healthy animal intestines (caggianiello et al., 2016; park et al., 2017) . thus, l. plantarum nc8 is more suitable to act as a mucosal delivery shuttle vector to express protective antigens. recently, poly-γ-glutamic acid synthetase a (pgsa) was identified as being a constitutively expressed protein of the polyglutamate acid (pga) synthetase system of bacillus subtilis. pgsa can stably anchor γ-pga synthetase on the cell membrane, acting as a transport carrier of γ-pga and playing a crucial role in the formation of γ-pga (sung et al., 2005) . the pgsa protein has a transmembrane region near its n-terminus (26-42 amino acid residues), providing the necessary characteristics for the establishment of a pgsa transmembrane expression system. the pgsa gene was combined with the target gene for fusion expression, after which l. plantarum was used as a mucosal vehicle to deliver an exogenous antigen. this experiment provided a theoretical basis for the construction of a novel, orally administered, genetic engineered vaccine to immobilize exogenous medicinal proteins on the cell wall surface of l. plantarum (cai et al., 2016; narita et al., 2006) . dendritic cells (dcs) are the most powerful functional antigen presenting cells (apcs) and are the exclusive apcs that are capable of inducing initial t and b cell activation . dcs can elicit t cell polarization and differentiation in t cells subsets, which also trigger secretion of ifn-γ (subramaniam et al., 2017) . dcs are involved in regulating the function of b cells and promoting the differentiation of b cells into plasma cells to produce igg . in the germinal center, dcs can boost b cell antibody affinity maturation and help to form and maintain memory b cells. in addition, siga can play an important role in maintaining intestinal integrity and intestinal symbioses (shaw et al., 2000) . dcs can restrict penetration of microorganisms into the mucosa and promote uptake of antigens in the intestinal mucosal system (owen et al., 2013) . it has been reported that nc8-psip409-pgsa'-s-dcpep can specifically conjugate dendritic celltargeting peptide (dcpep) and induce an immune response (apostolico et al., 2017) . to make dcs recognize the s-antigen more effectively in the intestinal mucosa, recombinant nc8-psip409-pgsa'-s-dcpep was constructed using standard molecular biology techniques, and its immunogenicity was further investigated. after balb/c mice were immunized by lavage administration of our recombinant l. plantarum, the results indicated that the in vivo antibody levels were greatly improved. the escherichia coli-lactobacillus shuttle vector psip409 (sorvig et al., 2005) and l. plantarum nc8 were kindly provided by professor a. kolandaswamy (madurai kamaraj university, india). the pm18-t-s-dcpep (dc peptide, fypsyhstpqrp) was stored at jilin provincial engineering research center of animal probiotics in jilin agricultural university. the pm18-t-s-dcpep plasmid was cut at xbai and hindiii sites within the primers using the appropriate restriction enzymes at 37°c for 3-4 h. next, the digested 2240 bp s gene fragment (spike protein gene genbank: jq257007.1 (source: 75-2313)/spike protein genbank: aew24858.1 (source: 26-771)) and dcpep (s-dcpep) were ligated into psip409-pgsa' using t4 dna ligase at 16°c overnight. the recombinant psip409-pgsa'-s-dcpep plasmid was detected and purified using a gel extraction kit (axygen). the recombinant plasmid was introduced into the l. plantarum nc8 strain by electroporation and erythromycin-resistant bacteria were selected for, after which the plasmid was sequenced. in addition, to meet the rigorous nature of the experimental study, a nc8-psip409-pgsa'-s-ctrlpep (control peptide, epihpettftnn) was constructed similar to nc8-psip409-pgsa'-s-dcpep and used as the experimental control group. to examine the expression of the s-protein on the surface of recombinant nc8-psip409-pgsa', nc8-psip409-pgsa'-s-ctrlpep and nc8-psip409-pgsa'-s-dcpep was cultured in mrs with 10 μg/ml erythromycin. as described in previous studies, we added sakacin p (sppip) to the culture medium when the (od 600 ) of the medium was between 0.3 and 0.4. after induction at 37°c for 10 h, cells from each strain were washed with pbs containing 1% bovine serum albumin. next, cells were resuspended in pbs and 1 × 10 5 cfu of bacteria were incubated with 1 ml of primary antibody (1/2000 anti-s-produced in rat) for 1 h at 37°c with shaking at 60 rpm. the pellets were washed with pbs containing 0.2% tween-20 (pbs-t), after which secondary antibodies (fitc, goat anti-rat igg (h + l), cst) were added and the mixture was incubated for one hour at 37°c with shaking at 60 rpm in the dark. finally, samples were washed with pbs-t and resuspended in pbs. the cells were examined under a laser scanning confocal microscope (lsm710; carl zeiss, germany) following the manufacturer's instructions. to detect the s-protein expression levels, we induced its expression in recombinant lab as described previously. strains were subsequently resuspended in 500 μl of pbs and were lysed by ultrasonic crushing as previously described (shi et al., 2014) . samples were assessed by 10% sds-page and transferred to pvdf membranes (millipore, usa). the s-ctrlpep and s-dcpep proteins were detected using an anti-s polyclonal primary antibody (polyclonal antibody was obtained from serum of mice which were immunized with the purified s protein) and a hrpconjugated goat anti-rat igg secondary antibody (a21040; abbkine, usa). the bands were visualized using enhanced chemiluminescence reagents in an ecl plus detection kit (thermo scientific) according to the manufacturer's instructions. for experiments involving animals, thirty-six female balb/c mice without specific pathogen (spf) were bought from beijing hfk bioscience co., ltd., china. according to the provisions of the animal care and ethics committees of jilin agriculture university. all animals were strictly raised with commercialized germ-free feed and sterilized water in the jilin provincial engineering research center of animal probiotics, which provided the pathogen-free environment facilities. six-week-old mice with no specific pathogens were randomly divided into four groups, with 6 mice in each group (n = 9). briefly, using oral gavage, four groups of mice were immunized with nc8-psip409-pgsa', nc8-psip409-pgsa'-s-ctrlpep, nc8-psip409-pgsa'-s-dcpep or saline. the entire immunization procedure was divided into three phases: prime immunization (days 1-3), booster immunization (days 8-10), and the last immunization (days 29-31), with 10 8 cfu/mouse administered by oral gavage. generally, experimental mice were sacrificed by cervical dislocation 30 days after being immunized. lamina propria (lp) cells of the small intestine, pps and mlns were acquired via a previously described method (kikuchi et al., 2014 ) on a clean sterile clean bench. small intestine samples were washed with pbs and cut into small pieces. next, samples were digested with lymphocyte separation medium, after which lp cell digestive juice was added to obtain single lp cells from the small intestine in an isotonic percoll solution. the cell suspension was filtered through a 70 μm sterile filter-film and the filtrate was centrifuged at 500g for 5 min at 4°c. subsequently, the cells were washed with 4°c pbs and were eventually resuspended in complete rpmi 1640 (containing 10% fbs, 1% penicillin and streptomycin). the method used for the analysis of s protein expression in recombinant l. plantarum was consistent with the immunofluorescence assay, with cells analyzed by flow cytometry (bd facs lsr fortessa™ flow cytometer). data were analyzed via flowjo 7.6.1 software. to test the activation of dc surface costimulatory molecules and b cells, flow cytometry was conducted using a previously published protocol . in brief, single lp cells from the small intestine suspensions obtained from immunized mice were diluted to 1 × 10 6 cells/ 100 μl and incubated with 10 μl anti-cd11c (clone hl3), cd80 (clone 16-10a1), cd86 (clone gl1), cd40 (clone 3/23) and mhcii (clone 3/ 23) antibodies to assess the dc cells. to analyze b lymphocyte cells activation, 10 μl of anti-b220 (clone ra3-6b2) and iga (clone c10-3) antibodies was added to stain b lymphocyte cells for 20 min at room temperature in the dark. afterwards, cell suspensions were washed twice with facs buffer. after staining, samples were examined using flow cytometry (bd facs lsr fortessa™ flow cytometer). data were analyzed using flowjo 7.6.1 software. blood samples were obtained on days 14, 35 and 42 after immunization and were centrifuged at 4000 rpm for 15 min at 4°c to obtain the serum, which was stored at −80°c. the fecal samples were prepared according to our previously published method, followed by centrifugation at 5000 rpm for 20 min at 4°c. the supernatants were acquired and stored at −80°c for further analysis. elisa was performed to determine the levels of specific igg and siga antibodies (yang et al., 2017d) . in brief, 100 μl of purified s protein (4 μg/ml) was added to each well of the 96-well plate and incubated overnight at 4°c. on the second day, the blocking solution was added and the plate was incubated overnight at 4°c. on the third day, the fecal samples (1:10) and serum samples (1:100) were diluted and added separately, after incubating at 37°c for 2 h, the wells were rinsed. subsequently, goat anti-mouese igg (h + l)-biotin and goat anti-mouse iga-biotin (southern biotechnology, birmingham, al) was added separately, and the plate was incubated at 37°c for one hour, after which streptavidin-hrp (southern biotechnology, birmingham, al) secondary antibody was added and the plate was incubated at 37°c for 30 min. finally, tmb substrate solution and stop solution was added, after which the color intensity was measured at 492 nm. the end-point titers (log2) were defined as the highest dilution yielding an absorbance that was two and three times higher than the background for the fecal and serum samples, respectively . the mln cells were stimulated by s protein (5 μg/ml) and the supernatant was collected 3 days later and examined for specific cytokines, including ifn-γ, il-4 and il-17a using an elisa kit (liuhebio, wuhan, china) according to the manufacturer's instructions. for determining the pedv neutralizing activity of the serum antibodies we detected sample obtained from mice which were orally immunized with recombinant l. plantarum by antibody neutralization assay (microdetermination) as described before (tian et al., 2014) with minor modifications. under aseptic conditions serum and fecal samples were collected at 14, 35 and 42 days respectively after immunization, quantitative virus and serial diluted serum were added to vero cells to evaluate serum neutralizing antibody titers, the neutralizing antibody titer pd50 (median protective dose refers to the dose of serum or vaccine that protects half (50%) of the experimental animals) was determined by karber's method at 72 h. all results were performed by two-tailed t-tests and one-way analyses of variance (anova) (graphpad prism 5.0, graphpad software) were used to analyze the significance of the difference between means. all data were obtained from at least three independent experiments, and the geometric mean titers were expressed as averages. all data were expressed as the means ± sem. the psip409-pgsa'-s-dcpep and psip409-pgsa' plasmids were introduced separately into l. plantarum nc8 strain by electroporation (fig. 1a) . using flow cytometry, s protein expression levels of recombinant l. plantarum nc8-psip409-pgsa'-s-dcpep and nc8-psip409-pgsa'-s-ctrlpep were determined (fig. 1b) . the green fluorescence marked by white arrows indicated that recombinant lactobacillus of nc8-psip409-pgsa'-s-dcpep and nc8-psip409-pgsa'-s-ctrlpep expressed s protein on the surface (fig. 1c ) than nc8-psip409-pgsa', which was barely observed with green fluorescence under a confocal microscope. western blot analysis showed that a specific band appeared at 80 kda, which demonstrated that the pedv s protein had been expressed. thus, the experimental results suggest that nc8-psip409-pgsa'-s-dcpep and nc8-psip409-pgsa'-s-ctrlpep were expressed in l. plantarum nc8. to determine the effect of nc8-psip409-pgsa'-s-dcpep on dcs, flow cytometry was performed to test the level of costimulatory molecules activated by dcs, which were induced by nc8-psip409-pgsa'-s-dcpep gavage immunization 3 days after the last immunization ( fig. 2a) . compared with the saline group, the expression level of cd11c + cd40 + of dcs surface molecules in the lp cells of the small intestine was significantly increased in the nc8-psip409-pgsa'-s-dcpep group (p < 0.01) and nc8-psip409-pgsa'-s-ctrlpep group (p < 0.05) experimental groups (fig. 2b) . moreover, our experimental results indicated that nc8-psip409-pgsa'-s-dcpep can promote greater expression of cd80 + in lp dcs than the nc8-psip409-pgsa'-s-ctrlpep (p < 0.05), nc8-psip409-pgsa' (p < 0.05) or saline (p < 0.01) groups (fig. 2c) . however, no obvious differences were observed in cd11c + cd86 + expression of dcs surface molecules in the lp cells of the small intestine. the number of iga + b220 + b cells in pp was determined by flow cytometry 3 days after the last immunization (fig. 3a) . the results showed that there was a remarkable increase in the percentage of iga + b220 + b cells within the b cell populations of balb/c mice having received lavage administration with nc8-psip409-pgsa'-s-dcpep compared with the nc8-psip409-pgsa', nc8-psip409-pgsa'-s-ctrlpep and saline groups. in addition, nc8-psip409-pgsa'-s-dcpep in pp significantly increased the production of iga + b220 + b cells compared to the nc8-psip409-pgsa' and saline group control (p < 0.01) (fig. 3b) , and the nc8-psip409-pgsa'-s-dcpep was significantly higher than the nc8-psip409-pgsa'-s-ctrlpep groups in pp (p < 0.05) (fig. 3b) . collectively these results demonstrated that vaccination with the nc8-psip409-pgsa'-s-dcpep can activate the b cell response in mice. the specific siga levels in fecal intestines were measured at days 14, 35 and 42 after the first immunization using elisa (fig. 4) . however, the concentration of siga in the excrement of mice treated with nc8-psip409-pgsa'-s-dcpep was higher than that of the nc8-psip409-pgsa' (p < 0.05) and saline (p < 0.05) groups at days 14 (fig. 4) , and nc8-psip409-pgsa'-s-dcpep was higher than that of the nc8-psip409-pgsa' (p < 0.05) and saline (p < 0.01) groups at day 35 (fig. 4) . in contrast, no obvious difference was observed with respect to slga was observed at days 14 and 35 in the nc8-psip409-pgsa'-s-dcpep and nc8-psip409-pgsa'-s-ctrlpep groups (fig. 4) . with respect to the 42 day lavage administration, the nc8-psip409-pgsa'-s-dcpep group was higher than that of the nc8-psip409-pgsa' (p < 0.01) and saline (p < 0.01) groups. higher expression of siga was also observed in nc8-psip409-pgsa'-s-dcpep group compared with nc8-psip409-pgsa'-s-ctrlpep group (p < 0.05) at day 42 (fig. 4) . these data suggested that lavage administration of nc8-psip409-pgsa'-s-dcpep could stimulate the mucosal immune system in mice. the specific igg-antibodies level after lavage administration of nc8-psip409-pgsa'-s-dcpep in mice was notably increased compared with the nc8-psip409-pgsa ', nc8-psip409-pgsa'-s-ctrlpep and saline groups at days 14, 35 and 42 after the first immunization. the results showed that the concentration of specific igg in the serum of mice treated with nc8-psip409-pgsa'-s-dcpep was significantly increased compared to the nc8-psip409-pgsa' (p < 0.05) and saline (p < 0.01) groups at days 14 and 35 (fig. 5 ). in addition, only nc8-psip409-pgsa'-s-dcpep showed higher levels than the nc8-psip409-pgsa'-s-ctrlpep (p < 0.05), nc8-psip409-pgsa' (p < 0.01) and saline (p < 0.001) groups at day 42 (fig. 5) , with an increasing trend from days 28 to 35 (fig. 5) . these data suggested that lavage administration of nc8-psip409-pgsa'-s-dcpep could increase iga antibody titers and produce humoral immune responses in mice. specific cytokines in the mln cell secretions were measured 3 days after the last immunization using elisa. the results showed that the concentration of il-4 in the supernatants of mln cells cultured from the nc8-psip409-pgsa'-s-ctrlpep and nc8-psip409-pgsa'-s-dcpep-treated mice were significantly increased compared to those from the saline group (p < 0.01) (fig. 6a) . higher level of il-17 was observed in the group of mice orally immunized with recombinant nc8-psip409-pgsa'-s-dcpep compared to the group of mice orally administered with saline (p < 0.01), nc8-psip409-pgsa'-s-ctrlpep and nc8-psip409-pgsa' groups (p < 0.05) (fig. 6c) . unexpectedly, the level of ifn-γ in the supernatant of mln cells cultured with the strains expressing s-dcpep was significantly higher in the group of mice orally immunized with recombinant nc8-psip409-pgsa'-s-dcpep compare to the group of mice orally administered with saline (p < 0.01), nc8-psip409-pgsa'-s-ctrlpep and nc8-psip409-pgsa' groups (p < 0.05) (fig. 6b ). fig. 3 . detection of activated b cells in pp of immunized mice using flow cytometry. the number of total cells for (a) the quadrants and (b) the bar graphs refer to iga + b220 + cells. the degree of activation of b cells was analyzed by counting the percentage of iga + b220 + expression. significant differences relative to the saline and nc8-psip409-pgsa' controls (p < 0.05, p < 0.01). significant differences are denoted by an asterisk (*) between saline, nc8-psip409-pgsa', nc8-psip409-pgsa'-s-ctrlpep and nc8-psip409-pgsa'-s-dcpep controls (*p < 0.05, **p < 0.01, ***p < 0.001). k.-y. huang et al. virus research 247 (2018) 84-93 3.7. nc8-psip409-pgsa'-s-dcpep induced production of serum/fecalneutralizing antibodies specific pedv antibodies titers in serum and faeces were measured by cell neutralization assays at different time after the first immunization. results showed that serum and fecal antibodies of mice orally immunized with recombinant nc8-psip409-pgsa'-s-dcpep was higher than that from mice orally administered with and saline at days 14 (p < 0.01) (fig. 7) . the serum antibody of mice orally immunized with recombinant nc8-psip409-pgsa'-s-dcpep was significantly increased compared with nc8-psip409-pgsa' at days 35 (p < 0.01) (fig. 7a) , however, the fecal antibodies of nc8-psip409-pgsa'-s-dcpep was no significant difference at days 35 compared with saline and nc8-psip409-pgsa' at days 14 (fig. 7b ). significant differences were observed in both serum and fecal antibodies of mice orally immunized with recombinant nc8-psip409-pgsa'-s-dcpep compared with the other three group at days 42 (fig. 7) , particularly, difference were also observed in serum and fecal antibodies between the group of mice orally immunized with recombinant nc8-psip409-pgsa'-s-dcpep and nc8-psip409-pgsa'-s-ctrlpep (p < 0.05). the results suggesting that recombinant l. plantarum nc8-psip409-pgsa'-s-dcpep has good pedv virus-neutralizing activity in humoral and mucosal immunity. porcine epidemic diarrhea (ped) caused by pedv is a contagious disease which seriously threaten pig production industry (huang et al., 2013) . ped characterized by high morbidity and mortality in piglets was reported in southern china in fall of 2010 (huang et al., 2013 . furthermore, in april 2013 ped has also spread throughout and to the highest degree destroyed the american pork industry, the homology between the genome of popular strain in american and china is as high as 97%-99%, which has caused immeasurable economic losses (huang et al., 2013) . the continuing outbreak of ped suggests that it is able elude current immunization strategies (curry et al., 2017) . currently, there is no effective drug for pedv treatment, although inactivated and live vaccines are the two most commonly used vaccines, although live and inactivated vaccines are already in widespread use, development of effective, safe and economical oral vaccines are still the trend of future. and researchs of developing oral vaccine to prevent pedv have achieved effective protection. l. plantarum is an intestinal predominant flora with immunomodulatory effects that enhance the chemotaxis and phagocytosis of neutrophils, prevent viral infection and maintain intestinal microbial homeostasis (li et al., 2017a,b) . therefore, l. plantarum with probiotic properties has become good exogenous protein vector platform. the anchorin expressed by pgsa gene can help exogenous proteins to locate to the surface of bacterium and display their biological function. other and our team have achieved good protection using pgsa to display exogenous antigens, hence, using pgsa to display exogenous antigens during vaccine development is a viable strategy. the feature of localized intestinal infection of pedv determined that the immunization and prevention is difficult, hence, intestinal mucosal immunity plays an important role in pigs except suckling piglets (temeeyasen et al., 2017) . resistance to pedv infection often relies on iga secreted by mucosa, which requires the development of vaccines that can effectively increase intestinal immunity. contemporary strategies in vaccination primarily focus on using different vectors to express the identified antigenic regions. antigens delivered by different routes might stimulate the intestinal mucosal immune system to achieve immunoprophylaxis effect. the use of probiotics to express antigens and proliferate them in the intestinal of host can promote the digestion of livestock and poultry; meanwhile, the protective antigen expressed can stimulate the intestinal mucosal immune system to produce protective effect. as a favorable mucosal immunization vaccine vector, l. plantarum can also be used as a natural adjuvant in the mucosal microenvironment and attenuate inflammatory response to enhance the body immunity (yu et al., 2015) . morever, vaccines constructed by dcpep could provide effective protection (yang et al., 2017a,b) . consequently, we constructed fusion protein by the immunogenic s-protein of pedv and the induced dcpep and expressed it on the surface of l. plantarum, which could activate dcs to initiate mucosal and humoral immune responses against microbial infection via secreting antibodies to clear the antigens. results showed that the dcpep fusion group had better immune protection compared with other groups. dcs are the foremost antigen presenting cells (apcs) and play an important role in inherent and adaptive immunity (sato et al., 2017) . cd11c + is a surface marker of dcs, and cd80 + and cd40 + are subsets of cd11c + . there was a difference in expression of cd11c + cd40 + between the group of mice orally immunized with recombinant nc8-psip409-pgsa'-s-dcpep and saline group but not nc8-psip409-pgsa'-s-ctrlpep and nc8-psip409-pgsa' groups, however, the mei of group nc8-psip409-pgsa'-s-dcpep was still higher than the above two groups ( fig. 2a) . the expression of the cd11c + cd80 + in nc8-psip409-pgsa'-s-dcpep group was significantly higher than the control group and the nc8-psip409-pgsa' group in the lp of small intestine (fig. 2b) . in this study, the expression of cd11c + cd86 + in dcs was no significant difference between nc8-psip409-pgsa'-s-dcpep and saline group, which should also increased according to previous studies. this difference in results might be due to different host strains and different immunization programs. however, cd11c + cd86 + did not affect the biological function of cd11c + cd80 + and cd11c + cd40 + dcs, which could also identify cd40 + , cd80 + , cd86 + and other co-stimulatory molecules, fig. 4 . s-specific siga titers levels in mouse feces examined by elisa se. the s-specific siga ab titers were detected after prime immunization, booster immunization, and the last immunization. end-point titer (log 2 ) were defined as the highest dilution yielding an absorbance that was two and three times higher than the background for the fecal and serum samples, respectively. significant differences are denoted by an asterisk (*) between stroke-physiological saline solution, nc8-psip409-pgsa', nc8-psip409-pgsa'-s-ctrlpep and nc8-psip409-pgsa'-s-dcpep controls (*p < 0.05, **p < 0.01, ***p < 0.001). s-specific igg titers levels in sera of mice detected by elisa. the s-specific igg ab titers were detected after prime immunization, booster immunization, and the last immunization. end-point titer (log 2 ) were defined as the highest dilution yielding an absorbance that was two and three times higher than the background for the fecal and serum samples, respectively significant differences are denoted by an asterisk (*) between stroke-physiological saline solution, nc8-psip409-pgsa', nc8-psip409-pgsa'-s-ctrlpep and nc8-psip409-pgsa'-s-dcpep controls (*p < 0.05, **p < 0.01, ***p < 0.001). present antigen to activated t cells or effector t cells. meanwhile, cd80 + and cd86 + of activated b cells could combine with cd28 expressed on the surface of t cells, which involved in co-stimulatory signal transduction. in addition, the results of s-specific igg in serum and s-specific siga in feces showed that l. plantarum strains expressing s protein could improve the secretion of igg and siga and further enhance immune function, particularly for nc8-psip409-pgsa . several studies showed that iga secreted by mucosal lamina propria could combine with mucus and cover on the surface of epithelial cells which inhibit the adhesion of microorganisms to epithelial cells and neutralization of toxins produced by microorganisms or enzymes. siga agglutinates and incapacitates pathogens by which it inhibits pathogen adhesion to the mucosal surface and easily cleared in the secretions. therefore, siga disenables pathogens interact with epithelial cell receptors and inhibits the assembly of viral particles within the host cell cytoplasm (kurashima and kiyono, 2017; motegi et al., 2000) . our study suggesting that the anti-s mucosal siga responses of recombinant lactobacillus nc8-psip409-pgsa'-s-dcpep group significantly increased. following four indicators are used to evaluate the effectiveness of a vaccine in mucosal system: connecting closely with lymphoid tissue in mucous membrane; the existence of scattered lymphoid tissues and lymphoid organs with a certain structure; dcs use a special mechanism to uptake antigen; and the presence of many activated and memory lymphocytes in the absence of an infection (chung et al., 2017; faria and reis, 2017) . currently, many investigators are focused on the development of mucosal vaccines. our study demonstrated that the recombinant l. plantarum nc8-psip409-pgsa'-s-dcpep could increase the activation of b cells and further improve the level of humoral immunity (fig. 3) . orally administration of recombinant nc8-psip409-pgsa'-s-dcpep in mice boosted the number and frequency of iga + b220 + b cells compared with the nc8-psip409-pgsa' and nc8-psip409-pgsa'-s-ctrlpep groups. similarly, we also detected anti-s systemic igg in the blood, the results showed that s-dcpep could effectively initiate a mucosal immune response and humoral immunity. subsequently, l. plantarum expressed s-dcpep fusion protein could enhance the secretion of mucosal antibodies against pathogens (mohamadzadeh et al., 2009; shi et al., 2016; yang et al., 2017a yang et al., ,b, 2016 . in the construction of the recombinant strain nc8-psip409-pgsa'-s-dcpep, pgsa has been used as an anchorin for its stability and safety in clinical use. besides, pgsa has a wide range of applications for surface expression on some types of lactobacillus compared with other anchorins. therefore, we used pgsa as a surface display element for applying to surfaced of l. plantarum. a recombinant vaccine constructed by s-protein expressed on the surface of l. plantarum and dcpep fusion protein can provide protection since pedv is primarily replicated in the small intestine. the results of western blot, facs and confocal microscopy analyses suggested that s protein was expressed on the surface of l. plantarum. compared with traditional intramuscular injection, gavage administration allows antigens to be presented through the gastrointestinal mucosa and obtain better immunological response, which is a simple, convenient and ideal immunization strategy. several genetically engineered bacteria (salmonella, escherichia coli, lactobacillus) could colonize in the intestines after inoculation that were regarded as promising oral vaccine vectors (garcia-fruitos, 2012 ). in addition, peptidoglycan expressed on the surface of l. plantarum, could function as a natural immune adjuvant and be utilized to design a novel vaccine (dieye et al., 2001) . previous studies have demonstrated that l. plantarum could act as a mucosal delivery system to induce immune response and tolerance (tauer et al., 2014) . the distinctive feature of the immune response triggered by oral mucosal immunization is the production of specific secretory iga. antigens were taken up by m cells after oral inoculation, then dendritic cells and t cell subsets were activated, cytokines and chemokine were released and mhci and mhcii were expressed (kurashima and kiyono, 2017) . finally the activation of b cells, the expression of specific integrin, phenotype transformation (especially to iga type conversion) are observed (perdigon et al., 2002) . th1 and th2 responses induced by recombinant l. plantarum were evaluated via cell-specific cytokines analysis. th1 cell response can helpcytotoxic t cell differentiation mediate cellular immune responses, secrete ifn-γ and activate macrophages, which enhance the ability of cells to kill phagocytized pathogens (maldonado-lopez and moser, 2001). besides, the primary function of th2 cells is to stimulate b cell proliferation and produce antibodies, which is related to humoral immunity (mosmann and coffman, 1989) . secretion of il-4 increased the ability of monocytes differentiate into macrophages . il-17 can increase the ability of costimulatory molecules, mhc, antigens and t lymphocytes to stimulate antigen peptide delivery and promote dc maturation . therefore, we analyzed cytokine system ifn-γ, il-4, and il-17 of th1/th2/th17 cells secreted by the mln of the immunized animals after treatment with recombinant bacteria. our results showed that nc8-psip409-pgsa'-s-dcpep could significantly increase cytokine secretion and stimulate th1 and th2 cells and th17 cell response, maintain the balance of th1, th2 immune response. the focus of our study was th1 and th2 immune responses induced by nc8-psip409-pgsa'-s-dcpep, which also stimulated the activation of th17 cells (fig. 6) . taken together, recombinant l. plantarum expressed s-dcpep could improve th1/th2/th17 immune response in balb/c mice and induce immune responses, such as the differentiation of b cells and dcs maturation. in this study, we detected pedv-specific neutralizing antibody in serum and fecal obtained from the group of mice orally administrated with l. plantarum expressed pedv-s protein on the surface, which could significantly inhibit the formation of plaque on vero cells. the results indicated that systemic immune response induced by recombinant lactobacillus provided an immunoprotective effect against the virus as shown in (fig. 7) . the constructed recombinant l. plantarum might contribute to the development of novel vaccines against pedv fig. 7 . nc8-psip409-pgsa'-s-dcpep induced mice to produce serum(a)/feces(b)-neutralizing antibodies against pedv. pd50 (median protective dose refers to the dose of serum or vaccine that protects half (50%) of the experimental animals).significant differences are denoted by an asterisk (*) between stroke-physiological saline solution, nc8-psip409-pgsa', nc8-psip409-pgsa'-s-ctrlpep and nc8-psip409-pgsa'-s-dcpep controls (*p < 0.05, **p < 0.01, ***p < 0.001). challenge. in conclusion, our study demonstrates that nc8-psip409-pgsa'-s-dcpep can modulate mucosal and humoral immunity and produce specific antibodies against s-protein. next, we will study the protective effect of recombinant bacteria against pedv challenge in piglet. exopolysaccharides produced by lactic acid bacteria: from health-promoting benefits to stress tolerance mechanisms 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antigen expressed by lactobacillus plantarum alleviation of enterotoxigenic escherichia coli challenge by recombinant lactobacillus plantarum expressing a faeg-and dc-targeting peptide fusion protein construction and immunological evaluation of recombinant lactobacillus plantarum expressing so7 of eimeria tenella fusion dc-targeting peptide protection of chickens against h9n2 avian influenza virus challenge with recombinant lactobacillus plantarum expressing conserved antigens protective efficacy of fc targeting conserved influenza virus m2e antigen expressed by lactobacillus plantarum lactobacillus protects the integrity of intestinal epithelial barrier damaged by pathogenic bacteria advances in reverse genetics to treat porcine epidemic diarrhea virus construction of a bivalent dna vaccine co-expressing s genes of transmissible gastroenteritis virus and porcine epidemic diarrhea virus delivered by attenuated salmonella typhimurium key: cord-290801-dv6aak01 authors: ivanyi-nagy, roland; darlix, jean-luc title: reprint of: core protein-mediated 5′–3′ annealing of the west nile virus genomic rna in vitro() date: 2012-09-27 journal: virus res doi: 10.1016/j.virusres.2012.09.009 sha: doc_id: 290801 cord_uid: dv6aak01 genome cyclization through conserved rna sequences located in the 5′ and 3′ terminal regions of flavivirus genomic rna is essential for virus replication. although the role of various cis-acting rna elements in panhandle formation is well characterized, almost nothing is known about the potential contribution of protein cofactors to viral rna cyclization. proteins with nucleic acid chaperone activities are encoded by many viruses (e.g., retroviruses, coronaviruses) to facilitate rna structural rearrangements and rna–rna interactions during the viral replicative cycle. since the core protein of flaviviruses is also endowed with potent rna chaperone activities, we decided to examine the effect of west nile virus (wnv) core on 5′–3′ genomic rna annealing in vitro. core protein binding resulted in a dramatic, dose-dependent increase in 5′–3′ complex formation. mutations introduced in either the uar (upstream aug region) or cs (conserved sequence) elements of the viral rna diminished core protein-dependent annealing, while compensatory mutations restored the 5′–3′ rna interaction. the activity responsible for stimulating rna annealing was mapped to the c-terminal rna-binding region of wnv core protein. these results indicate that core protein – besides its function in viral particle formation – might be involved in the regulation of flavivirus genomic rna cyclization, and thus virus replication. flaviviruses constitute a large and diverse group within the flaviviridae family of positive-strand enveloped rna viruses (lindenbach et al., 2007) . medically important flaviviruses include the mosquito-borne west nile virus (wnv), yellow fever virus (yfv), japanese encephalitis virus (jev), and dengue virus (denv serotypes 1-4), as well as tick-borne encephalitis viruses (tbev). infection by arthropod-borne flaviviruses is associated with significant morbidity and mortality worldwide (gould and solomon, 2008; griffin, 2011; guzman et al., 2010) . increased urbanization, intercontinental travel, failure of vector mosquito control and increasing global temperatures have collectively resulted in the recent emergence or re-emergence of mosquitoborne flaviviruses in previously disease-free areas (gould and higgs, 2009; kilpatrick, 2011) . west nile virus first appeared in the new world in 1999, causing a viral encephalitis outbreak in new york city (petersen, 2009) . since then, the virus has rapidly spread throughout the us, central and south america, reaching argentine by 2006 (petersen, 2009 ). due to the increased geographic dispersal of the asian tiger mosquito aëdes albopictus, dengue virus is currently re-emerging in southern europe (gjenero-margan et al., 2010; la ruche et al., 2010) , after almost a hundred-year hiatus (rosen, 1986) . flaviviruses are small, enveloped viruses with a single-stranded, positive sense rna genome of ∼11 kb. a single open reading frame (orf) encodes a large polyprotein precursor which is co-and post-translationally processed to yield the viral structural proteins (c-prm-e), and the non-structural proteins (ns1-ns2a-ns2b-ns3-ns4a-ns4b-ns5) directing genome replication (lindenbach et al., 2007) . the orf is flanked by a short (∼100 nt) 5 untranslated region (utr) and a longer (∼400-800 nt) 3 utr, both of which contain highly conserved rna secondary structures and rna sequences involved in the regulation of viral translation and rna replication [reviewed in (brinton, 2002; markoff, 2003) ; see fig. 1 ]. schematic representation of the linear and circular forms of wnv genomic rna. flavivirus infected cells accumulate two major rna species, the viral genomic rna and the small sfrna (subgenomic flavivirus rna). rna secondary structures were predicted by mfold (zuker, 2003) , using constraints on experimentally characterized rna structures (see references in text). the conserved rna structures and sequences include: sla-stem-loop a; uar-upstream aug region; dar-downstream aug region; cs-conserved sequence; chp-capsid region hairpin; shp-small hairpin; db1 and db2-dumbbell structures; 3 sl-3 stem-loop. 5 and 3 uar sequences are highlighted in dark red and magenta, respectively. 5 and 3 cs elements are highlighted in dark and light blue. conserved pseudoknot structures are illustrated in green. the core protein-coding sequence partly overlaps with rna secondary structures in the 5 region of the genome. note that the lower stem of 3 sl is opened in the circular rna form, facilitating binding of the viral replication complex. cyclization sequences of representative mosquito-borne flaviviruses are shown below the circular rna structure (genbank accession for west nile virus-af260968; japanese encephalitis virus-nc 001437.1; yellow fever virus-nc 002031.1; dengue virus type 1-nc 001477). (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.) the 5 utr begins with a y-shaped stem-loop (sla) (brinton and dispoto, 1988) necessary for viral replication (filomatori et al., 2006) . the initiator aug codon for polyprotein synthesis is situated in a second stem-loop structure (slb), which is followed by the capsid-coding region hairpin (chp) regulating start codon selection (clyde et al., 2008; clyde and harris, 2006) . the 3 utr consists of a variable region, followed by tandem dumbbell-like structures (db1 and db2), and a highly stable and conserved terminal stem-loop (3 sl) (brinton et al., 1986) . in addition, a complex network of pseudoknot interactions is thought to influence the topology and function of the 3 utr (funk et al., 2010; olsthoorn and bol, 2001; pijlman et al., 2008; shi et al., 1996; silva et al., 2010) . the positive-sense genomic rna of flaviviruses is first copied by the viral rna-dependent rna polymerase (rdrp) to generate a complementary minus-strand rna that, in turn, serves as a template for the amplification of plus-strand viral genomes. interestingly, in vitro polymerase assays demonstrated that minusstrand rna synthesis requires the presence of both the 5 and 3 utrs (you et al., 2001; you and padmanabhan, 1999) . the promoter element for minus-strand rna synthesis was later identified as stem-loop a (sla, fig. 1 ) in the 5 utr, which binds the viral rdrp with high affinity (filomatori et al., , 2006 lodeiro et al., 2009 ). according to current models, based mostly on work in dengue viruses, panhandle formation through complementary sequences in the 5 -core and 3 utr regions would reposition the 3 end of the genome in the vicinity of the promoter-rdrp complex, coupling transcription initiation to genome cyclization (reviewed in gebhard et al., 2011) . at the same time, the long-distance rna interaction is believed to be accompanied by structural rearrangements in the highly conserved 3 stem-loop (3 sl, fig. 1 ) , allowing rdrp binding to the terminal nucleotides of the genome (gebhard et al., 2011) . genomic rna cyclization in mosquito-borne flaviviruses is mediated by three pairs of long distance rna interactions: 5 -3 uar (upstream aug region) (alvarez et al., 2005b) , 5 -3 dar (downstream aug region) friebe and harris, 2010) , and 5 -3 cs (conserved sequence) (hahn et al., 1987) (fig. 1) . mutations disrupting complementarity in any of these elements abrogate viral replication in subgenomic replicon model systems (alvarez et al., 2008 (alvarez et al., , 2005b bredenbeek et al., 2003; corver et al., 2003; friebe and harris, 2010; friebe et al., 2011; khromykh et al., 2001; lo et al., 2003; men et al., 1996; villordo et al., 2010; zhang et al., 2008) , while compensatory mutations rescue viability (alvarez et al., 2008 (alvarez et al., , 2005b friebe and harris, 2010; friebe et al., 2011; khromykh et al., 2001; lo et al., 2003; zhang et al., 2008) . flavivirus genomic rna is encapsidated by the small, highly basic core protein. core corresponds to the n-terminus of the polyprotein, from where it is released in its mature form by the action of the ns2b-ns3 serine protease complex (amberg et al., 1994; lobigs, 1993; yamshchikov and compans, 1994) . despite relatively little sequence conservation, all flavivirus core proteins share a common functional and structural domain organization. the n-and c-terminal extremities contain a high concentration of basic amino acids, and bind to the viral genomic rna independently (khromykh and westaway, 1996) . flanked by the rna-binding regions, an internal hydrophobic domain is responsible for the dimerization/oligomerization (bhuvanakantham and ng, 2005; wang et al., 2004) , as well as for the membrane/lipid association of core protein (markoff et al., 1997; samsa et al., 2009) . structural studies by circular dichroism, x-ray crystallography and nuclear magnetic resonance (nmr) on denv, wnv, and yfv core proteins (dokland et al., 2004; ivanyi-nagy et al., 2008; jones et al., 2003; ma et al., 2004) also support a conserved structure, with a highly flexible n-terminal rna-binding region followed by three or four alpha-helices. interestingly, deletion analyses demonstrated a remarkable functional flexibility of core protein, suggesting that precisely folded three-dimensional structures are not required for rna binding and membrane association, and these functions are rather determined by the overall physicochemical nature of the domains. large deletions are well tolerated in the internal hydrophobic region of both mosquito-borne and tick-borne flaviviruses (kofler et al., 2002 (kofler et al., , 2003 schlick et al., 2009; zhu et al., 2007) . similarly, the n-and c-terminal extremities can function (at least partly) redundantly in rna packaging (patkar et al., 2007) . core proteins of wnv (ivanyi-nagy et al., 2008) , dengue virus (pong et al., 2011) , as well as cores in the related hepaciviruses (cristofari et al., 2004; ivanyi-nagy et al., 2006) and pestiviruses (ivanyi-nagy et al., 2008) , were shown to facilitate nucleic acid rearrangements without atp consumption, acting as efficient rna chaperones in vitro. the rna chaperone activity of wnv core was mapped to the c-terminal rna-binding region of the protein (ivanyi-nagy et al., 2008) . in this study, we examined the effect of wnv core protein chaperoning on viral 5 -3 utr annealing, using an in vitro model system with separate 5 and 3 rnas. we found that core protein binding greatly increases the rate of 5 -3 complex formation, and is required for the interaction when full-length 3 utr rnas are used (fig. 2) . mutations abolishing either the uar or cs interaction diminished, but did not completely abrogate core-protein induced annealing, while compensatory mutations restored the interaction (fig. 3 ). in agreement with the results of in vitro chaperone assays (ivanyi-nagy et al., 2008) , stimulation of rna annealing was mapped to the c-terminal rna-binding region of core protein (fig. 4 ). for cloning the 3 utr of west nile virus (eg101 strain, gen-bank accession af260968), total rna was extracted from virus infected vero cells and reverse transcribed using the thermoscript rt-pcr system (invitrogen). reverse transcription was carried out at 60 • c, using odn eg101-3 utr-ss as a primer (table 1) . cdna was amplified by eg101-3 utr-ss and eg101-3 utr-as and cloned between the sali and hindiii sites of psp64 (promega), resulting in psp64-3 utr vector. deletions in the 3 utr were introduced by amplifying the desired regions in psp64-3 utr by pcr, and re-cloning the fragments between the sali and hindiii sites of psp64. the 5 utr-core region (nt 1-164 of the viral genome) was cloned by annealing of overlapping odns (eg101-5 utr-01 to -05), followed by ligation between the xmai and ecori sites of psp64 vector (promega). mutations were introduced in the uar or cs region of psp64-3 utr or psp64-5 utr-core by using a pcr-based mutagenesis protocol (mikaelian and sergeant, 1992) , with three common odns (psp64, psp64-t7, and sp6) and one mutation-specific odn (mut-uar or mut-cs) for each mutant. all plasmid constructs were verified by sequencing. full length wnv core protein (amino acids 2-105; genbank accession number af481864) was expressed in escherichia coli and purified as previously described (ivanyi-nagy et al., 2008) . core peptides wnv c(1-24) and wnv c(80-105), corresponding to the n-and c-terminal rna-binding regions of the core protein, were synthesized by fmoc-oh/dcc/hobt chemistry and purified as previously described (ivanyi-nagy et al., 2008) . plasmids containing the 5 utr-core region or fragments of the 3 untranslated region of wnv strain eg101 were linearized by digestion with xmai or hindiii restriction enzymes, respectively. in vitro transcription was carried out using t7 rna polymerase, according to the manufacturer's instructions (promega). 5 utrcore rnas were labelled by incorporation of ␣ 32 -p ump during in vitro transcription. rnas were purified on 6% denaturing polyacrylamide gels containing 7 m urea in 50 mm tris-borate (ph 8.3)-1 mm edta (0.5× tbe), and recovered by elution in 0.3 m sodium acetate-0.1% sds overnight at 37 • c, followed by ethanol precipitation. in vitro synthesized rnas were heat denatured for 2 min at 95 • c and chilled on ice. 0.1 pmol of each 5 utr-core and 3 utr rna were mixed with annealing buffer to a final concentration of 20 mm tris-cl, ph 7.0, 30 mm nacl, 0.1 mm mgcl 2 , 10 m zncl 2 , fig. 2 . core protein facilitates the annealing of wnv 5 utr-core and 3 utr rnas. (a) position and secondary structure of rna molecules used in this study. (b) core protein enhances 5 -3 wnv rna interactions. radioactively labelled rna 1-164 was incubated with the full-length 3 fl-utr rna (lanes 1-5), 3 vr rna (lanes 6-10), or 3 cyc rna (lanes 11-15) in the presence of increasing concentrations of full-length wnv core protein, as described in section 2. protein-to-nucleotide molar ratios were 1/40 (lanes 2, 7, 12), 1/20 (lanes 3, 8, 13), 1/10 (lanes 4, 9, 14), or 1/5 (lanes 5, 10, 15). lanes 1, 6, and 11 show rna annealing in the absence of core protein. (c) kinetics of 5 -3 rna annealing without core protein. radioactively labelled rna 1-164 was incubated with 3 fl-utr rna, 3 vr rna, or 3 cyc rna at 37 • c for different length of time, as indicated below the lanes. (d) kinetics of 5 -3 rna annealing in the presence of core protein. the experiment was carried out the same way as in (c), but a constant 1 protein/5 nucleotide molar ratio of full-length core protein was added to the rna molecules. (e) quantification of the annealing reactions shown in (c) and (d). annealing curves were fitted using graphpad prism © , as described in doetsch et al. (2011). 10 u rnasin and 5 mm dtt in 10 l final volume. wnv core protein or core peptides were added to final protein to rna nucleotide molar ratios as indicated in the figure legends (typically between 1/40 and 1/5 protein/nucleotide ratios). reactions were incubated at 37 • c for 10-15 min and quenched by adding stop solution (0.5% sds-25 mm edta). proteins were removed by proteinase k digestion and phenol-chloroform extraction. the purified rna samples were resolved by 8% native polyacrylamide gel electrophoresis in 0.5× tbe and analysed by autoradiography and phosphorimager quantification. cyclization of the genomic rna is essential for viral replication in all mosquito-borne flaviviruses (alvarez et al., 2008 (alvarez et al., , 2005b corver et al., 2003; khromykh et al., 2001; lo et al., 2003; zhang et al., 2008) , but the determinants and regulation of panhandle formation are still poorly understood. although complex formation between 5 and 3 utr rnas can be readily detected in vitro in the absence of protein cofactors (alvarez et al., 2008 (alvarez et al., , 2005b villordo et al., 2010; zhang et al., 2008 zhang et al., , 2010 , these interactions usually require high magnesium and rna concentrations and involve short rna molecules, thus minimizing the possibility for the rna to become kinetically trapped in non-functional conformation(s). but in the cellular milieu, rna folding and rna-rna interactions are perhaps universally facilitated by rna chaperones and/or specific rna-binding proteins (cristofari and darlix, 2002) , either encoded by viruses or hijacked from the host for chaperoning viral translation, replication, and packaging. we have previously shown that the core protein of wnv possesses potent rna chaperone activities in vitro (ivanyi-nagy et al., 2008) , facilitating nucleic acid annealing and rna structural rearrangements. in order to analyse the possible effect of core protein on panhandle formation in the wnv genome, rna molecules corresponding to nucleotides 1-164 of the genomic rna (rna 1-164; fig. 2a ) were in vitro synthesized, radioactively labelled and incubated with equal amounts of non-labelled 3 fl-utr rna ( fig. 2a) in the presence of varying amounts of full-length wnv core protein. following proteinase k digestion of proteins and phenol-chloroform purification of rnas, 5 -3 rna complex formation was assessed by electrophoretic mobility shift assays (fig. 2b, lanes 1-5) . wnv core induced a dose-dependent increase in rna-rna interactions, resulting in almost complete annealing at 1 protein to 5 nt molar ratio (fig. 2b , lane 5; compared to lane 1 in the absence of core protein). in order to examine the potential effect of intramolecular interactions on annealing in the 3 utr, 5 deleted 3 utr rnas, lacking either the variable region (3 vr rna), or most of the utr except for the cyclization sequences and the 3 stem-loop (3 cyc rna) were examined (fig. 2b, lanes 6-10 and 11-15, respectively) . for all rna molecules, incubation with core protein induced efficient annealing in a dose-dependent manner (fig. 2b, lanes 7-10 and 12-15) . interestingly, core protein-independent annealing was more pronounced for the shortest 3 cyc rna (lane 11 vs lanes 6 and 1), suggesting that rna sequences 5 to the cyclization signals might change the topology of the 3 utr and interfere with the annealing reaction. to further characterize these differences, a time-course analysis of annealing, either without core protein (fig. 2c) , or in the presence of full-length core (fig. 2d ) was carried out. without core protein, annealing for the 3 fl-utr rna and 3 vr rna was hardly detectable during the 1 h incubation period, while for the shortest 3 cyc rna, around one third of the molecules formed 5 -3 rna interaction ( fig. 2c and e) . in contrast, core protein dramatically increased complex formation, resulting in high levels of annealing as early as 30 s (fig. 2d and e). indeed, based on second-order kinetics, the initial annealing rate was estimated to increase by ∼500-fold in the presence of core (data not shown). nevertheless, annealing of the longer rnas was still significantly delayed compared to 3 cyc rna, the sequence of wnv c(1-24) and wnv c(80-105) peptides is indicated below the rna-binding domains. the 3d structure of the wnv core dimer, determined by x-ray crystallography, is shown to the right (based on pdb entry 1sfk (dokland et al., 2004) , rendered by jmol). (b) radioactively labelled rna 1-164 was incubated with 3 fl-utr rna in the presence of increasing concentrations of full-length wnv core (lanes 2-6), wnv c(1-24) (lanes 8-12), or wnv c(80-105) (lanes 14-18). protein-to-nucleotide molar ratios were 1/40 (lanes 2, 8, 14), 1/20 (lanes 3, 9, 15), 1/10 (lanes 4, 10, 16), 1/5 (lanes 5, 11, 17), and 1/2.5 (lanes 6, 12, 18). lanes 1, 7, and 13 show rna annealing in the absence of core protein. suggesting that a kinetic barrier must be overcome for the reaction to proceed. phylogenetic analyses suggest that the current discontinuous cyclization sequence of mosquito-borne flaviviruses originatedpossibly by template switching -as a perfectly complementary continuous region, conserved to this day in the tick-borne members of the flavivirus genus (gritsun and gould, 2007) . thus, the uar-dar-cs region probably acts as a single regulatory sequence, where replication is determined by the overall stability of the long distance interaction between the 5 and 3 regions. in support of this, replication of wnv with a complete deletion of the 3 cs can be rescued by second-site mutations stabilizing the uar and dar interactions (zhang et al., 2010) . in order to examine whether the core protein-dependent annealing of the 5 and 3 utr rnas depends on the uar and/or cs interaction, mutations were introduced separately in these elements of rna 1-164 (fig. 3a) , and cyclization with wild-type 3 fl-utr rna was assessed by electrophoretic mobility shift assays. mutations in either the 5 uar or 5 cs led to a significant decrease in annealing (fig. 3b, lanes 6-10 and 11-15 ), while cyclization was rescued by compensatory mutations introduced in the 3 uar or 3 cs of the 3 fl-utr rna (lanes 16-20 and 21-25, respectively). these results suggest that the core protein-dependent in vitro annealing recapitulates the features of the cyclization sequence analysed in subgenomic or genome-length cellular models. interestingly in vitro rna annealing was readily detectable even with 5 mutations at high core protein levels, while even single point mutations result in a lethal phenotype in subgenomic replicons. this suggests that chaperoning by core protein may stabilize the weak interaction present in the mutants, thus partially relaxing the complementarity requirements of the long distance interaction (basu and brinton, 2011; zhang et al., 2010; see section 4). flavivirus core proteins have two independent rna-binding regions at the n-and c-terminal extremities of the protein (khromykh and westaway, 1996; fig. 4a) . we have previously shown that the two rna-binding domains do not act in synergy and that only the c-terminal basic region was active in in vitro nucleic acid chaperone assays (ivanyi-nagy et al., 2008) . in order to delineate the requirements of core protein for facilitating 5 -3 utr interaction, synthetic peptides corresponding to the n-and c-terminal rna-binding regions (wnv c(1-24) and wnv c(80-105), respectively; fig. 4a ) were used in the annealing reactions with wild-type rnas. while wnv c(1-24) did not influence 5 -3 annealing (fig. 4b, lanes 7-12) , wnv c(80-105) induced an increase in 5 -3 complex formation (fig. 4b, lanes 13-18) . nevertheless, at equal molar ratios, incubation with full-length core protein resulted in higher levels of complex formation than with wnv c(80-105), indicating that sequences or structural features outside the c-terminal region contribute to full chaperoning activity. complementarity between the genome-terminal cyclization sequences, presumably resulting in panhandle formation in vivo, is absolutely required for flavivirus rna replication (khromykh et al., 2001) . although formation of the long-distance interaction is believed to be thermodynamically favoured, the annealing reaction is prohibitively slow (or may not even reach completion) under physiologically relevant conditions (fig. 2) . rna chaperones are able to disrupt transient or non-functional rna interactions, table 1 oligonucleotides used in this study. sense sequence thereby decreasing the kinetic barrier hindering the formation of the most stable rna conformation (reviewed in cristofari and darlix, 2002; schroeder et al., 2004) . indeed, rna chaperoning might increase the speed of intramolecular rna rearrangements or intermolecular rna-rna interactions several thousand-fold without atp consumption. we have previously shown that core proteins in the flaviviridae family, including that of wnv, hepatitis c virus and bovine viral diarrhoea virus, possess potent rna chaperone activities (cristofari et al., 2004; ivanyi-nagy et al., 2006 , facilitating nucleic acid annealing reactions with various dna and rna substrates. the molecular mechanism of chaperone action is still poorly understood, and it probably involves a combination of charge neutralization, molecular crowding, and local melting of nucleic acid structures by entropy transfer (tompa and csermely, 2004) . high affinity binding of a protein to the substrate dna/rna is not sufficient, in itself, to trigger the conformational changes observed upon core protein chaperoning (cristofari et al., 2004; ivanyi-nagy et al., 2008) . in this study, we analysed the rna chaperone activity of wnv core using its cognate target molecules, corresponding to the highly structured terminal regions of the viral genomic rna. wnv core protein was found to induce a dramatic acceleration in the 5 -3 utr annealing reaction (figs. 2-4) , suggesting a possible link between genome cyclization, replication, and packaging. paradoxically, with the exception of a short n-terminal sequence which contains the 5 cs rna element, the structural protein-coding region is not essential for wnv genomic rna replication (khromykh and westaway, 1997) . the discrepancy between the in vitro activity of core protein and its in vivo dispensability suggests that flaviviruses might hijack cellular rna-binding proteins that may compensate for the loss of core chaperoning. indeed, an rna-protein-rna co-immunoprecipitation assay identified seven distinct proteins (with molecular weights of 35, 37, 40, 45, 52, 76, and 97 kda) interacting with both the 5 utr and 3 utr rnas of dengue virus (garcia-montalvo et al., 2004) . although most of these cellular proteins remain to be identified and their interaction with the genomic rna verified, the 52 kda band was confirmed as being la protein, an abundant cellular rna chaperone (belisova et al., 2005; chakshusmathi et al., 2003) . la protein was shown to interact with the utrs of dengue virus and japanese encephalitis virus (de nova-ocampo et al., 2002; garcia-montalvo et al., 2004; vashist et al., 2009 vashist et al., , 2011 yocupicio-monroy et al., 2007) , and to bind to the viral replication proteins ns5 and ns3 (garcia-montalvo et al., 2004) . at present, the effect of la binding on genomic rna circularization is still controversial. recombinant la protein led to decreased rna synthesis in an in vitro viral replicase assay, leading to the suggestion that its binding inhibited rna cyclization (yocupicio-monroy et al., 2007) . however, co-precipitation of the 5 and 3 genomic rna regions was stimulated by the presence of increasing amounts of la protein (vashist et al., 2011) . a possible explanation for these seemingly contradictory findings might be provided by the results of villordo and co-workers, who have shown that a balance between the linear and circular conformations of the genomic rna, rather than cyclization per se, is important for efficient viral replication (villordo et al., 2010) . thus, overly efficient rna cyclization, as well as the lack of it, might both be deleterious for rna synthesis. besides la protein, a number of other cellular rna chaperones, including heterogeneous nuclear ribonucleoprotein a1 (hnrnp a1), hnrnp a2, hnrnp q, y-box binding protein (yb-1), and polypyrimidine tract binding protein (ptb), have been described to interact with flaviviral genomic rna (agis-juarez et al., 2009; de nova-ocampo et al., 2002; katoh et al., 2011; paranjape and harris, 2007) . although the effect of these chaperones on genome cyclization is currently unknown, they might participate in the regulation of panhandle formation in the absence of (or in addition to) core protein, thus masking the in vivo effect of core on genome replication. nevertheless, recent mutagenesis studies have provided indirect evidence for the involvement of the core protein region in genome circularization and rna replication (basu and brinton, 2011; zhang et al., 2010) . genome-length viral rnas with multiple adjacent mutations in cs or with a complete deletion of the 3 cs element were replication competent and generated revertants or second-site mutations upon passaging, restoring efficient panhandle formation (basu and brinton, 2011; zhang et al., 2010) . in contrast, disruption of the 5 -3 cs interaction invariably resulted in a lethal phenotype when analysed in subgenomic replicons lacking core protein (corver et al., 2003; khromykh et al., 2001; lo et al., 2003) . similarly, a deletion in the core coding region of a genome-length replicon also precluded the rescue of the cs mutant (zhang et al., 2010) , although it is still debated whether this was due to the lack of core protein expression or to the absence of rna secondary structures in the core-coding region (friebe et al., 2012) . these results, together with our findings (fig. 3b) , suggest that cellular proteins can only partially substitute for core protein chaperoning in genome cyclization, and the requirements for panhandle formation might be more relaxed in the presence of core. this is especially important since mutations in the cyclization sequences are considered for the design of liveattenuated flavivirus vaccines, with the aim to reduce the risk of recombination with naturally circulating viruses (suzuki et al., 2008) . the annealing efficiency of 3 utr rnas was found to be highly dependent on the length of the rna used (fig. 2) . while 3 cyc rna, containing the cyclization sequences and the 3 sl structure, could interact with the 5 region even in the absence of core protein, the longer 3 vr and 3 fl-utr rnas required core for 5 -3 complex formation, suggesting that the presence of the dumbbell structures (db1 and db2) might interfere with annealing. pseudoknot interactions, conserved in mosquito-borne flaviviruses, have been suggested to form between the loop of the dumbbells and a single-stranded region, including the cs element, in the linear form of the viral rna (olsthoorn and bol, 2001; figs. 1 and 2) . total or partial deletion of the individual dumbbell structures was found to seriously compromise rna replication (alvarez et al., 2005a; manzano et al., 2011; men et al., 1996) , resulting in attenuated viruses that are actively pursued as a vaccine candidate for dengue viruses troyer et al., 2001; whitehead et al., 2003) . our results suggest that core protein chaperoning might be required to resolve the pseudoknots, yielding an rna conformation competent for 5 -3 annealing. thus, the pseudoknot structures may constitute an additional layer of regulation in genome cyclization, contributing to the delicate balance between the linear and circular rna forms. in addition to the genomic rna, a small subgenomic rna (sfrna), corresponding to the last 300-500 nts of the positivestrand viral rna, also accumulates in cells infected by flaviviruses (lin et al., 2004; pijlman et al., 2008; urosevic et al., 1997) . sfrna is generated by the incomplete degradation of the viral genome by the host exonuclease xrn1, where the 3 rna region is protected by a conserved pseudoknot structure (funk et al., 2010; pijlman et al., 2008; silva et al., 2010) . kunjin virus sfrna was not required for viral replication but was found to be essential for cytopathicity in cell culture and for pathogenicity in infected mice (pijlman et al., 2008) . 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structures in the 3 non-coding region of japanese encephalitis virus genome: role in virus replication la protein can simultaneously bind to both 3 -and 5 -noncoding regions of japanese encephalitis virus genome a balance between circular and linear forms of the dengue virus genome is crucial for viral replication identification of the homotypic interaction domain of the core protein of dengue virus type 2 a live, attenuated dengue virus type 1 vaccine candidate with a 30-nucleotide deletion in the 3 untranslated region is highly attenuated and immunogenic in monkeys processing of the intracellular form of the west nile virus capsid protein by the viral ns2b-ns3 protease: an in vitro study mosquito la protein binds to the 3 untranslated region of the positive and negative polarity dengue virus rnas and relocates to the cytoplasm of infected cells in vitro rna synthesis from exogenous dengue viral rna templates requires long range interactions between 5 -and 3 -terminal regions that influence rna structure a novel in vitro replication system for dengue virus. initiation of rna synthesis at the 3 -end of exogenous viral rna templates requires 5 -and 3 -terminal complementary sequence motifs of the viral rna west nile virus genome cyclization and rna replication require two pairs of long-distance rna interactions genetic analysis of west nile virus containing a complete 3 csi rna deletion attenuated dengue 2 viruses with deletions in capsid protein derived from an infectious full-length cdna clone mfold web server for nucleic acid folding and hybridization prediction work was supported by anrs, inserm and finovi. thanks are due to hervé zeller for providing wnv infected cells used to clone the 5 and 3 regions of the wnv genome. key: cord-267363-5qri915n authors: shi, mang; zhang, yong-zhen; holmes, edward c. title: meta-transcriptomics and the evolutionary biology of rna viruses date: 2018-01-02 journal: virus res doi: 10.1016/j.virusres.2017.10.016 sha: doc_id: 267363 cord_uid: 5qri915n metagenomics is transforming the study of virus evolution, allowing the full assemblage of virus genomes within a host sample to be determined rapidly and cheaply. the genomic analysis of complete transcriptomes, so-called meta-transcriptomics, is providing a particularly rich source of data on the global diversity of rna viruses and their evolutionary history. herein we review some of the insights that meta-transcriptomics has provided on the fundamental patterns and processes of virus evolution, with a focus on the recent discovery of a multitude of novel invertebrate viruses. in particular, meta-transcriptomics shows that the rna virus world is more fluid than previously realized, with relatively frequent changes in genome length and structure. as well as having a transformative impact on studies of virus evolution, meta-transcriptomics presents major new challenges for virus classification, with the greater sampling of host taxa now filling many of the gaps on virus phylogenies that were previously used to define taxonomic groups. given that most viruses in the future will likely be characterized using metagenomics approaches, and that we have evidently only sampled a tiny fraction of the total virosphere, we suggest that proposals for virus classification pay careful attention to the wonders unearthed in this new age of virus discovery. metagenomics is transforming the study of virus evolution, allowing the full assemblage of virus genomes within a host sample to be determined rapidly and cheaply. the genomic analysis of complete transcriptomes, so-called meta-transcriptomics, is providing a particularly rich source of data on the global diversity of rna viruses and their evolutionary history. herein we review some of the insights that meta-transcriptomics has provided on the fundamental patterns and processes of virus evolution, with a focus on the recent discovery of a multitude of novel invertebrate viruses. in particular, meta-transcriptomics shows that the rna virus world is more fluid than previously realized, with relatively frequent changes in genome length and structure. as well as having a transformative impact on studies of virus evolution, meta-transcriptomics presents major new challenges for virus classification, with the greater sampling of host taxa now filling many of the gaps on virus phylogenies that were previously used to define taxonomic groups. given that most viruses in the future will likely be characterized using metagenomics approaches, and that we have evidently only sampled a tiny fraction of the total virosphere, we suggest that proposals for virus classification pay careful attention to the wonders unearthed in this new age of virus discovery. our knowledge of the virosphere is scant. although viruses are the most abundant source of nucleic acid on earth, with every species of cellular life likely harboring multiple viruses, until recently most studies of virus biodiversity and evolution were of limited scope, with a strong focus on aquatic environments and prokaryotic dna viruses (angly et al., 2006; culley et al., 2006; desnues et al., 2008; paez-espino et al., 2016; philosof et al., 2017) . far less is known about the diversity of rna viruses in terrestrial organisms. this has begun to change following advances in bulk genome sequencing that have initiated a new age of virus discovery, in which it is now possible to rapidly document the entire virome of groups of host organisms (li et al., 2015; shi et al., 2016a) . as well as greatly expanding our knowledge of virus diversity, including the 'dark matter' of highly divergent viruses that often elude characterization, these new data will enable us to determine the fundamental evolutionary and ecological processes that shape the virosphere, and better understand the virus-host interactions that lead to disease emergence. it is also clear that the virus diversity generated from these genomic studies will radically shake-up attempts to classify the virus world (simmonds et al., 2017a) . one genomic technique that is already having a major impact on studies of virus diversity and evolution is rna-seqa whole transcriptome shotgun sequencing approach that enables enormous amounts of rna sequence to be generated rapidly (palacios et al., 2008; we describe the technique in more detail below). as the transcriptome data generated by rna-seq is able to provide an unbiased and likely comprehensive view of all the viruses present within a host samplethat is, their complete viromeit can also be thought of as 'metatranscriptomics'. the data generated by meta-transcriptomics is a rich source of evolutionary and ecological information. as a case in point, meta-transcriptomic studies of invertebrates have unearthed remarkable levels of untapped virus genetic diversity, such that the virosphere is evidently far broader and more complex than previously anticipated (li et al., 2015; shi et al., 2016a; webster et al., 2015) . for example, an analysis of 220 species from nine invertebrate phyla identified a remarkable 1445 novel rna viruses, as well as potentially novel genera and families (or orders) (shi et al., 2016a) . aside from its evolutionary utility which we will discuss in more detail below, meta-transcriptomics allows the identification of novel microbial pathogensthat is, those associated with overt disease in their hostson clinically actionable time-scales (wilson et al., 2014) . indeed, it is possible that with a continually declining cost meta-transcriptomics may eventually be used for routine microbiological diagnostics. a key advantage of this over other diagnostic techniques is that it has the potential to detect, in an unbiased fashion, any pathogen that produces an rna molecule (dna viruses, bacteria, fungi, eukaryotes) , as well as the obvious case of rna viruses. hence, if appropriate tissues are analyzed meta-transcriptomics may provide a one stop diagnostic shop. as much as metagenomics is transforming studies of virus evolution, it is also the case that it has shone a bright light on fundamental gaps in our understanding of the virus world. most obviously, it is evident that we have only just begun to scratch the surface of the true diversity of viruses that make up the virosphere, and the factors that shape this diversity and evolution within ecosystems and over long-term evolutionary scales are largely unknown. herein, we will review what, in our opinion, meta-transcriptomics has told us about virus diversity, evolution and taxonomy, and provide some suggestions for future work in this area. before the advent of dna sequencing, new viruses were discovered using a variety of approaches, including filtration, cell culture, electron microscopy, and serology. many of these techniques remain important in virology (leland and ginocchio, 2007) . indeed, the propagation of viruses in cells, accompanied by the visualization of virus particles by electron microscopy and the successful replication of infection in animal models, can still be considered the gold standard for virus discovery. however, the substantial time and effort required for work of this kind means that it is often impossible. in addition, most viruses are not culturable and there are not enough cell lines to meet the diversity of viruses. more modern approaches of virus discovery involve the determination and comparison of viral nucleic acids. this combination of pcr and sequencing can be used to screen for infectious agents using degenerative primers targeting conserved genomic regions, thereby identifying novel, but related, viruses with great sensitivity. this approach has been very successful in virus discovery, with notable examples including bat influenza a virus (tong et al., 2012) and rodent hepaciviruses (drexler et al., 2013) . however, the drawback of consensus pcr is that it is heavily dependent on currently available sequences and hence has limited capability to detect more divergent viruses. it can also be tedious to design and run consensus pcr for a large number of different virus families. the most robust, although costly, method of virus discovery is through a coupling of metagenomics and high-throughput sequencing technology. indeed, metagenomics provides an unbiased survey of the genetic material within a sample, and has revolutionized virus discovery in terms of speed, accuracy, sensitivity, and the amount of information generated (firth and lipkin, 2013) . among the various metagenomics approaches are available, meta-transcriptomics has recently come to the fore. this approach involves gathering total transcriptome information from a host sample after depletion of ribosomal (r) rna, as this is the dominant component of the host transcriptome. compared to metagenomics protocols that involve viral particle enrichment (reviewed in kumar et al., 2017) , this method is far simpler yet still achieves a high level of sensitivity, generality, and efficiency for virus discovery (fig. 1) . previous methodologies were often based on removing as much nucleic acid outside viral particles as possible by filtering, centrifugation, lysis, and nuclease treatment, although this seldom results in a complete depletion of host rna (firth and lipkin, 2013; mokili et al., 2012) . in contrast, in meta-transcriptomics total rna (i.e. the transcriptome) is directly extracted from untreated homogenates and used for library preparation without filtering and nuclease digestion steps. another benefit of meta-transcriptomics is that it provides a ready way to quantify each virus present in a sample. specifically, the percentage of reads that map to a particular virus genome is a good indication of how abundant any virus is, especially in the context of conserved host genes (shi et al., 2016a; shi et al., 2017) . in turn, abundance level can provide important pointers to disease associations, whether viruses are segmented (such that genomic components have similar or different expression levels), and help identify those viruses that are in fact derived from other eukaryotic organisms present in the host sampled, such as in undigested food or prey, gut micro flora, and parasites, or simply contamination (and the greater the virus abundance, the more likely that active viral infection has occurred in the host under consideration). in addition, compared to genomic nucleic acid, the transcriptome comprises compact information that is more balanced across domains of life, thereby preventing the over-dominance of genetic information from large cellular organisms. those meta-transcriptomic studies undertaken to date have transformed our understanding of the extent and nature of viral biodiversity, making it abundantly clear that we have only sampled a tiny fraction of rna virus biodiversity (as will also be true of dna viruses). indeed, it is likely that the diversity of uncharacterized viruses far exceeds that of those that have been classified to date (fig. 2) . these studies also highlight the inherent bias toward studying viruses that can be cultured, or associated with overt disease, which in turn reflects a longerstanding historical preference to studying viral infections in humans and economically important plants and animals. as is discussed in more detail below, it is possible that such highly biased sampling has distorted our view of virus evolution. what is perhaps more daunting is that these studies have only been conducted in a small number of sampling locations, often in china. it is therefore simple to predict that we will identify a legion of new viruses in the near future, especially given that only a minuscule fraction of the perhaps eight million eukaryotic species (many of which are marine) have ever been sampled for viruses. indeed, it was recently estimated that approximately 99.995% of the eukaryotic virosphere remains undiscovered or unclassified . the reality, therefore, is that our study of virus diversity and evolution, and hence taxonomy, has only just begun. a powerful example of how meta-transcriptomics is changing our understanding of virus diversity was the discovery of chuviruses in 2015 (li et al., 2015) , that have recently and rapidly been accepted as a new family of negative-sense rna viruses by the international committee on taxonomy of viruses (ictv). although the chuviruses form a monophyletic group in phylogenetic trees of the rna-dependent rna polymerase (rdrp), they contain a diverse array of genome structures, including both segmented and unsegmented representatives, as well as a potentially circular form that would be unique among rna viruses. it is highly likely that similarly diverse new families will be identified in the future. there are also huge differences between the diversity revealed by previous culturing and pcr-base methods and by metagenomics, again highlighting the biases that detection method may have introduced into our understanding of natural viromes. for example, considerable effort has been directed toward isolating and culturing mosquito viruses that are relevant to humans, such as flaviviruses, alphaviruses and orthobunyaviruses. in reality, however, these disease agents represent a tiny fraction of the mosquito virome (hall et al., 2017; junglen and drosten, 2013; vasilakis and tesh, 2015) , which in fact comprises representatives from every major virus group, that are more prevalent in the mosquito population, have much higher abundance, and are often transmitted vertically (cook et al., 2013; vasilakis and tesh, 2015; shi et al., 2017) . the new wealth of diversity revealed by meta-transcriptomics also shows that the virus world is far more connected than we previously thought. new broad-scale rdrp phylogenies have shown that virus families, orders, floating genera, and undefined lineages can often be amalgamated into larger groups, such that they exhibit an evolutionary continuity (shi et al., 2016a) , in turn providing compelling evidence for their common origin (koonin et al., 2015) . it is obvious that the increasing number of newly described viruses from diverse hosts will continue to fill 'gaps' in phylogenetic diversity (i.e. the long branches present in inter-virus phylogenies) resulting in a more robust and stable depiction of virus evolutionary history. it is now clear that invertebrates carry a huge diversity of rna viruses, including the potential ancestors of many those viruses found in vertebrates (junglen and drosten, 2013; li et al., 2015; marklewitz et al., 2015; nga et al., 2011; shi et al., 2016a; webster et al., 2015) . given their vast diversity, abundance and often huge population sizes, it is no surprise that invertebrates harbor such a high number and diversity of rna viruses. although they are the most sampled group, arthropods may be especially important in this evolutionary arena because of their strong ecological relationship with both plants and vertebrates, and a phylogenetic mix between these taxa is becoming increasingly apparent (li et al., 2015; shi et al., 2016a) . what is far less clear is how frequently this huge array of invertebrate viruses is associated with overt disease in their hosts and, if invertebrates are largely refractory to disease, how this is mediated. the orthomyxo-like viruses provide an informative example of how the sampling of invertebrate viruses has changed our perspective on virus evolution. prior to 2015 the orthomyxoviruses comprised a small group of vertebrate (mammal and bird) and tick-associated rna viruses that were best known through influenza virus and classified into five genera (allison et al., 2015; presti et al., 2009) . however, subsequent studies have revealed a remarkable diversity of orthomyxo-like viruses in invertebrates, including mosquitoes, cockroaches and earthworms, that fell both basal to, and interleaved among, the previously known genera on phylogenetic trees (li et al., 2015) . hence, the gaps on the tree have been dramatically filled and the previous genera no long appear as phylogenetically distinct groups. in addition, that all orthomyxo-like viruses currently sampled are segmented shows that this form of genome organization is an ancient innovation in this group. despite the recent dramatic expansion in the number of invertebrate viruses, it is striking that some families rna viruses remain vertebratespecific and contain no invertebrate viruses, with the arenaviridae, paramyxoviridae and picornaviridae providing important examples. clearly, the monophyletic nature of vertebrate-specific viruses implies that have had a long-term evolutionary association with vertebrate hosts. also, although some invertebrate viruses appear basal to vertebrate viruses, the distance between them are often substantial and phylogenetic relationships are not always stable. therefore, while it is tempting to conclude that most, if not all, families of vertebrate viruses will have their ultimate ancestry with invertebrates, particularly as so very few of the latter have been sampled, it would be wrong to think that this a forgone conclusion. determining the host range of viruses is essential to understanding the process of cross-species transmission that underpins disease emergence. meta-transcriptomic data provide a ready means to determine what viruses are present in which hosts and allows a simple measure of virus abundance. equally important is that the meta-transcriptomic sampling of an increasing number and diverse set of hosts has fundamentally changed the view of the host structure of major virus groups. fig. 1 . comparisons of virus enrichment and meta-transcriptomics approaches for rna virus discovery. the workflow of a typical virus enrichment approach is marked in blue, whereas that of a metatranscriptomics approach is marked in red. before the metagenomics revolution the virus diversity within a specific family was often dominated by particular host groups; so, for example, vertebrate, insect, and plant viruses often fell into distinct taxonomic groups. this has changed dramatically with meta-transcriptomics. for example, the family totiviridae, previously thought to be largely associated with fungi, are now commonly found in metazoa. similarly, some previously defined families of plant viruses, such as the tombusviridae and luteoviridae, have expanded to include viruses from arthropods, nematodes, molluscs, and protists (shi et al., 2016a) . given such a complexity of host structure, combined with still very sparse sampling, it is dangerous to construct detailed ancestor-descendant relationships on the currently available data. for example, arthropods were initially proposed to be the ancestral hosts of bunyaviruses (marklewitz et al., 2015) , although more divergent viruses in this group have now been discovered in other invertebrates, fungi, and protists (akopyants et al., 2016; shi et al., 2016a) . the combination of meta-transcriptomics and phylogenetics has also told us that virus evolution is a complex interaction between cross-species transmission and virus-host co-divergence, with the evolutionary history of many virus groups reflecting an interweaving of both processes . however, given their complexity and the often great genetic distances between virus genomes, determining the precise sequence of cross-species transmission and codivergence events that have shaped the evolutionary history of a particular group will undoubtedly be challenging and require a denser sampling of host taxa. indeed, the greater diversity of hosts sampled, the more cases of species jumping we are likely to document . although the occurrence of virus-host co-divergence has long been suggested, meta-transcriptomic-based studies indicate that this may extend even further back in time than previously suspected. for example, one interpretation of the evolutionary relationships within the narna-levi clade of rna viruses is that there has been virus-host co-divergence since the î±-proteobacteria became endosymbionts (shi et al., 2016a) . at the same time, however, it is clear that cross-species transmission has occurred frequently, even among phylogenetically divergent taxa, and is likely the dominant mode of fig. 2 . current taxonomy of rna viruses in the context of the genetic diversity revealed by meta-transcriptomics. the phylogenies are based on rdrp amino acid sequences from a broader analysis as performed by shi et al. (2016a,b) (and see this paper for a description of branch lengths and rooting schemes). the taxonomic groups (i.e. genus, family, and order) established by ictv are shown to the left of each phylogeny. finally, although meta-transcriptomics has profound implications for our understanding of virus evolution, it likely undermines biodiversity-based attempts to predict the virus source of the next major disease pandemic (olival et al., 2017) . although the bulk sequencing of potential animal reservoir species as been proposed as a way to better predict of what types of virus may emerge in human populations in the future, and where this may occur, in reality disease emergence is a nuanced process that entails a complex interaction of ecological and genetic factors (parrish et al., 2008; plowright et al., 2017) . metatranscriptomics tells us that there are so many viruses in nature that trying to establish which will ultimately appear in a new host from diversity sampling alone is almost certainly a futile exercise. this is apparent in the current vogue to study bat viruses. since the emergence of sars coronavirus in humansa pathogen that has its ultimate ancestry in batssampling bat viruses as a means to determine which next might emerge in humans has received considerable attention (smith and wang, 2013) . while these studies have made it clear that bats indeed harbor an enormous number of viruses (anthony et al., 2017; luis et al., 2013; olival et al., 2017) , at the same time they clearly show that the vast majority of these viruses have not jumped to humans. the true goal of studies of disease emergence should therefore be to reveal that combination of genetic and ecological factors that underpins successful cross-species transmission and emergence. one of the most important impacts of metagenomic data has been to change our understanding of the structure of virus genomes and the evolutionary processes that have given rise to them. suffice to say, rna virus genomes are more diverse, have more complex structures, and a wider range of lengths than previously anticipated. although the reasons for this diversity and the birth of individual genes are uncertain, one process of undoubted importance is inter-specific recombination, including lateral gene transfer (krupovic et al., 2012) . this evidently occurs more frequently than previously anticipated, and can involve both structural and non-structural genes, with even evidence that cellular genes can be integrated into viral genomes (shi et al., 2016a) . indeed, an emerging view is that rna viruses experience as complex processes of genome evolution as in dna organisms. to better determine the evolutionary processes that shape viral genome structures, and hence how new viruses are created, it is important to use the new wealth of meta-transcriptomic data to carefully determine the frequency, pattern and history of gene duplications and losses, lateral gene transfers, and genomic rearrangements; combined, these will provide a more complete picture of genome-scale evolutionary processes obtained. another component of rna virus genome organization that has proven more fluid than previously envisioned is segmentation. families of rna viruses were generally thought to be characterized by a specific segmentation type, such as the presence/absence of segmented genomes or certain number of segments. however, segmentation no longer appears to be a strong taxon defining trait, and a combination of segmented and unsegmented genomes has now been observed within families of rna viruses. an informative example is presented by the flaviviridae and their relativesthe so-called 'flavi-like' viruses. traditionally, flaviviruses were considered to be small (â�¼10 kb) unsegmented positive-sense rna viruses that infected vertebrates; if invertebrates were involved then it was as vectors of these viruses among vertebrates, particularly mosquitoes and ticks (simmonds et al., 2017b) . meta-transcriptomic studies have radically changed this view, including the identification of a large number of 'insect-specific' flaviviruses (bolling et al., 2015; may et al., 2013; qin et al., 2014; shi et al., 2016b) . indeed, flavi-like viruses now appear to be a group of predominantly invertebrate rna viruses with the potential to have very large genomes (â�¼26 kb) and which can be arranged in four or five segments (ladner et al., 2016; qin et al., 2014) . even more dramatic is that some of these flavi-like viruses appear to comprise distinct virus particles such that they are multipartite, a form of genome organization that was previously thought to be the exclusive domain of plant rna viruses (ladner et al., 2016) . despite such a data revolution, one key feature of rna virus genomes that has held firm in the metagenomics revolution is an upperlimit on genome length of < 35 kb, with ball python nidovirus exhibiting the largest rna virus genome reported to dateat 33.5 kb (stenglein et al., 2014) . although there is still debate as to the cause of this size limit, it is tempting to think that it reflects the high rate of rna virus evolution and the mutational burden this entails, particularly since single-stranded dna viruses, that also mutate rapidly, similarly possess small genomes (holmes, 2009) . of course, it is possible that the length profiles of viruses will radically change with increased sampling, and an rna virus with the length and complexity of a large doublestrand dna virus stands represents something of a virological holy grail. the lessons learned from evolutionary studies of meta-transcriptomic data clearly have important implications for rna virus taxonomy and classification, and we will consider some of these here. most obviously, that the virosphere is vast and we have only searched a tiny fraction of it leads us to believe that the 'traditional' way to perform virus taxonomy is dead. given the huge number of viruses that exist in nature , it is both practically impossible and inherently pointless to isolate of all these, determine their structure, and measure their ability of replicate in cells of different types. indeed, there is now a growing recognition that the primary way in which viruses will be characterized in the future will be through metagenomic surveys (simmonds et al., 2017a) , with complete 'classical' virological investigations only being performed on that subset of viruses that may be of special interest or that can be considered as markers of specific groups. metagenomics has already revealed the challenges facing current virus classification, with increased sampling challenging the criteria proposed to define many groups (simmonds et al., 2017a) . a key issue is that the genome structures that have been used as criteria for classification, such as segmentation and orf arrangement, are no longer 'conservative' enough over broad evolutionary timescales. an informative example is provided by the mononegaviralesan order of viruses originally characterized by unsegmented negative-sense rna genomes and which has recently been the subject of considerable attention from the ictv. although use of the taxonomic term 'mononegavirales' is growing in popularity, it now makes little sense in its strict literal definition as rdrp-based phylogenies show that this group contains segmented viruses, so that they no longer fulfil the criterion of possess a single ('mono') negative-sense rna molecule (li et al., 2015) , with genome segmentation evolving a number of times independently. similar stories can be told for the flaviviridae and the totiviridae that were originally defined based on single segment but are now found to be closely related to viruses with multiple segments (li et al., 2015; qin et al., 2014; sasaya et al., 2002) , and the partitiviridae and picobirnaviridae that were thought to be bisegmented yet now include viruses containing one to six segments. these growing number of these 'exceptions' have often been classified as separate families or floating genera, in doing so ignoring their evolutionary relationships. another important limitation of the current classification system is that equivalent taxonomic groups can vary enormously in their component genetic diversity. although this is a common problem in classification, and in large part reflects the fact that some families have a much longer evolutionary history than others, it is especially prominent in rna viruses. the reason for such imbalance again points to the sometimes shaky criteria used for viral classification. for example, the 'hepe-virga' clade (also known as the alpha-like supergroup) are relatively closely related in rdrp phylogenies yet the ictv divides them into one order (tymovirales), eight families (the virgaviridae, togaviridae, bromoviridae, closteroviridae, endoranviridae, alphatetraviridae, hepeviridae, and benyviridae), and three floating genera (negevirus, idaeovirus, and cilevirus) . although this clade does possess some divergent genome structures, with differences in segmentation, orf arrangement, genome length, and even the genome sense, its rdrp diversity is no larger than that of reoviruses that are still classified as a single family thanks to a stable genome plan. in other cases these taxonomic differences appear to be largely arbitrary. for example, in the newly established order bunyavirales (https://talk. ictvonline.org/taxonomy/), the jonviridae, feraviridae and phasmaviridae are defined as separate families, although they form a single rdrp cluster whose diversity is significantly smaller than those of some individual families, such as the phenuiviridae and the peribunyaviridae. although there have been clear improvements in making virus classifications more compatible with underlying phylogenetic relationships, there are notable exceptions. for example, the togaviridae comprise two genera, alphavirus and rubivirus, that do not share common ancestry in phylogenies of either their replicase or structural proteins. at the very least proposals for individual taxonomic groups should be monophyletic, which is not always the case (kuhn et al., 2013) . we also contend that it is naive to think that the structure of virus diversity in nature, and the phylogenetic analysis of this data, will necessarily produce a simple and stable classification scheme. first, the boundaries we draw to mark higher virus taxa are inherently arbitrary, rather than reflecting a hard evolutionary 'rule', and we should not expect nature to provide neat boundaries for classification. as noted above, the gaps apparent in many phylogenetic trees will likely be filled by newly discovered lineages as our sampling becomes more extensive. hence, phylogenetic gaps do not necessary reflect a fundamental evolutionary process, but are likely an artefact of sparse and inadequate sampling. indeed, from a metagenomic perspective virus species will simply be points in phylogenetic space, and viruses 'species' differ fundamentally from those in diploid outcrossing animals in which the term has a real biological meaning. at a lower taxonomic level, using genetic distance cut-offs to determine taxonomic differences within virus species, particularly genotypes, is also fraught with difficulties as different schemes are used in different viruses and all such rules of distance may break down if there is extensive rate variation among taxa and if our sampling is biased toward specific geographic locations. it is also important to recall that virus gene trees are not the same as species trees, such that phylogeny-based classifications will often be only genic in nature. because of high levels of sequence divergence it is necessarily the case that most deep (particularly inter-family) virus phylogenies are based on the analysis of rdrp alone. however, given the dynamic nature of virus genome organization, particularly the occurrence of lateral gene transfer, it is certain that in many cases the phylogeny of the rdrp will not match that of the virus genome as a whole. for example, the luteoviridae are currently defined based on the relatedness of the structural proteins, although the replicase sequences of these viruses do not form a monophyletic group. unfortunately, phylogenetic analyses of other genes, particularly those that encode structural proteins, often present an unsurmountable challenge for sequence-based analytical methods because of the huge sequence distances involved (holmes, 2009; zanotto et al., 1996) . it is therefore an inconvenient truth that while phylogenies based on the rdrp can sometimes accurately depict the evolutionary history of that gene, they do not necessary reflect that of the virus as a whole. although there are pros and cons to using either replicase or structural genes to determine phylogenetic relationships, the fact that they often give contrasting views of evolutionary history clearly complicates virus classification. most importantly, phylogenetic trees are only ever able to depict the relationship among those viruses that are present in the sample of viruses under study; as our sample is likely negligible, so our classification is necessarily incomplete. a more fundamental question is whether the current classification scheme can withstand the onslaught of metagenomic data? the proliferation of 'family-like' viruses revealed from meta-transcriptomic surveys amply highlights the scale of the challenge facing taxonomists. as emphasized throughout this paper it is clear that we are still only scratching the surface of the virosphere and that we evidently have a great deal to learn about virus diversity and evolution. as well as revealing an abundance of new virus taxa, and determining the evolutionary processes that have shaped this diversity, it is undoubtedly the case that viruses exist in hosts that have not been screened for rna viruses or that are so divergent in sequence that they cannot readily be detected by standard homology-based methods (such as blast) or included in phylogenetic analyses. if the nature of this dark matter can be resolved it will surely shed new light on the ultimate origins of viruses as well as their deep phylogenetic relationships. the situation is particularly acute in the case of the archaea in which only a single putative rna virus has been described to date (bolduc et al., 2012) , and which in large part may reflect our current inability to identify viruses that possess highly divergent genome sequences. it is therefore of critical importance to perform unbiased metagenomics surveys of prokaryotic taxa that have not been examined to date, followed by novel bioinformatics analyses that are able to accurately identify viruses and reveal their phylogenetic relationships. this will entail the characterization of the unknown biodiversity of rna viruses in prokaryotes and basal eukaryotes and, in parallel, developing and utilizing new computational tools to robustly extract sequence information from highly divergent genome sequences. similarly, the increasingly frequent detection of recombination and lateral gene transfer also poses a major challenge to current phylogenetic protocols and may require a new computational tool-kit (iranzo et al., 2016; koonin and dolja, 2014) . our knowledge of the evolutionary processes that have generated the diversity of the virosphere has been strongly skewed by a focus on those viruses that act as agents of disease in economically important animals and plants and those that can be easily cultured. importantly, recent work has shown that animals harbor enormous uncharacterized viral diversity, only some of which has been associated with disease. however, these viruses still only reflect a tiny proportion of those in nature and therefore provide an incomplete picture of the major processes of virus ecology and evolution. key questions for future research that can be addressed with the new wealth of meta-transcriptomic data include (i) determining the flow of viruses between host taxa and the processes that shape virus ecosystems; (ii) revealing the mechanisms of long-term virus macroevolution, particularly lineage birth and death, and (iii) revealing the mechanisms and evolutionary processes that structure viral genomes. rather than simply surveying biodiversity and classifying, the goal for the future should be to perform more ecologyfocused studies to reveal fundamental patterns and processes. it is critical that studies of virus diversity evolution shape our attempts to classify these infectious agents, rather than classification schemes guiding how we think that viruses have evolved. finally, we contend that is perhaps premature to construct inflexible and overly hierarchical classification schemes for rna viruses when we have clearly sampled so little of what is there in nature. the new age of virus discovery will undoubtedly provide many 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distribution, and evolution of viruses associated with drosophila melanogaster actionable diagnosis of neuroleptospirosis by next-generation sequencing a reevaluation of the higher taxonomy of viruses based on rna polymerases this study was supported by the special national project on research and development of key biosafety technologies (2016yfc1201900), the 12th five-year major national science and technology projects of china (2014zx10004001-005), and national natural science foundation of china (grants 81290343, 81672057). ech is funded by an nhmrc australia fellowship (gnt1037231). key: cord-286658-9kco7qad authors: jiang, lei; han, zongxi; chen, yuqiu; zhao, wenjun; sun, junfeng; zhao, yan; liu, shengwang title: characterization of the complete genome, antigenicity, pathogenicity, tissue tropism, and shedding of a recombinant avian infectious bronchitis virus with a ck/ch/ljl/140901-like backbone and an s2 fragment from a 4/91-like virus date: 2018-01-15 journal: virus res doi: 10.1016/j.virusres.2017.11.007 sha: doc_id: 286658 cord_uid: 9kco7qad in this study, we isolated an infectious bronchitis virus, designated i1101/16, from broiler breeders in china. analysis of the s1 gene showed that isolate i1101/16 was genetically close to strain ck/ch/ljl/140901, which belongs to the tw i genotype (also known as lineage gi-7 based on the recent ibv classification), however the s2 gene showed genetic diversity comparing to that of s1 gene. comparison of the genomic sequences showed that the genome of isolate i1101/16 was similar to that of strain ck/ch/ljl/140901 from the 5′ end of the genome to the 5′ end of the s2 gene and from the 5′ end of the 3a gene to the end of the genome, whereas the remaining parts of the genome sequences were more closely related to those of strain 4/91 than those of ck/ch/ljl/140901, thereby suggesting that recombination might have occurred during the origin of the virus. simplot and bootscan analysis of the complete genomic sequence confirmed this hypothesis, where it showed that isolate i1101/16 arose through recombination events between ck/ch/ljl/140901and 4/91-like viruses. isolate i1101/16 and strain ck/ch/ljl/140901 shared identical amino acids in almost all five of their b cell epitopes, but the two viruses had a serotype relatedness value of 65, which is well below 80, i.e., the lower cutoff value for viruses of the same serotype. in addition, pathogenicity tests demonstrated that isolate i1101/16 was more pathogenic to 1-day-old specific-pathogen-free chickens than strain ck/ch/ljl/140901, according to analysis of the clinical signs, whereas strain ck/ch/ljl/140901 exhibited prolonged replication and shedding after challenge compared with isolate i1101/16. this study did not provide evidence that recombination can directly alter the antigenicity, virulence, replication, shedding, and tissue tropism of a virus, but it did show that recombination events are likely to be major determinants of viral evolution. avian infectious bronchitis virus (ibv), the prototype gammacoronavirus species in the family coronaviridae, is a positivesense, single-stranded rna virus with a genome of approximately 27.6 kb (5′-untranslated region (utr)-1a/1ab-s-3a-3b-e-m-5a-5b-n-3′-utr). it causes an acute and highly contagious disease in chickens, which is responsible for high economic losses in the poultry industry. the current control strategies are based mainly on mass vaccination strategies. nevertheless, vaccine-induced immunity generally give poor protection because the current vaccine offers only limited cross protection among strains (cook et al., 2012; de wit et al., 2011) mainly due to the antigenic diversity caused by the variability of the s1 protein. recently, numerous ibv strains have been identified and new genotypes/serotypes have emerged from existing viruses via point mutations, insertions, and deletions in the viral genome, especially in the s1 subunit of the spike protein gene. at least six ibv genotypes together comprise 34 distinct viral lineages and a number of unassigned interlineage recombinants have been recognized worldwide according to a simple and repeatable phylogeny-based classification system that uses the complete nucleotide sequence of the s1 gene and an unambiguous and rationale lineage nomenclature for the assignment of ibvs (valastro et al., 2016; chen et al., 2017; jiang et al., 2017) . there is also considerable variation in the virulence and tropism of ibvs, and in some cases the novel ibv strains emerged from point mutations, insertions, and/or deletions in the s1 gene. another important mechanism that underlies the emergence of novel ibv strains is genomic recombination. in australia, three recently isolated novel subgroup ibv strains were shown to be derived from recombination between subgroup 1 and 2 strains (mardani et al., 2010) . more recently, the complete genome analysis of newly emerged strains found multiple recombination events throughout the genome between wild-type viruses and vaccine strains (quinteros et al., 2016) . in the usa, the emergent virulent strain ark dpi appears to have originated from recombination among four different ibv strains (ammayappan et al., 2008) . in addition, the emergence of new strains in the usa has been reported to have arisen from recombination events between the massachusetts (mass), connecticut (conn), and holte strains as well as field strains (thor et al., 2011) . recently in europe, it has been shown that emergent viruses, such as the xdn-like virus in spain and italy (moreno et al., 2016) , and the γcov/ck/italy/i2022/13 virus in italy (franzo et al., 2015) , may have arisen from recombination events. there have been several episodes of infectious bronchitis (ib) in chinese chicken flocks, and the genotypes/serotypes of ibvs were previously classified based mainly on the nucleotide sequences of genes encoding the s1 subunit of the spike protein (han et al., 2011) , and in some cases based on cross virus-neutralization chen et al., 2017) in china. since 1995, the predominant ibv type in china has been lx4 (also known as qx-like viruses), but molecular studies have shown that new types and variants are emerging continually mo et al., 2013; zhao et al., 2013; liu et al., 2014; zhou et al., 2014; chen et al., 2015; xu et al., 2016; zhang et al., 2015; leghari et al., 2016; chen et al., 2017; zhao et al., 2017; zhou et al., 2017) . it has been suggested that the emergent ibv strains in china may have different origins. it is considered that some of the ibv types circulating in china, such as mass and 793/b, are the most widely distributed types worldwide and they may have infected chinese chickens from an exogenous source, probably due to the use of live vaccines (chen et al., 2015; han et al., 2017) . by contrast, some ibv types, such as lx4 and ck/ch/ldl/97i (q1-like), are believed to have originated in china and spread to other regions of the world (valastro et al., 2016) . the remaining ibv types, such as ck/ch/lsc/99i (liu et al., 2006a,b) , nrtw i , and gi-28 , are considered to be indigenous to china. the origins of most of the ibv types in china are still unknown, although it has been shown that some of these types have arisen from recombination events (chen et al., 2015; xu et al., 2016; chen et al., 2017) . the results of many of these previous studies are based on analyses of the available s1 gene sequences, but it is impossible to fully understand the origins and evolutionary processes related to these emerging viruses by only analyzing this small part of the genome. in this study, we isolated and identified an nrtw i type ibv from an h120 and 4/91 vaccinated chicken flock with respiratory signs of ib. we sequenced the complete genome and compared it with other ibv sequences available in genbank, and we then subjected these sequences to phylogenetic, molecular, and recombination analyses. we also investigated the antigenicity, pathogenicity, replication, and shedding of the nrtw i type ibv in chickens. trachea samples were collected from five suspected ibv-infected broiler breeders and they were submitted to our laboratory in 2016 for routine diagnosis. chickens in this flock had been vaccinated against ibv using the live attenuated h120 vaccine at 7 days of age, and the chickens were then boosted at 25 and 60 days with the live attenuated 4/91 and h120 vaccines, respectively. the birds were also vaccinated with a bivalent, inactivated vaccine, newcastle disease virus la sota/ ibv m41, at 120 days. some of the birds exhibited respiratory signs of ib at 150 days, which was accompanied by decreased egg production and abnormal shell quality in the breeding hens, and the clinical signs disappeared approximately 20 days later. the morbidity was approximately 5% and only a few birds died during this outbreak. gross lesions were mainly associated with tracheitis and proventriculitis, and hepatitis and hydropericardium were observed in some chickens. kidney lesions were not observed in the chickens examined. to isolate the virus, the tracheal samples from the dead chickens were pooled and inoculated into 9-day-old specific pathogen-free (spf) embryonated chicken eggs via the allantoic sac route, as described previously . the virus recovered from the third passage was named γcov/ck/china/i1101/16 (i1101/16) (ducatez and the european unit cost action fa1207, 2016) and it was used in the following experiments. the virus stock was prepared after an additional passage by inoculating the allantoic cavity of 9-day-old embryonated spf chicken eggs. after incubation for 48 h, the allantoic fluid was collected aseptically and clarified by centrifugation at 2500 × g for 20 min, as described previously . fertile white leghorn spf chicken eggs and white leghorn spf chicks were obtained from the laboratory animal center, harbin veterinary research institute, the chinese academy of agricultural sciences, china. the birds were kept in isolation units with negative pressure throughout the experiment, and water and feed were provided ad libitum. the complete genomes of the i1101/16 isolate and the ck/ch/lsc/ 99i strain were sequenced in this study. ibv strain ck/ch/lsc99i was isolated from preventriculus of a layer hen in sichuan province in china in 1999 (liu et al., 2006a,b) . genomic rna was extracted from virusinoculated allantoic fluids using trizol reagent (invitrogen corporation, carlsbad, ca, usa) according to the manufacturer's instructions. first-strand cdna synthesis and subsequent pcr were performed using a primescript™ one-step rt-pcr kit ver. 2 (takara bio inc., shiga, japan) according to the manufacturer's instructions. the complete genomes of the i1101/16 isolate and ck/ch/lsc/99i strain were amplified with primers used for amplifying the complete genomes of other chinese ibv strains . the pcr profiles comprised initial denaturation for 5 min at 94°c, followed by 30 cycles at 94°c for 30 s, 50-60°c (depending on the primer set) for 30 s, and 72°c for 2 min. a 3′/5′ rapid amplification of cdna ends kit (takara bio inc.) was used to determine the 3′ and 5′ ends of the viral genomes according to the manufacturer's instructions . the amplified products were sequenced directly or cloned into the pmd18-t vector (takara bio inc.) according to the manufacturer's instructions. each fragment of the viral genome was sequenced at least three times. the complete genomic sequences of the i1101/16 isolate and the ck/ch/lsc/99i strain were mapped using the genome sequences of the beaudette and ck/ch/ljl/140901 ibv strains, respectively, in order to determine a consensus sequence. 2.4. comparison and analysis of the s1 subunit of the spike and spike protein genes the region coding for the s1 subunit of the spike protein is used for genotyping and classification purposes. comparisons with published sequences were first performed using the nucleotide blast search tool in genbank with the s1 gene nucleotide sequence from the i1101/16 isolate. the s1 nucleotide sequences from 70 ibv reference strains were downloaded from genbank, where most of these ibvs were isolated in china and they represented the previously reported types (valastro et al., 2016; chen et al., 2017) . phylogenetic analyses were conducted based on the s1 gene nucleotide sequences using the neighbor-joining method (jukes-cantor model) with 1000 bootstrap replicates (mega software version 5.0; available at http://www.megasoftware.net/). to analyze the genetic diversity of the i1101/16 isolate, the percentages of similarity were calculated for both the s1 subunit and the entire spike protein at the nucleotide and amino acid levels using the i1101/16 isolate and seven reference strains, which were selected based on the phylogenetic analysis results. the seven reference strains included two vaccine strains, h120 and 4/91, which are used commonly in chicken flocks in china and they represented two genotypes, and five ibv field strains that circulate in chicken flocks in china, where they represented different genotypes (i.e., ck/ch/ljl/140901 [the nrtw i type] , ck/ch/ldl/091022 [the lx4 type] , ck/ch/lgx/111119 [the gi-28 type] , ck/ ch/lsc/99i [the ck/ch/lsc/99i type] (liu et al., 2006a,b) , and ck/ ch/ldl/97i [the ck/ch/ldl/97i type] (liu et al., 2006a) ). in addition, we compared the amino acid sequences of the five conformationdependent neutralizing antigenic sites (epitopes) mapped to the s1 subunit of the spike protein as well as another immunodominant region in the amino-terminal region of s2 lenstra et al., 1989; koch et al., 1990 ) in a pairwise manner in isolate i1101/16 and the seven reference strains. multiple sequence alignment of the complete genome sequences of the i1101/16 isolate and the reference strains was performed with multiple alignment with fast fourier transformation (mafft) v6 (http://mafft.cbrc.jp/alignment/software/). the alignment included seven sequences from the strains mentioned above. to refine the recombination event pattern analysis, similarity plots (simplots) and bootscan analysis were used to evaluate the possible recombinant nature of the isolate using the on-line multiple sequence local alignment (mulan) tool (ovcharenko et al., 2005) with the complete genome sequences of the ck/ch/ljl/140901 and 4/91 strains, where we used a window size of 500 bp and a step size of 20 bp. the analyses were performed using simplot version 3.5.1 (lole et al., 1999) where the complete genome sequence of the ck/ch/lsd/110851 strain (accession number kp118884) was used as the query because it was a representative of 4/91 vaccine sequence and supposed parental recombinant sequence of strain i1101/16. the deduced sequences of the recombination breakpoints and flanking sequences of the i1101/16, ck/ch/ljl/140901, and 4/91 viruses were compared in a pairwise manner to accurately detect the positions of the recombination breakpoints. in addition, we performed pairwise comparisons of the nucleotide sequences of the s2 subunit of the spike gene between i1101/16 isolate, and 4/91 vaccine (genbank accession kf377577) and a pathogenic 4/91 strain (genbank accession jn192154), in order to identify which was the possible parental virus strain of i1101/16 isolate. the ibv i1101/16 isolate and the seven reference ibv strains mentioned above were used in virus-neutralization tests. stocks of these viruses were prepared by passaging them in the allantoic cavity of 9day-old embryonated spf chicken eggs. viral titers were determined by inoculating 10-fold dilutions into groups of five 9-day-old embryonated chicken eggs and the 50% embryo infectious dose (eid 50 ) was calculated using the method of reed and muench (1938) . serotype-specific antisera against all the viruses listed in table 1 were prepared in chickens, and cross virus-neutralization tests were conducted in 9-day-old spf chicken eggs as described previously (jackwood et al., 2007) . the β virus neutralization method with constant virus and diluted serum was employed. titers were calculated using the method of reed and muench (1938) , and relatedness values were calculated using the method of archetti and horsfall (1950) . viruses with an archetti and horsfall relatedness value greater than 50 were considered to be related serotypes. ethical approval for the animal experiments was granted by the ethical and animal welfare committee of the harbin veterinary research institute, china. forty-five 1-day-old spf layer chickens were assigned randomly to three groups each containing 15 birds. the i1101/16 isolate and the ck/ch/ljl/140901 strain were administered by eyedrop inoculation to 1-day-old birds in groups 1 and 2, respectively, at a dosage of 1 × 10 5.5 eid 50 in a 0.1-ml volume, and the remaining groups served as negative controls. five days after challenge all chickens were evaluated blindly for respiratory rales, such as nasal and/or tracheal, by closely listening to each bird. signs were scored as 0 (absent), 1 (mild), 2 (moderate), or 3 (severe). at 5 days post-challenge, five birds from each group were killed and necropsied, and the trachea and kidneys were collected. measurements of infectivity and viral replication levels in the chicks were performed by determining the viral titers in 9-day-old spf embryonated chicken eggs. in addition, the ibv antigen was detected by immunohistochemistry using monoclonal antibody 6d10 . blood samples and nasopharyngeal and cloacal swabs were collected from the birds in each of the groups on days 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 , and 44 post-challenge. the blood samples were used to detect antibodies against ibv with a commercial enzyme-linked immunosorbent assay (idexx corporation, westbrook, me, usa) according to the manufacturer's instructions . the swabs were placed in individual tubes containing sterile phosphate-buffered saline (ph 7.4) with antibiotics and stored at −80°c for virus recovery, as described previously . the chickens were observed for disease symptoms, death, and kidney lesions for 45 days. the s1 gene phylogenetic tree contained seven clusters (fig. 1) , which corresponded to the seven types of ibv, i.e., the gi-28 , lx4 (qx or gi-19) , ck/ch/lsc/99i (gi-22) (liu et al., 2006a,b) , 793/b (gi-13) , ck/ch/ ldl/97i (q1 or gi-16) (liu et al., 2006a) , mass (gi-1), and tw i (gi-7) types . among these, two vaccine strains, h120 and 4/ 91, are used commonly in china and they belong to the mass (gi-1) and 793/b (gi-13) types. the remaining five ibv types are field strains that circulate in chicken flocks in china. the i1101/16 isolate clustered into the tw i group (gi-7), which was genetically close to the ck/ch/ljl/ 140901 strain and slightly distant from other strains in the tw i group. in agreement with the results of the phylogenetic analysis, the i1101/16 isolate shared the highest genetic similarity (95.7% and 95.4% at the nucleotide and amino acid levels, respectively) with the ck/ch/ljl/140901 stain. by contrast, the i1101/16 isolate shared low genetic similarity with the other types of viruses, with nucleotide and amino acid identities ranging among 76.1-80.7% and 76.7-80.6%, respectively. similarly, the i1101/16 isolate also shared the highest similarity with the ck/ch/ljl/140901 strain at both the nucleotide (90.9%) and amino acid (93.5%) levels based on the spike protein gene, whereas it shared no more than 90% similarity with the other strains at either the nucleotide or amino acid levels. interestingly, the genetic similarities between the i1101/16 isolate and the ck/ch/ljl/140901 strain based on the s1 gene were obviously higher than those based on the spike protein gene, which is the opposite result compared with those obtained between the i1101/16 isolate and most of the reference strains (table 1) . pairwise comparisons were performed of the amino acid sequences of b cell epitopes d (residues 24-61), e (residues 132-149), c/a/b (residues 291-398), and f (residues 497-543) in the s1 subunit and the g region (residues 548-574) of the s2 subunit of the spike protein, and the results are summarized in fig. 2 . the i1101/16 isolate shared similar amino acids in all epitopes with the ck/ch/ljl/140901 strain, whereas it exhibited amino acid differences compared with the ck/ch/ lgx/111119, ck/ch/lsc/99i, ck/ch/ldl/97i, conn, 4/91, and h120 strains. the i1101/16 isolate shared similar amino acids with the ck/ ch/ldl/091022 strain in the f and g epitopes, but the amino acid residues differed in the d, e, and c/a/b epitopes of these strains. the genome sequences of the i1101/16 isolate and the ck/ch/lsc/ 99i strain were determined and deposited in genbank under accession numbers ky620116 and ky799582, respectively. the genomes of the viruses comprised 27,674 bp and 27,665 bp, respectively, excluding the poly (a) tail. the complete genome sequences of the i1101/16 isolate and the ck/ch/lsc/99i strain had the typical ibv gene order of 5′-utr-1a-1b-s-3a-3b-e-m-5a-5b-n-3′-utr, and the gene lengths are shown in after comparing the genomic sequence of the i1101/16 isolate with those of the reference strains using the mafft alignment, we found that the genome of the i1101/16 isolate was similar to that of the ck/ ch/ljl/140901 strain from the 5′ end of the genome to the 5′ end of the s2 gene, whereas the sequences differed significantly from that of the ck/ch/ljl/140901 strain from the 5′ end of the s2 gene to the end of the s2 gene. in the i1101/16 isolate, this region was most closely related to the 4/91 strain. interestingly, the sequence of the i1101/16 isolate was more closely related to that of the ck/ch/ljl/140901 strain than that of the 4/91 strain from the 5′ end of the 3a gene to the end of the genome (fig. 3) . simplot and bootscan analyses also confirmed these results (fig. 4) (fig. 5 ). in addition, 12 nucleotides differences were identified in the s2 subunit of the spike protein gene of isolate i1101/16 and the two 4/91 strains; pairwise comparisons demonstrated that isolate i1101/16 shared seven nucleotide identities with the 4/91 vaccine strain and only two with the pathogenic 4/91 strain (table 3) , thereby indicating that this region of the i1101/16 isolate is more similar to that of 4/91 vaccine strain than that of the pathogenic strain. based on the method of archetti and horsfall (1950) , serotype-relatedness values were calculated in cross virus-neutralization studies using embryonating chicken eggs (table 1) , which showed the i1101/ 16 isolate and the ck/ch/ljl/140901 strain were related serotypes with a serotype relatedness value of 65. none of the other ibv strains tested in this study were related serotypically to isolate i1101/16. the pathogenicity of the i1101/16 isolate was tested in 1-day-old spf chickens and compared with that of the ck/ch/ljl/140901 strain, and the results are presented in table 4 . at 5 days post-challenge, clinical signs were observed in 2/10 birds and 1/10 birds inoculated with the i1101/16 isolate and the ck/ch/ljl/140901 strain, respectively. at 11 days post-challenge, 10/10 birds inoculated with the i1101/16 isolate had severe clinical signs including tracheal rales, watery eyes, nasal mucus, and occasional sneezing, whereas 9/10 birds inoculated with the ck/ch/ljl/140901 strain had only mild clinical signs. the mean scores of the clinical signs for the birds challenged with the i1101/16 isolate and the ck/ch/ljl/140901 strain were listed in table 4 . no birds died in any of the groups during the experiments. no clinical signs were observed in the non-challenged birds. at 8 days post-challenge, antibodies against ibv were detectable by an enzyme-linked immunosorbent assay in seven and four birds challenged with the i1101/16 isolate and the ck/ch/ljl/140901 strain, respectively. antibodies were detected in all the challenged birds at 12 days post-challenge. antibodies were not detected in the non-challenged birds. table 1 antigenic relatedness a and s1/s gene similarities between the i1101/16 isolate and seven reference strains. percentage of relatedness/s1 or s similarities archetti and horsfall (1950) with neutralization indices derived from the results of reciprocal virus-neutralization tests performed in 9-day-old embryonated eggs. b the percentages of relatedness for the first 1665 nucleotides starting at the aug translation start codon in the s1 protein genes between the i1101/16 isolate and seven ibv reference strains. c the percentages of relatedness for the amino acid of the s1 subunit in the spike protein between the i1101/16 isolate and seven ibv reference strains. d the percentages of relatedness of the nucleotides encoding the spike protein between the i1101/16 isolated and the seven ibv reference strains. e the percentages of relatedness of amino acids in the spike protein between the i1101/16 isolate and seven ibv reference strains. as illustrated in fig. 6 , replication of the ck/ch/ljl/140901 strain was detected in the trachea and kidneys of chickens, with titers of 3.83-4.83 log 10 eid 50 and 3.5-4 log 10 eid 50 , respectively. by contrast, replication of the i1101/16 isolate was detected in the trachea and kidneys, with titers of 1.7-2.83 log 10 eid 50 and 1.38-1.68 log 10 eid 50 , respectively. in addition, viral antigens were detected by fig. 1 . phylogenetic tree based on the s1 nucleotide sequences (from the atg start codon to the cleavage site of the spike protein). the phylogeny contains a total of 71 ibv strains, including the i110116 isolate and 70 ibv reference strains representing seven genotypes from china. bars denoting the genotype and lineage designations are shown. the ibv strain and genbank accession number are given for each strain. the scale bar represents the number of nucleotide substitutions per site. the ibv strains used for further comparisons and virus-neutralization testing are highlighted in bold. immunohistochemistry in the trachea and kidneys of the birds challenged with both viruses (fig. 7) . no virus was detected in the trachea and kidneys of birds in the control group. shedding of the challenge viruses in the respiratory and digestive tracts was determined by virus recovery using nasopharyngeal and cloacal swabs from 9-day-old spf chicken eggs. as illustrated in table 4 , the challenge viruses were recovered from the nasopharyngeal swabs of all the birds at 4 days post-challenge, whereas viruses were not detectable in some of the birds at 8 and 12 days post-challenge with the i1101/16 isolate and the ck/ch/ljl/140901 strain, respectively. viral shedding was not detectable in the nasopharyngeal swabs of all the birds at 24 days post-challenge with the ck/ch/ljl/140901 strain. by contrast, viral shedding was still detectable at 28 days post-challenge in the nasopharyngeal swabs from one bird challenged with the i1101/16 isolate. based on the cloacal swabs, viral shedding from some of the birds lasted until 40 and 28 days post-challenge with the i1101/16 isolate and the ck/ch/ljl/140901 strain, respectively. virus was not recovered from the non-challenged control birds. ib is among the most important diseases in both vaccinated and non-vaccinated chickens in china, and it has been monitored continuously in chicken flocks since it was first identified. an outbreak possibly caused by a respiratory pathogen occurred at the end of 2016 in a broiler breeder flock in neimenggu province in china, and only a very small number of diseased chickens died during the outbreak. however, the egg production decreased and the egg quality was adversely affected. the main lesions were tracheitis and proventriculitis at the early stage of the outbreak, and severe bacterial infections were observed at the late stage, but no obvious kidney lesions were observed in the dead chickens throughout the outbreak. an ibv strain designated as i1101/16 was isolated from the tracheas of the dead chickens. both the ib live vaccines h120 and 4/91 as well as an inactivated vaccine were used to vaccinate the chicken flock before the occurrence of the ib outbreak, so we initially sequenced the s1 gene to identify, differentiate, and genotype the ibv isolate. based on the s1 gene analysis, the i1101/16 isolate clustered most closely with reference strains of the ntw i type , and it has been diverging as an independent genetic clade with the ck/ch/ljl/140901 strain (fig. 1) . our analysis showed that the i1101/16 isolate and the ck/ch/ljl/ 140901 strain were more closely related serologically compared with other ibv serotypes (table 1) , but the serotype relatedness value was only 65 between these strains, which is greater than 50% but well below 80%, i.e., the lower cutoff value for the same serotype and the fig. 2 . multiple alignment of the deduced amino acid sequences of the five conformation-dependent neutralizing antigenic sites (epitopes) mapped to the s1 subunit of the spike protein and another immunodominant region in the amino-terminal region of the s2 protein using the i1101/16 isolate and the seven reference strains. the sequences of the i1101/16 isolate are listed; only the amino acids that differed from those of the i1101/16 isolate are depicted and the amino acids that were same as those of the i1101/16 isolate are represented by dots. deleted amino acid residues are represented by dashes. 3 . alignment of the complete genome sequences of the i1101/16 isolate and the seven reference strains performed using mafft. the genome sequence of the ibv i1101/16 isolate was set as the reference sequence. the reference sequence was represented by black and gray gaps in the reference sequence indicated the nucleotide insertion occurred in the genomes sequence of other viruses. the nucleotide sequences of other viruses which disagreement to the reference sequence at indicated positions were represented by black, while the nucleotide sequences of other viruses which agreement to the reference sequence at indicated positions were represented by gray. the genbank accession numbers for these genome sequences are the same as those in fig. 1. maximum value defining serotype relatedness, respectively (wadey and faragher, 1981) . the low antigenic relatedness between the i1101/16 isolate and the ck/ch/ljl/140901 strain was not due to differences in their b cell epitopes because the sequences of the b cell antigenic epitopes located in the spike protein were nearly identical in both viruses, although it has been reported that only a few amino acid changes in the s1 subunit of the spike protein can result in serologically distinct viruses (cavanagh et al., 1992a,b) . hence, we focused on the entire spike gene of the two viruses, especially the s2 subunit of the spike gene. interestingly, there was greater genetic diversity in the spike gene (90.9%) between the i1101/16 isolate and the ck/ch/ljl/140901 strain compared with that of s1 gene (95.7%), thereby indicating the greater divergence of the s2 region, which led us to conclude that possible recombination events may have occurred in the genome of the i1101/16 isolate. next, recombination events were analyzed using the complete genomic sequences of these strains. according to mafft, only the s2 gene of the i1101/16 isolate was highly similar to that of the 4/91 strain, whereas the remainder of the genome was highly similar to that of the ck/ch/lhlj/140901 strain. this suggests that for the emergent i1101/16 isolate, the donor of the s2 gene was probably a 4/91-like virus, whereas the remainder of the genome came from a ck/ch/lhlj/ 140901-like virus. the first prerequisite for recombination is considered to be co-infection. ibv strain ck/ch/lhlj/14090 is one of the deduced parental viruses of the i1101/16 isolate and it has an nrtw serotype, where it originated from recombination events between tw i-like and lx4-like viruses in china around 2009 . in the commercial poultry industry in china, ibv is controlled by the use of live attenuated and inactivated vaccines. vaccine 4/91 is another deduced parental virus of the i1101/16 isolate and it has been used widely in china for a long time. previously, we found that vaccination with the 4/91 strain cannot provide complete protection against nrtw viruses , but vaccination with the 4/91 strain allows prolonged replication and shedding of the vaccine virus . hence, it is reasonable to speculate that vaccination with the 4/91 vaccine produces an environment where co-infections between field and vaccine strains can occur, which may enhance the likelihood of recombination. high frequencies of recombination between vaccines (such as the 4/91 strain) and field strains have been reported frequently in china and other parts of the world (cavanagh et al., 1992a,b; jackwood et al., 2012; liu et al., 2013; han et al., 2017; jiang et al., 2017) . it has been suggested that recombination may play an important role in viral evolution. the exchange of part of a region of the genome allows viruses to rapidly explore areas of sequence space, potentially leading to the emergence of variants with different features in terms of their virulence, cross-protection, and cell and host tropisms (simon-loriere and holmes, 2011) . it is considered that the emergence of the australian n1/88 strain was driven by antigenic differences between the virus and other circulating ibvs, but also by the enhanced capacity of the recombinant virus to replicate in chickens (quinteros et al., 2016) . we found that the recombinant i1101/16 isolate was serologically related to but not the same as strain ck/ch/lhlj/140901. we cannot conclude that this antigenic change was due to recombination because the b cell antigenic epitopes located in the spike protein (including that located in the s2 subunit of the spike gene) were nearly identical in the two viruses. in addition, it is very interesting to note that the/i1101/16 isolate exhibited decreased replication levels in both the tracheal and kidney tissues (two target tissues for most ibvs) compared with one of its parental viruses (the ck/ch/lhlj/140901 strain, which does not cause severe clinical disease in spf chickens), but it exhibited prolonged replication and shedding post-challenge in a table 3 pairwise comparisons of the nucleotide sequences of the s2 subunit of the spike genes between the 4/91 vaccine strain, i1101/16 isolate, and pathogenic 4/91 strain a . a nucleotides shared between the i1101/16 isolate and 4/91 vaccine strain are shown in gray. b nucleotide positions corresponding to those in the spike gene in the 4/91 vaccine strain. the genbank accession number of the pathogenic 4/91 strain is jn192154. the genbank accession numbers of the other viruses are the same as those shown in fig. 1 . similar manner to one of its parental viruses, the 4/91 vaccine . meanwhile, the high viral titer but low clinical score of chickens infected with ck/ch/ljl/140901 was also observed in this study although the exact mechanism was unknown and needed to be further investigated. it is believed that the coronavirus s1 domain is involved with host cell receptor binding and that the conserved s2 domain mediates fusion between the virus and cellular membranes (bosch et al., 2003) . the s2 4 8 12 16 20 24 28 4 8 12 16 20 24 28 32 36 40 44 i1101/16 10/10 3 10/10 b 9/10 7/10 6/10 2/10 1/10 1/10 7/10 8/10 10/10 7/10 7/10 5/10 6/10 6/10 6/10 2/10 0/10 ck/ch/ljl/ 140901 9/10 0.9 10/10 10/10 6/10 2/10 2/10 0/10 -c 10/10 10/10 10/10 10/10 6/10 6/10 6/10 0/10 ---negative 0/10 0 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 a two procedures were used for virus recovery after challenge. first, lesions were observed in individual tissue samples (trachea or kidneys) from inoculated embryos. second, rt-pcr was conducted using the oligonucleotide primers n(+) and n(−) on rna recovered from the allantoic fluid in the same eggs. the results obtained using the two procedures were identical. b number of chickens yielding virus/number of chickens tested. c not done. fig. 6 . replication of the i1101/16 isolate and ck/ch/ljl/140901 strain in the trachea and kidneys of chickens. one-day-old spf layer chickens were inoculated with a dosage of 1 × 10 5.5 eid 50 of the i1101/16 isolate or the ck/ch/ljl/140901 strain in a 0.1-ml volume, and the trachea and kidneys from each bird were collected at 5 days post-challenge for virus titration in eggs. each bar represents the virus titer from an individual bird. membrane fusion unit of the ectodomain contains two heptad repeat regions, which interact to form the coiled-coil structure of the stalk (de groot et al., 1987; wickramasinghe et al., 2011) , and a putative fusion peptide. after endocytosis, conformational changes in the s protein are triggered by exposure to acidic ph in the endosomes (chu et al., 2006) , thereby resulting in fusion of the viral envelope with the cellular membrane. the s2 domain is not involved mainly with binding to the host cell receptor, but the interplay between s1 and s2 might synergistically determine the avidity and specificity of viral attachment (de hann et al., 2006; promkuntod et al., 2013) . it is unknown whether the changes in the spike protein caused by the recombination event altered the antigenic and pathogenic features of the i1106/16 isolate, or whether it is more likely that these changes as well as other changes in polymerase peptides are jointly rather than individually responsible for the differences in the features of the two ibv strains. however, it has been reported that recombination is likely to have a high fitness cost because of the destruction of optimized intra-and inter-protein interactions (simon-loriere and holmes, 2011), where the fitness of recombinant ibv strains must be superior to that of the parental strains if they are to emerge and spread in the environment. thus, the fitness of recombinant ibvs requires further investigation. this study does not provide evidence that recombination can directly alter the antigenicity, virulence, replication, shedding, and tissue tropism of a virus, but because the backbone of the investigated virus differs substantially, we showed that gene(s) exchange between ibvs is likely to be the major determinant of viral evolution. our previous results liu et al., 2014; chen et al., 2015) and the results obtained in this study provide evidence that recombination events are occurring frequently in the field in china. high chicken densities in poultry farms and the co-circulation of multiple ibv strains in a given flock as well as the use of different live vaccine strains may explain the high frequency of recombination. researchers should be aware of the effect of virus recombination, especially recombination events between field and vaccine strains, which may cause changes in viral 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host factors reflects virus tropism and pathogenicity emergence of novel nephropathogenic infectious bronchitis viruses currently circulating in chinese chicken flocks serotype shift of a 793/b genotype infectious bronchitis coronavirus by natural recombination analysis of a qx-like avian infectious bronchitis virus genome identified recombination in the region containing the orf 5a, orf 5b, and nucleocapsid protein gene sequences origin and evolution of lx4 genotype infectious bronchitis coronavirus in china complete genome sequence of a novel infectious bronchitis virus strain circulating in china with a distinct s gene identification of a novel recombinant virulent avian infectious bronchitis virus this work was supported by grants from the china agriculture research systerm (no. cars-40-k18) and national "twelfth five-year" plan for science and technology support (2015bad12b03). the authors declare that they have no competing interests. key: cord-271568-qgpi2kcs authors: jackwood, m.w.; rosenbloom, r.; petteruti, m.; hilt, d.a.; mccall, a.w.; williams, s.m. title: avian coronavirus infectious bronchitis virus susceptibility to botanical oleoresins and essential oils in vitro and in vivo date: 2010-01-21 journal: virus res doi: 10.1016/j.virusres.2010.01.006 sha: doc_id: 271568 cord_uid: qgpi2kcs anti-coronaviral activity of a mixture of oleoresins and essential oils from botanicals, designated qr448(a), was examined in vitro and in vivo. treatment of avian infectious bronchitis virus (ibv) with qr448(a) reduced the virus titer as measured in two laboratory host systems, vero e6 cells and embryonating eggs. the effect of qr448(a) on ibv in chickens was also investigated. administering qr448(a) to chickens at a 1:20 dilution by spray, 2 h before challenge with ibv was determined to be the most effective treatment. treatment decreased the severity of clinical signs and lesions in the birds, and lowered the amount of viral rna in the trachea. treatment with qr448(a) protected chickens for up to 4 days post-treatment from clinical signs of disease (but not from infection) and decreased transmission of ibv over a 14-day period. anti-ibv activity of qr448(a) was greater prior to virus attachment and entry indicating that the effect is virucidal. in addition, qr448(a) had activity against both massachusetts and arkansas type ib viruses, indicating that it can be expected to be effective against ibv regardless of serotype. to our knowledge, this is the first report on the in vivo use of a virucidal mixture of compounds effective against the coronavirus ibv. avian infectious bronchitis virus (ibv), a coronavirus, causes a highly contagious upper-respiratory tract disease in chickens. it can also be found in other species of birds including peafowl (pavo spp.) and teal (anas crecca) (cavanagh, 2005) . the virus is worldwide in distribution causing huge economic losses to the poultry industry that result from a reduction in feed conversion and weight gain, and condemnations at the processing plant (cavanagh and gelb, 2008) . losses are also due to drops in egg production and poor egg quality in layer type chickens, and for some strains of the virus that are nephropathogenic extensive mortality can occur (cavanagh and gelb, 2008) . infectious bronchitis virus, presents unique challenges regarding its control in commercial chickens. genomic diversity and the abbreviations: cpe, cytopathic effects; eid50, 50% embryo infectious dose; elisa, enzyme linked immunosorbent assay; hma, hexamethylene amiloride; ibv, infectious bronchitis virus; mhv, mouse hepatitis virus; pbs, phosphate buffered saline; rflp, restriction fragment length polymorphism; rt-pcr, reverse transcriptasepolymerase chain reaction; sars-cov, severe acute respiratory syndrome virus; spf, specific pathogen free; tcid50, 50% tissue culture infectious dose. * corresponding author. tel.: +1 706 542 5475; fax: +1 706 542 5630. e-mail address: mjackwoo@uga.edu (m.w. jackwood). ability of ibv to rapidly change have created different serotypes of the virus that do not cross protect (cavanagh and gelb, 2008) . consequently, attenuated live vaccines, used to control the disease, must be selected specifically for the serotype of ibv present in the flock. compounding this situation is the presence of serologically different variant strains for which no vaccines exist. we were interested in exploring alternative methods of controlling ibv that could potentially be effective across all serotypes. antiviral compounds have been used to target proteins involved in coronaviral replication. coronaviruses have a single stranded positive sense rna genome that is approximately 27.5 kb in length for ibv. during coronavirus replication, a 3 nested set of subgenomic viral mrnas are produced by the viral rna-dependent rna-polymerase, and four structural proteins are translated by this enveloped virus: spike, envelope, membrane and nucleocapsid (cavanagh and gelb, 2008) . two large genes, 1a and 1ab from the genomic viral rna encode the viral-replication complex. those genes are translated into polyproteins that are processed by the main protease (m pro ) as well as a papain-like protease 2 (pl2) to form the viral polymerase and associated nonstructural proteins (ziebuhr et al., 2007) . the main protease of coronaviruses appears to be the most attractive target for antiviral compounds (haagmans and osterhaus, 2006; niu et al., 2008; xue et al., 2008; yang et al., 2005 yang et al., , 2006 . inhibitors of m pro include a michael acceptor inhibitor (designated n3), chloromethyl ketones, epoxides, and ag7088 an inhibitor shown to be effective against the rhinovirus m pro homolog (anand et al., 2003; niu et al., 2008; xue et al., 2008) . other antiviral compounds like hexamethylene amiloride (hma) target ion channel conductance, and have been shown to have an antiviral effect against human coronavirus (hcov)-229e and mouse hepatitis virus (mhv) coronavirus in vitro (wilson et al., 2006) . in addition, specific inhibition of ibv has been reported in vitro using lithium chloride (harrison et al., 2007) . however, lithium chloride is a toxin and its usefulness as an antiviral against ibv in vivo remains to be determined. a major problem surrounding antiviral compounds targeting specific viral genes or proteins is the ability of the virus to rapidly mutate during replication to become resistant. some examples include acyclovir resistant human immunodeficiency viruses and herpes simplex viruses (mcmahon et al., 2008) , oseltamivir resistant influenza viruses (collins et al., 2008) , and nucleoside/nucleotide analog resistant hepatitis b viruses (delaney and borroto-esoda, 2008) . alternatively, compounds that have a virucidal effect work like a disinfectant, and do not require replication to inactivate the virus (reichling et al., 2005; schuhmacher et al., 2003) . thus, resistance to virucidal compounds due to mutations generated in the viral genome during replication is unlikely (schnitzler et al., 2007) . virucidal activity of essential oils from botanicals has been reported for a number of viruses including herpes simplex virus, dengue virus, and junin virus (duschatzky et al., 2005; koch et al., 2008; schnitzler et al., 2007) . virucidal activity of essential oils, which are lipophilic by nature, is probably due to disruption of the viral membrane or interference with viral envelope proteins involved in host cell attachment. although selection of resistant mutants is possible, it has been reported that inactivation of virus by lipophilic essential oils is time dependent and that infectious virus remaining after treatment are still sensitive to the essential oils making selection of resistant mutants unlikely (schnitzler et al., 2007) . in this study we examined the effect of a natural based product consisting of a synergistic blend of botanical oleoresins and essential oils in a liquid emulsion designated qr448(a) against ibv in vitro and in vivo. to our knowledge this is the first report on the virucidal activity of botanical extracts against a coronavirus in vivo. the data are important because these types of compounds might also be useful to control other coronaviruses like the severe acute respiratory syndrome coronavirus (sars-cov) as well as other enveloped viruses like avian influenza virus and newcastle disease virus. strains of ibv used in this study are; the vero cell adapted beaudette strain (american type culture collection (atcc, rockville, md) vr-22), the pathogenic massachusetts 41 (mass41) strain (atcc, vr-21), and the arkansas dpi (arkdpi) vaccine strain (intervet, schering-plough animal health, millsboro de). the beaudette strain of ibv was propagated in vero e6 cells (crl-1586), and the mass41 and arkdpi strains of ibv were propagated by injecting the chorioallantoic sac of specific pathogen free (spf) 10day-old embryonating chicken eggs (sunrise farms inc., catskill, ny) using standard methods (gelb and jackwood, 2008) . the vero e6 cells were obtained from the american type culture collection (rockdale, md) and propagated in minimum essential medium (eagle) with 2 mm l-glutamine, 1.5 g/l sodium bicarbonate, 0.1 mm non-essential amino acids, 1.0 mm sodium pyruvate, and 10% fetal bovine serum (invitrogen corp [gibco] , carlsbad, ca) at 37 • c and 5% co 2 . 2.2. rna extraction and real-time rt-pcr for ibv viral rna was extracted with the magmax-96 rna isolation kit (ambion inc., austin, tx) according to the manufacturer's protocol, using a kingfisher magnetic particle processor (thermo scientific, waltham, ma). real-time rt-pcr was conducted using a smart cycler ii (cepheid, sunnyvale, ca) and the agpath-id tm one-step rt-pcr kit (ambion inc.) according to the manufacturer's recommendations. the real-time rt-pcr primers and probe were previously published (callison et al., 2006) and consist of a forward primer ibv5 gu391 (5 -gct ttt gag cct agc gtt-3 ), a reverse primer ibv5 gl533 (5 -gcc atg ttg tca ctg tct att g-3 ) and a taqman ® dual-labled probe ibv5 g probe (5 -fam-cac cac cag aac ctg tca cct c-bhq1-3 ). the rt-pcr primers were synthesized by integrated dna technologies (coralville, ia), and the taqman ® probe was obtained from biosearch technologies (novato, ca). components of the real-time rt-pcr test and the thermocycler parameters were previously described, and a standard curve for the assay, was used to calculate the approximate genome copy number for each sample (callison et al., 2006) . chicks were hatched from spf fertile leghorn chicken eggs (sunrise farms, inc.) at the poultry diagnostic and research center (college of veterinary medicine, university of georgia, athens, ga). meat-type commercial broiler chickens were obtained from harrison poultry (bethlehem, ga). for each experiment, the chicks were randomly divided into different groups, and housed in positivepressure horsfal isolation units or on litter in 10 × 10 colony houses. feed and water were given ad libitum. the experiments described below were designed to determine the effect of qr448(a) obtained from quigley pharma, inc. a subsidiary of the quigley corporation (doylestown, pa), on ibv in vitro and in vivo. qr448(a) is a synergistic blend of botanical oleoresins and essential oils in a liquid emulsion of pharmaceutical grade excipients. the exact composition of qr448(a) is available from quigley pharma, inc. toxicity of qr448(a) on vero e6 cells, and embryonating eggs was examined. then, the minimum effective dose of qr448(a) against ibv propagated in those laboratory systems was determined. the vero e6 cells were grown to 85% confluence in a t75 flask (bd biosciences, franklin lakes, nj) and transferred to two 96-well plates (bd biosciences). when the cells reached 90% confluence the media was removed and 100 l of undiluted and each of the10-fold dilutions of qr448(a) or the diluent (quigley pharma, inc.) prepared in cell culture maintenance medium (containing 1% fetal bovine serum) was added to each of 4 different wells. control wells receiving cell culture maintenance medium only were also included in the experiment. the cells were incubated for 7 days at 37 • c and 5% co 2 and examined twice daily for cytopathic effects (cpe). the experiment was repeated 3 times. specific pathogen free fertile chicken eggs were obtained from sunrise farms (catskill, ny) and incubated at 37 • c for 10 days. the embryonated eggs were inoculated in the chorioallantoic sac with 100 l of undiluted and each of the 10-fold dilutions of qr448(a) or the diluent prepared in pbs (ph 7.4). four eggs were inoculated with each dilution and the experiment was repeated 3 times. negative control eggs that received pbs only were also included. the eggs were incubated at 37 • c and candled daily for 7 days to record mortality. any mortality occurring within the first 24 h was considered to be due to trauma associated with inoculation and disregarded. on the 7th day, all the remaining eggs were chilled to 4 • c and opened to examine the embryos for clinical signs of toxicity. to test the effect of qr448(a) on ibv propagated in vero e6 cells, a constant amount of virus, 1 × 10 3 50% tissue culture infectious dose (tcid 50 ) of the beaudette strain of ibv was added to seven 10-fold serial dilutions beginning with a dilution of 1 × 10 −3 (determined to be nontoxic for the vero e6 cells) of qr448(a) or the diluent prepared in cell culture maintenance medium. the mixtures were incubated at room temperature for 30 min then nine 10-fold serial dilutions (in maintenance medium) of each mixture were prepared for inoculation onto cells. the cell culture media was removed from the cells in a 96-well tissue culture plate and the mixtures were inoculated onto the monolayers. negative control wells receiving cell culture maintenance medium only were also included in the experiment. the cells were incubated for 7 days at 37 • c and 5% co 2 and examined twice daily for cytopathic effects (cpe). the experiment was repeated 3 times. the experimental design to test the effect of qr448(a) on ibv grown in embryonating eggs was the same as above, except that the beginning concentration of qr448(a) and the diluent was undiluted since neither compound was toxic for the embryos. a constant amount of virus, 1 × 10 4 50% embryo infectious dose (eid 50 ) of the ibv beaudette strain, was mixed with undiluted and each of the 10-fold dilutions of qr448(a) or the diluent prepared in pbs. negative control eggs that received pbs only were also included. the mixtures were incubated at room temperature for 30 min then nine 10-fold serial dilutions (in pbs) of each mixture were prepared and 200 l of each dilution was inoculated into the chorioallantoic sac. four eggs were inoculated for each dilution incubated at 37 • c and candled daily for 7 days to record mortality. since mortality occurring within the first 24 h could be due to trauma associated with inoculation, they were disregarded. on the 7th day, all the remaining eggs were chilled to 4 • c and opened to examine the embryos for lesions, and the eid 50 titer was calculated by the reed and muench method (villegas, 2008) . the experiment was repeated 2 times. two-week-old spf leghorn chickens were divided into 11 groups of 10 birds each and housed in horsfal isolation units. the undiluted qr448(a) compound was given intranasally (200 l per bird), by spray (1 ml per bird), or in the drinking water (1 ml mixed with an equal volume of reconstituted non-fat dry milk per bird after 1 h of water starvation to promote drinking). the spray was delivered with a preval ® sprayer (precision valve corp., yonkers, ny) while blocking the fresh air delivery to the isolator. the preval ® spray applicator consists of a 475 ml refillable reservoir and a compressed air power unit. although not directly measured, the average particle size for this type of sprayer is in the range that would be deposited in the naso-oro-pharyngo-laryngeal region of the respiratory tract (phalen and mendez, 2009 ). the spray treatment lasted approximately 10 s and fresh air was resumed 10 min following treatment. for each of the delivery methods, qr448(a) was administered to the birds at either 6 h before challenge, 2 h before challenge, or 2 h after challenge. two non-treated groups of birds served as a challenged control and a non-challenged negative control. all of the treated birds and one of the non-treated groups were challenged with 1 × 10 4 eid 50 of the pathogenic mass41 strain of ibv per bird. the challenge virus was given intranasally and the birds were examined twice daily for clinical signs of the disease. at 5 days post-challenge the birds were killed and necropsied. at necropsy, tracheal swabs were collected and placed in ice cold pbs. the presence of virus was determined by extracting rna directly from the pbs, and quantitative real-time rt-pcr was conducted on the rna. clinical signs and gross lesions were recorded and tracheas were harvested for histopathology. clinical signs were scored as follows: 1 = normal, 2 = watery eyes or mucus in the nares, 3 = watery eyes and mucus in the nares, and 4 = watery eyes, mucus in the nares and tracheal rales. the tracheas were collected in 10% neutral buffered formalin, routinely processed into paraffin, and 5-m sections were cut for hematoxylin and eosin staining. epithelial hyperplasia, lymphocyte infiltration, and the severity of epithelial deciliation were scored for each trachea with 1 being normal and 4 being severe. for this experiment, 2-week-old spf leghorn chickens were divided into 20 groups of 10 birds each and housed in horsfal isolation units. half of the birds were treated 2 h before challenge and the other half were treated 2 h after challenge. the birds were treated with either undiluted or a 2-fold serial dilution of qr448(a) starting at a 1:5 dilution to a dilution of 1:1280. dilutions were prepared in sterile distilled deionized water, and 1 ml/bird was delivered via spray with a preval ® sprayer (precision valve corp.) as described in experiment 2. additional control groups of 10 birds each included in the experiment were birds that were not treated and challenged (challenge control group), birds treated by the spray route with undiluted qr448(a) and not challenged, and birds that were not treated and not challenged (negative control group). all of the treated birds unless otherwise indicated above and one of the non-treated groups were challenged with 1 × 10 4 eid 50 of the pathogenic mass41 strain of ibv per bird as described in experiment 2. all of the birds were examined for clinical signs, and necropsied 5 days post-challenge as described for experiment 2. clinical signs were scored as described above. for this experiment 290 1-day-old commercial broiler chickens that were not vaccinated, were divided into different groups and housed in horsfal isolation units. serum was collected from several birds at the beginning of the experiment to determine the level of maternal antibodies to ibv. antibodies were detected using a commercial enzyme linked immunosorbent assay (elisa) test (idexx, inc., westbrook, me). the birds were treated with a 1:20 dilution of qr448(a) in sterile distilled deionized water by spray with a preval ® sprayer (precision valve corp.) then challenged with 1 × 10 3.5 eid 50 of the mass41 strain of ibv at 2 h, 6 h, 1 day, 2 days, 4 days and 7 days after treatment. the birds were challenged intranasally as described in experiment 2. controls included birds that were not treated and challenged at those times, and a negative group that was not treated or challenged. birds challenged at 2, 4, 6, and 12 h were necropsied on the same day, 5 days after challenge, along with one non-treated/nonchallenged control group. birds challenged at 1, 2, 4, and 7 days after treatment were necropsied along with a negative (non-challenged) control group at 5 days after each of those challenge times. all of the birds were scored for clinical signs, and necropsied as described above. in this experiment, 240 commercial broiler chickens that were not vaccinated, were equally divided and housed in two 10 × 10 colony houses on pine shavings on the floor. colony houses were used so an adequate number of birds could be maintained together for the transmission experiment and to mimic as closely as possible commercial chicken house conditions. at 2 weeks of age 20 birds from each house were removed and wing-banded (for future identification) and given a full dose of modified live ibv arkansas type vaccine in the eye according to the manufacturers recommendations (intervet, schering-plough animal health, millsburo, de). the birds were held in isolation for 2 h before reintroducing them back into the colony houses. immediately after reintroducing the birds into the colony houses, all of the birds in one house were treated by spray using a preval ® sprayer (precision valve corp.) with 1 ml of a 1:20 dilution (in sterile deionized-distilled water) of qr448(a) per bird. we monitored the transmission of the virus to contact exposed birds by removing and swabbing the trachea of 25 birds at 3, 7, 10 and 14 days posttreatment. we also removed and swabbed 5 birds given the vaccine at each sample time. birds that were swabbed were removed from the experiment to avoid promoting the spread of the virus by inducing damage to the trachea and to prevent swabbing the same bird twice during the experiment. virus detection in the tracheal swabs was determined by real-time rt-pcr. serum was collected from 10 birds at the beginning of the experiment (2 weeks of age) to determine the level of maternal antibodies to ibv. in addition, we collected sera from all of the birds (n = 12) necropsied on the last day of the experiment and tested them for antibodies to ibv, newcastle disease virus, and infectious bursal disease virus. antibodies were tested using a commercial elisa test (idexx, inc.). virus titers were calculated by the method of reed and muench (villegas, 2008) . means and standard deviations were calculated for ct values and analyzed statistically with student's t-test, and the histopathological scores were analyzed by the kruskal-wallis test coupled with dunn's post-test, and transmission experiment data was analyzed with fisher's exact test using jmp statistical discovery software (sas institute, inc., cary, nc). 3.1. experiment 1: toxicity of qr448(a) in cell culture and in embryonating eggs, and effect on ibv in vitro undiluted and a 1 × 10 −1 dilution of qr448(a) was toxic to vero e6 cells and cells inoculated with a 1 × 10 −2 dilution of qr448(a) had cpe affecting about 50% of the monolayer. cells inoculated with a 1 × 10 −3 dilution or higher of qr448(a) were unaffected at 7 days post-treatment. the vero e6 cells inoculated with undiluted and a 1 × 10 −1 dilution of the diluent had cpe in >90% of the cells, whereas cells inoculated with higher dilutions of diluent did not have any cpe following 7 days of incubation. none of the negative control cells had cpe. disregarding embryo deaths within 24 h of inoculation, none of the embryonating eggs died during the course of the experiment. at 7 days post-treatment, none of the embryos that received qr448(a) or the embryos that received diluent had gross lesions. as expected, none of the negative control embryos had lesions. to determine the decrease in titer of infectious virus, we examined the effect of qr448(a) on ibv (beaudette strain) as determined by titration in vero e6 cells and the data are presented in fig. 1 . treatment with qr448(a) at a 1 × 10 −3 dilution reduced the titer of ibv from an average of 1 × 10 3.0 to 1 × 10 0.8 tcid 50 /ml. treatment at a dilution of 1 × 10 −4 reduced the ibv titer from 1 × 10 3.0 to 5 × 10 2.0 tcid 50 /ml. none of the dilutions of qr448(a) higher than 1 × 10 −4 reduced the titer of ibv. no reduction in virus titer was observed for the diluent at any dilution and, none of the cells in the negative control wells (cell culture maintenance medium alone) had cpe (data not shown). data showing the effect of qr448(a) on ibv (beaudette strain) as determined by titration in embryonating eggs also measured the decrease of infectious virus and the data are presented in fig. 1 . titration of ibv beaudette strain in vero e6 cells following treatment with 10-fold serial dilutions of qr448(a) starting at 1 × 10 −3 to 1 × 10 −9 in cell culture maintenance medium and cell culture maintenance medium plus ibv alone (control) are presented. the 50% tissue culture infectious dose (tcid50) titers reflect the average of three replicates. no reduction in virus titer was observed for ibv treated with the diluent (data not shown). fig. 2 . treatment with undiluted qr448(a) reduced the titer of ibv from 1 × 10 4 to 1 × 10 2 eid 50 /ml. dilutions of qr448(a) at 1 × 10 −1 and 1 × 10 −2 reduced the titer of ibv from 1 × 10 4 to 1 × 10 1.7 and 1 × 10 3 eid 50 /ml respectively. no reduction in ibv titer was observed when dilutions of qr448(a) from 1 × 10 −3 to 1 × 10 −7 were used or when the diluent at any dilution was used. the lesions in the embryos, were typical of those reported for ibv (gelb and jackwood, 2008) . clinical signs were observed in all of the mass41 virus challenged groups of birds regardless of treatment but in the intranasal and spray treated groups, fewer birds had signs and the signs were milder, as reflected by lower average scores (table 1) . virus was detected in the birds treated by the intranasal route 6 h before challenge but no virus was detected in the other intranasal treatment fig. 2 . titration of ibv beaudette strain in embryonating eggs following treatment with 10-fold serial dilutions of qr448(a) starting at 0 (no dilution) to 1 × 10 −7 in pbs, and pbs plus ibv (control) are presented. the 50% embryo infectious dose (eid50) titers reflect the average of two replicates. no reduction in virus titer was observed for ibv treated with the diluent (data not shown). clinical signs and virus detection in 2-week-old spf chickens following treatment with qr448(a) before or after ibv challenge a . water 6 h before challenge 10/10 (3.5) 9/10 23.13 3.50 a water 2 h before challenge 10/10 (3.2) 10/10 23.31 3.40 a water 2 h after challenge 10/10 (3.5) 10/10 24.72 3.39 a challenge control g 10/10 (4.0) 10/10 26.01 3.52 a negative control h 0/10 (1.0) 0/10 neg 1.06 b a the birds were intranasally challenged with 3.1 × 10 4 embryo infectious dose50/bird of pathogenic ibv strain mass41. b clinical signs were recorded 5 days following challenge and were scored as follows: 1 = normal, 2 = watery eyes or mucus in the nares, 3 = watery eyes and mucus in the nares, and 4 = watery eyes, mucus in the nares and tracheal rales. c virus was detected directly from tracheal swabs collected 5 days following challenge by real-time rt-pcr. d the average cycle threshold (ct) value for only the positive samples was calculated and indicates the relative amount of virus detected in the trachea (higher numbers = less virus). e the average histopathology score for only the birds with clinical signs was based on epithelial hyperplasia, lymphocyte infiltration, and epithelial deciliation with 1 being normal and 4 being severe. numbers with different superscripts are statistically different (kruskal-wallis test, p ≤ 0.05). f negative. g challenge control birds were not treated and were challenged. h negative control birds were not treated or challenged. groups or in the spray treated groups. clinical signs were observed in all of the birds treated with qr448(a) in the water, and the clinical sign scores were higher than the intranasal and spray treated birds. virus was detected in all but one bird (6 h before challenge group, see table 1 ) receiving qr448(a) in the water. severe clinical signs were observed and virus was detected in all of the ibv challenged birds that did not receive treatment. in addition, all of the negative control birds remained negative for clinical signs and virus detection. tracheas were scored as described in section 2 with 1 being normal and 4 being severe, and the birds that did not receive challenge virus had an average lesion score of 1.06. histopathology scores for all of the treated and challenged birds that had clinical signs were statistically different from the non-challenged controls but not from the challenged controls (p ≤ 0.05). birds that did not have clinical signs regardless of the group were not statistically different from the negative control birds (data not shown). a dose response was observed in the birds treated 2 h before mass41 virus challenge (fig. 3) . a less pronounced response was observed in birds treated 2 h after virus challenge. virus was detected in the trachea of all of the challenged birds, but treatment with the lower dilutions of qr448(a) 2 h before challenge, had a more pronounced effect on lowering the amount of virus detected in the trachea. in general, the clinical signs were more severe and involved more birds at the higher dilutions or qr448(a) (see average scores, table 2 ). tracheas were scored as described in section 2 with 1 being normal and 4 being severe, and all of the birds given undiluted qr448(a) were scored as normal (average score = 1.10). statistically lower (p ≤ 0.05) lesion scores (table 2) were observed for qr448(a) at a 1:20 dilution or lower when it was administered by spray treatment 2 h before challenge. spray treatment with qr448(a) did not statistically lower the tracheal table 2 experiment 3: dose titration of qr448(a) spray treatment in 2-week-old specific pathogen free chickens 2 h before or 2 h after challenge with pathogenic ibv. lesion scores when given 2 h after challenge. birds that were not treated and received the challenge virus had an average tracheal lesion score of 3.00. ten birds were bled at 1 day of age and as expected all of the nonvaccinated commercial broilers were positive for maternal antibodies to ibv (average titer = 2542) by elisa (idexx laboratories). the numbers of birds showing clinical signs of ibv infection and the average clinical sign scores are shown in table 3 . the number of birds with clinical signs that were treated with qr448(a) and challenged with mass41 within 4 days of treatment ranged from 0 to 50%. in birds challenged at 7 days after treatment, 90% of the birds had clinical signs. at each of the sample days, we observed 80-100% of the birds (78% at day 1) in the non-treated mass41 challenge control group with clinical signs indicating that maternal antibodies did not prevent the disease. none of the non-challenged birds had clinical signs. in general, clinical signs in the treated challenged birds consisted of watery eyes or mucus in the nares with an occasional bird having both. the signs were not unlike a vaccine reaction, and were considered to be extremely mild as evidenced by the average clinical sign scores. in comparison, the non-treated challenged birds had more severe signs consisting of watery eyes, mucus in the nares, swollen sinuses, and excessive mucus in the trachea resulting in tracheal rales. challenge virus was detected in all of the challenged birds, even in the qr448(a) treated birds (table 4 ), indicating that there was no protection from infection. although the data were not statistically different, qr448(a) treated birds that were challenged at 2 h, 6 h, 1 day, 2 days, 4 days, and 7 days had fewer viral genome copies than non-treated challenge groups at those same times respectively. no virus was detected in the tracheas of birds that were not challenged. tracheas were scored as described in section 2 with 1 being normal and 4 being severe, and the birds that did not receive challenge virus had a normal 1.00 average score ( table 5 ). birds that were not challenged did not have any appreciable lesions in the trachea, and the tracheal lesion scores in the non-challenged birds that were treated with qr448(a) were not statistically different from the nontreated, non-challenged birds (p ≤ 0.01). the tracheal lesion scores for the treated and challenged group were statistically higher than the non-challenged birds but were not as high as the non-treated challenged birds (p ≤ 0.01). the positive control (non-treated challenged) birds all had tracheal lesion scores statistically higher than the other groups (p ≤ 0.01). no virus was detected in the birds vaccinated with arkansas type ibv at 3 and 14 days post-infection (fig. 4) . virus was detected in 1 of 5 vaccinated birds in the treated group at 7 days post-vaccination table 3 experiment 4: clinical signs a in broiler chickens challenged with ibv at various times after treatment with qr448(a) at 1 day of age. clinical signs consisted of watery eyes, tracheal rales, and mucus in the nares and the trachea. b each bird was given 1 × 10 3.5 eid50 of the mass41 strain of ibv intranasally. c number of birds with clinical signs per total (average score). clinical signs were scored as follows: 1 = normal, 2 = watery eyes or mucus in the nares, 3 = watery eyes and mucus in the nares, and 4 = watery eyes, mucus in the nares and tracheal rales. average scores with different capital letter superscripts are statistically significant at p ≤ 0.01 (kruskal-wallis test). d clinical sign scores for negative control birds were all normal (1.0). birds were necropsied at 5 days post-challenge, and tracheas were collected in 10% neutral buffered formalin and routinely processed and stained (see section 2), then scored for epithelial hyperplasia, lymphocyte infiltration, and epithelial deciliation with 1 being normal and 4 being severe. scores with different capital letter superscripts are statistically significant, kruskal-wallis test (p ≤ 0.01). b birds that received challenge virus were given 1 × 10 3.5 eid50 of the mass41 strain of ibv intranasally. and in 3 of 6 and 2 of 5 vaccinated birds in the treated and non-treated groups respectively at 10 days post-infection. transmission of ibv from vaccinated to contact exposed birds was monitored by detecting the virus in the trachea using real-time rt-pcr and the percent of contact exposed birds positive for ibv is presented in fig. 4 . in the treated group, virus was not detected in the contact exposed birds at 3 and 7 days post-exposure. in the nontreated group 1 of 25 birds was positive at 7 days post-exposure. at 10 days post-exposure, 1 of 24 contact exposed birds was positive in the treated group whereas 3 of 25 birds were positive in the nontreated group. at 14 days post-exposure, 1 of 25 contact exposed and treated birds was positive for virus. this is in contrast to the non-treated group where 5 of 23 contact exposed birds were positive at 14 days post-exposure. the data were statistically different at 14 days (p ≤ 0.03) but not at any other day post-exposure. serum collected from 10 of the nonvaccinated commercial broilers at 1 day of age (these are the same birds bled in experiment 4) had an average maternal antibody titer against ibv of 2,542. antibodies to ibv were not detected by elisa (n = 12) at 2 weeks of age when the birds were vaccinated. serum was collected from all of the birds necropsied at the end of the experiment and none of the birds were positive for antibodies to ibv, none were positive for antibodies to ndv, and two birds in the treated contact group were positive for ibdv (average titer = 1038.5). the virucidal activity of essential oils, which are lipophilic by nature, likely act to disrupt the viral membrane or interfere with viral envelope proteins involved in host cell attachment (schuhmacher et al., 2003) . in this report, we examined the effect of a natural based product consisting of a synergistic blend of botanical oleoresins and essential oils in a liquid emulsion designated qr448(a) on ibv. the minimum effective concentration against ibv was determined in vitro using the beaudette strain of ibv because it is adapted to grow in cell culture. the optimal route and timing of administration, most effective dose, and the duration of effect on ibv in chickens were investigated using the widespread and pathogenic mass41 strain of the virus. because arkansas vaccine type viruses are frequently found circulating in commercial chicken flocks (jackwood et al., 2005) , we also examined the effect on transmission of modified live arkansas type ibv vaccine in 2-week-old broiler chickens. the lowest dilution of qr448(a) that did not cause toxic effects in vero e6 cells was capable of reducing the titer of ibv from 1 × 10 3 to 1 × 10 0.8 tcid 50 /ml. undiluted qr448(a) was not toxic for 10-dayold embryonating eggs and reduced the titer of ibv from 1 × 10 4 to 1 × 10 2 eid 50 /ml. the diluent did not reduce the titer, indicating that the oleoresins and essential oils in qr448(a) were responsible for the effect. next we sought to determine if the effects of qr448(a) are biologically significant in chickens infected with ibv. both positive and negative control groups were included in the in vivo studies however; we did not include a diluent control group because, in the first experiment, the diluent was shown not to have an effect on the virus titer. based on clinical signs, lesions and virus detection, the best routes of administration were determined to be intranasal and spray inoculation 2 h before challenge. in addition, it appears treatment intranasally or by spray 2 h after challenge was somewhat better than intranasal or spray treatment at 6 h before challenge. administration of qr448(a) by water had little effect regardless of treatment timing. based on microscopic lesions, it appears that qr448(a) alone does not adversely affect the epithelium of the trachea. it is interesting that we could not detect virus in some of the groups treated intranasally or by spray in this experiment. it appears that qr448(a) reduced the titer of ibv below an infectious threshold. infection often does not occur when birds are challenged with ≤1 × 10 2.5 eid 50 of the mass41 strain of ibv (jackwood, unpublished data) . it is also possible that inhibitors to the real-time rt-pcr reaction prevented detection; however, we were able to detect challenge virus in the other groups suggesting the presence of inhibitors are inconsistent or that another explanation is likely. since spray is suitable for mass application of commercial chickens, we used that route of administration to examine the minimum effective dose, duration of activity, and effect on transmission of ibv in chickens. birds treated by spray with qr448(a) 2 h before challenge showed a clear dose response between undiluted qr448(a) and a dilution of 1:40 (table 2 and fig. 3) . treatment 2 h after challenge had a less pronounced dose response, indicating that qr448(a) is less effective following virus attachment and entry into the host cell. this data and the timing of administration data above suggests that qr448(a) activity is virucidal rather than antiviral in nature. one-day-old broiler chickens treated with qr448(a) were protected from clinical signs of disease following challenge with pathogenic ibv up to 4 days post-treatment (table 3) . although maternal antibodies in chicks, which generally last from 1 to 2 weeks of age, and can provide some protection against ibv (mondal and naqi, 2001) , we observed clinical signs in most of the nontreated virus challenged control birds indicating that maternal antibodies did not play a role in protection from clinical signs. protection from infection was not observed since challenge virus was detected in all of the qr448(a) treated and challenged birds. however, at all but two challenge times (4 and 12 h) the amount of challenge virus in the trachea was slightly lower in the qr448(a) treated groups but the values were not statistically significant. in a previous study, where different amounts of ibv were given to chickens, it was reported that the amount of virus detected in a clinical sample from trachea correlated with dose at 1 day post-challenge but by 5 days post-challenge all of the birds had similar levels of virus in the trachea regardless of challenge dose (callison et al., 2006) . treatment with qr448(a) may have lowered the amount of virus in the birds but by 5 days post-challenge when we collected samples, little or no differences in viral load were observed. examining birds at 5 days post-challenge is the usda recommended procedure (usda, aphis title 9, code of federal regulations http://www.access.gpo.gov/nara/cfr/waisidx 08/9cfrv1 08.html) for evaluating protection from ibv challenge. since challenge virus was detected in the trachea of the qr448(a) treated birds it is possible that clinical signs would have been observed in more of the treated birds if they were observed beyond 5 days post-challenge. it is also possible that fewer or no clinical signs would be observed in those birds beyond 5 days post-challenge, since a delay in clinical signs may represent additional time for the birds to mount a protective immune response to the challenge virus. protection of broiler chickens for up to 4 days post-treatment from clinical signs of disease but not from infection may provide an opportunity to induce some level of local immunity due to the presence of the challenge virus. although we did not measure local immunity in these studies, protecting birds from severe upperrespiratory disease with qr448(a) and at the same time allowing field viruses to infect and induce an immune response may provide a mechanism to specifically protect chickens from resident field virus(es) regardless of the virus type. transmission of arkansas type ibv vaccine from infected to contact control pen mates was examined and we observed 22% of the non-treated contact exposed birds positive for ibv whereas only 4% of the treated contact exposed birds were positive for virus at 14 days post-exposure. clearly qr448(a) treatment diminished transmission of ibv to contact exposed birds and the effect was statistically significant (p ≤ 0.03). to ensure that maternal antibodies did not play a role in this experiment, we vaccinated the birds at 2 weeks of age when maternal antibodies were no longer detected. previously reported studies have shown that arkansas type vaccine is the most prevalent virus in the commercial broilers and is capable of persisting in the flock (jackwood et al., 2005; mckinley et al., 2008; nix et al., 2000; van santen and toro, 2008) . vaccine virus was used in this experiment because colony houses (necessary to house the number of birds needed for the experiment) are not biologically secure, preventing us from using pathogenic isolates of ibv. although this experiment was designed to be a model for the prevention of spread of pathogenic ibv isolates, the data directly address an extremely important problem of arkansas vaccine virus persistence in commercial broiler flocks. persistence of ibv in a flock can cause a severe vaccine reaction and provides opportunity for the vaccine virus to mutate and revert to pathogenicity as well as change antigenically avoiding the immune response of the bird. a reduction in transmission afforded by treatment with qr448(a) is extremely important since it will limit ibv transmission in the flock, and likely lessen or even prevent persistence of arkansas vaccine virus in flocks. since qr448(a) is effective against the pathogenic mass41 strain of ibv it is expected that it will also limit transmission of pathogenic field strains of ibv in commercial broilers and thereby decrease the severity of the disease. this is the first report on the use of a mixture of all natural ingredients, specifically botanical oleoresins and essential oils, as a control measure for ibv. mass delivery of qr448(a) by spray is compatible with application in a commercial setting and it is effective against both mass and ark type ib viruses, indicating that it ought to be effective against ibv regardless of serotype. the effect appears to be more pronounced on cell free virus indicating that the activity is likely virucidal, which is important because it may also be effective against other enveloped respiratory viruses like avian influenza virus and newcastle disease virus in commercial poultry, as well as other coronaviruses in animals and humans. in addition, since virucidal compounds work by inactivating viruses, rather than targeting specific virus genes or proteins, it reduces the likelihood that resistance will develop from mutations during virus replication. coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens coronaviruses in 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blocks e protein ion channels and inhibits coronavirus replication structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design drug design targeting the main protease, the achilles' heel of coronaviruses design of wide-spectrum inhibitors targeting coronavirus main proteases human coronavirus 229e papain-like proteases have overlapping specificities but distinct functions in viral replication key: cord-268930-y1cm58r6 authors: van aken, danny; benckhuijsen, willemien e.; drijfhout, jan w.; wassenaar, alfred l.m.; gorbalenya, alexander e.; snijder, eric j. title: expression, purification, and in vitro activity of an arterivirus main proteinase date: 2006-03-09 journal: virus res doi: 10.1016/j.virusres.2006.01.025 sha: doc_id: 268930 cord_uid: y1cm58r6 to allow the biochemical and structural characterization of the chymotrypsin-like “main proteinase” (non-structural protein 4; nsp4) of the arterivirus prototype equine arteritis virus (eav), we developed protocols for the large-scale production of recombinant nsp4 in escherichia coli. the nsp4 proteinase was expressed either fused to maltose binding protein or carrying a c-terminal hexahistidine tag. following purification, the nsp4 moiety of mbp-nsp4 was successfully used for structural studies [barrette-ng, i.h., ng, k.k.s., mark, b.l., van aken, d., cherney, m.m., garen, c, kolodenko, y., gorbalenya, a.e., snijder, e.j., james, m.n.g, 2002. structure of arterivirus nsp4—the smallest chymotrypsin-like proteinase with an alpha/beta c-terminal extension and alternate conformations of the oxyanion hole. j. biol. chem. 277, 39960–39966]. furthermore, both forms of the eav proteinase were shown to be proteolytically active in two different trans-cleavage assays. recombinant nsp4 cleaved the cognate nsp6/7and nsp7/8 site in in vitro synthesized substrates. in a synthetic peptide-based activity assay, the potential of the recombinant proteinase to cleave peptides mimicking the p9–p7′ residues of six nsp4 cleavage sites was investigated. the peptide representing the eav nsp7/8 junction was used to optimize the reaction conditions (ph 7.5, 25 mm nacl, 30% glycerol at 30 °c), which resulted in a maximum turnover of 15% of this substrate in 4 h, using a substrate to enzyme molar ratio of 24:1. the assays described in this study can be used for a more extensive biochemical characterization of the eav main proteinase, including studies aiming to identify inhibitors of proteolytic activity. many viruses with a single-stranded, positive-sense rna genome regulate their genome expression by synthesizing large precursor polypeptides (or polyproteins) that are subsequently subjected to limited proteolysis to produce functional viral proteins (for reviews, see dougherty and semler, 1993; gorbalenya and snijder, 1996; krausslich and wimmer, 1988; spall et al., 1997) . rna virus polyproteins that include replicative proteins are often processed autocatalytically, although in some virus groups cellular proteinases are also involved. in this manner, the expression of the non-structural proteins (or "replicase" complex) can be regulated in time and space, e.g. to produce alternative cleavage products or stable processing intermediates with unique functions (de groot et al., 1990; jore et al., 1988; lemm et al., 1994; ypma-wong et al., 1988) . viruses in the order nidovirales, which unifies the enveloped, positive-stranded arteriviridae, coronaviridae, and roniviridae spaan et al., 2005) , have a similar polycistronic genome organization, share a conserved array of homologous replicase domains, and use common transcriptional and (post)-translational strategies to regulate their genome expression. among these strategies, one of the most crucial is the proteolytic maturation of the replicase polyproteins pp1a and pp1ab that are translated from the incoming genome (den boon et al., 1991; ziebuhr et al., 2000) . equine arteritis virus (eav) is the prototype of the arterivirus family and has a genome of approximately 12.7 kb, of which fig. 1 . proteolytic processing of the eav replicase. (a) processing map of the 3175-amino acid eav replicase polyprotein pp1ab. the three eav proteinases (pcp␤, cp and sp), their cleavage sites and the eav nsp nomenclature are depicted. pcp␤, nsp1 papain-like cys proteinase; cp, nsp2 cys proteinase; sp, nsp4 ser proteinase; rdrp, rna-dependent rna polymerase; z, zinc finger; hel, helicase; n, nidovirus-specific endoribonuclease (nendou). (b) overview of the two alternative processing pathways that apply to eav pp1a (wassenaar et al., 1997) . the association of cleaved nsp2 with nsp3-8 (and probably also with nsp3-12) was shown to be a cofactor in the cleavage of the nsp4/5 site by the nsp4 proteinase (major pathway). alternatively, in the absence of nsp2, the nsp5/6 and nsp6/7 sites are processed and the nsp4/5 junction remains uncleaved (minor pathway). the status of the small nsp6 subunit (fully cleaved or partially associated with nsp5 and/or nsp7) remains to be elucidated. adapted from barrette-ng et al., 2002. the 5 -proximal three quarters contain the replicase gene. the 3 -proximal region of the genome includes seven open reading frames encoding the viral structural proteins, which are expressed from a nested set of subgenomic mrnas (de vries et al., 1990; snijder and meulenberg, 2001) . replicase gene expression ultimately yields (at least) 12 non-structural proteins ( fig. 1a ; snijder et al., 1994; van dinten et al., 1996; wassenaar et al., 1997) , which are produced from two primary genome translation products, the large polyproteins pp1a (1728 amino acids) and pp1ab (3175 amino acids), with the latter being expressed following a ribosomal frameshifting event (den boon et al., 1991) . the replicase subunits are released from the polyproteins by three virus-encoded proteinases (fig. 1) , of which the one located in nsp4 is responsible for the processing of the polypeptides that remain after nsp1 and nsp2 have been autocatalytically released from pp1a and pp1ab (snijder et al., 1992 (snijder et al., , 1994 (snijder et al., , 1995 van dinten et al., 1999; wassenaar et al., 1997) . since nsp4 controls the production of the viral rna-dependent rna polymerase and rna helicase, it has been termed the eav "main proteinase" (gorbalenya et al., 1991; ziebuhr et al., 2000) . the arterivirus nsp4 main proteinase belongs to the 3c-like serine proteinases, a distinct group of viral chymotrypsin-like proteolytic enzymes (barrette-ng et al., 2002; snijder et al., 1996; ziebuhr et al., 2000) . the arterivirus proteinase combines the catalytic triad his/asp/ser of canonical chymotrypsin-like proteinases with the substrate specificity of the 3c-like cysteine proteinases, a subgroup of chymotrypsin-like enzymes named after the picornavirus 3c proteinases. in the cleaved, 204-residue eav nsp4 (which equals gly-1065 to glu-1268 of the eav replicase polyproteins) the catalytic triad is formed by his-39, asp-65, and ser-120. cleavage sites recognized by the nsp4 proteinase carry a glu at the p 1 position (gln in one case) and a small amino acid (gly, ser, ala) at the p 1 position (using the cleavage site nomenclature of schechter and berger, 1967) . furthermore, nsp4 possesses a unique c-terminal domain (ctd) extension of unknown function, which is not found in most other chymotrypsin-like proteinases and might be involved in modulation of nsp4 activity (barrette-ng et al., 2002) . arterivirus replication depends on the regulation of replicase gene expression in time and space, in which nsp4 plays a key role. notably, an elegant (presumably) regulatory mechanism connected to nsp4-driven proteolysis was documented in the form of the differential processing of the nsp4-8 part of the eav replicase polyproteins ( fig. 1b ; wassenaar et al., 1997) . together, the two pathways yield a variety of products, whose role in the viral life cycle remains to be studied in detail. the biochemical characterization of the arterivirus main proteinase requires the large-scale expression and purification of an active form of the enzyme. in this paper, we describe the production of active, recombinant eav nsp4 from escherichia coli (e. coli), allowing the design of an in vitro cleavage assay, which was used to characterize the properties of nsp4 and its interaction with substrates. the expression system also formed the basis for structural studies, which led to the elucidation of the three-dimensional structure of eav nsp4 by x-ray crystallography (barrette-ng et al., 2002) . together, these studies are a major step towards dissecting the structure-function relation-ships of this key arterivirus enzyme and the future design of inhibitor drugs. expression plasmids pmaltnsp4 and pet-nsp4his were constructed using standard recombinant dna techniques and sequenced. the pmalt2i vector, a derivative of expression plasmid pmal-c2 (new england biolabs; wassenaar and snijder, unpublished data) uses the strong tac promoter and the male translation initiation signals to give high-level expression of the cloned sequences. target genes in the pet plasmid are under control of the strong bacteriophage t7 promoter. (i) construction of pmaltnsp4. a ncoi-xhoi restriction fragment from plasmid pl3440, encoding eav nsp4 (wassenaar et al., 1997) , was cloned into plasmid pbln+ (molenkamp, 2000) . the resulting plasmid (pl3440i) was digested with bamhi and xhoi and the desired fragment was cloned between the unique bamhi and sali restriction sites of pmalt2i. this plasmid (pmaltnsp4) encoded the eav nsp4 proteinase fused to the c-terminus of the e. coli maltose binding protein (mbp), with a (asn) 10 spacer and a thrombin cleavage site (leu-val-pro-arg/gly-ser) separating the mbp and nsp4 moieties of the fusion protein, which will be further referred to as mbp-nsp4. due to the cloning strategy, mbp-nsp4 lacked glu-204 (nsp4 numbering) and contained three additional amino acids at its c-terminus (leu-ala-ser; fig. 2c ). after digestion with thrombin, also the n-terminus of the protein carried three foreign amino acids (gly-ser-met; fig. 2c ). (ii) construction of pet-nsp4his. pl3440-e1268p (wassenaar et al., 1997) after cell lysis and ultracentrifugation (100,000 × g) the s100 fraction (lane 3) was loaded on an amylose affinity column and bound protein was eluted with buffer a supplemented with 10 mm maltose (lane 4). after an overnight incubation at 4 • c with 1 unit of thrombin per mg of mbp-nsp4 (lane 5), cleaved nsp4 was further purified by gel filtration (sephadex g-75), anion exchange chromatography, and repeated loading of the sample on an amylose column until a purity of more than 90% was reached (lane 6). although mass-spectrometry indicated that the purified nsp4 was homogeneous, the sample migrated as a double band, a repeatedly observed phenomenon with nsp4. proteins were separated by sds-page on a 10% polyacrylamide gel, which was stained with coomassie brilliant blue r-250. (b) expression and purification of nsp4his. samples were total lysates from non-induced (lane 1) and iptg-induced (lane 2) cultures of e. coli bl21(de3) cells transformed with pet-nsp4his or fractions made during purification (lanes 3-6). after cell lysis and ultracentrifugation, the s100 fraction was loaded on a talon affinity column (clontech) charged with cobalt chloride. samples were taken from the flow-through (lane 3), after washing the column with buffer d (lane 4), and with buffer d supplemented with 10 mm imidazole (lane 5). the bound nsp4his was eluted from the column with buffer e (lane 6). for buffer composition see section 2. as judged from the gel, the purity of the sample was more than 90%. (c) nucleotide-and amino acid sequences corresponding to the termini of the nsp4 gene in the mbp-nsp4 and nsp4his expression constructs. foreign amino acids are shown in bold face and indicated with "n" or "c". the top panel shows n-and c-termini of wild-type nsp4, which is produced from the eav replicase polyproteins by autoproteolysis of the nsp3/4 and nsp4/5 sites (indicated with ↓). both pp1a and nsp4 amino acid numbers are given. the middle panel shows the mbp-nsp4 fusion protein. after cleavage by thrombin, compared to native eav nsp4, three foreign amino acids (gly-ser-met) remain at the n-terminus. the amino acid sequence recognized by thrombin is underlined and the position of the thrombin cleavage site is indicated (↓). at the c-terminus of mbp-nsp4, due to the cloning strategy, the native glu-204 was replaced by a leu residue and two additional amino acids were introduced (ala-ser). the bottom panel shows the nsp4his protein. a translation initiation codon was added, resulting in an n-terminal foreign met residue upstream of the native p 1 gly residue. at the c-terminus of nsp4his, the native glu-204 was replaced by a gly residue, which is followed by the (his) 6 tag. was digested with restriction enzymes ncoi and stui and the desired fragment was cloned into a ncoi and bamhi-digested pet34h vector (wassenaar and snijder, unpublished data) together with a bamhi-digested gly-(his) 6 -encoding linker (5 -gggcatcaccatcaccatcactaagggctgcagcc-aagctgacggatcc-3 ). the resulting plasmid encoded nsp4 with an additional met at its n-terminus and a c-terminal (his) 6 tag ( fig. 2c) . as a precaution, glu-204 (the p 1 residue of the nsp4/5 cleavage site) was substituted with gly to ensure that the (his) 6 tag would not be cleaved off as a result of nsp4 activity. the resulting protein will be further referred to as nsp4his. the active site mutation ser-120 to ile, which was documented to inactivate the nsp4 proteinase , was transferred to pet-nsp4his and the mutant protein (nsp4his-s120i) was used as a negative control in the activity assays. (iii) construction of plnsp6-8 and plnsp6-7. plnsp6-8 was constructed by self ligation of a ncoi and smai digested pl1ae1451p plasmid of which the ncoi site was filled in using the 5 → 3 polymerase activity of the large (klenow) fragment of dna polymerase i. plnsp6-7 was a derivative of plnsp6-8, in which the sequence (5 -tatgaaggccta-3 ) encoding the p 2-p 2 residues of the nsp7/8 cleavage site was replaced (by 5 -ctagctagctaga-3 ), changing tyr-glu-gly-leu to leu-ala-ser-stop. e. coli strain bl21(de3), which carries an iptg-inducible t7 rna polymerase gene in its chromosome, was transformed with the expression plasmid and grown overnight in 3 ml luria-bertani liquid medium supplemented with 100 g/ml of ampicillin (lba). subsequently, 1 ml of the overnight culture was used to inoculate a 3-1 flask containing 1 l of lba. cultures were grown at 37 • c to an od 600 of 0.7 and subsequently protein expression was induced by adding 1 mm of isopropyl-␤-d-thiogalactopyranoside (iptg). bacteria were harvested 5 h after induction by centrifugation at 8000 × g for 15 min and cell pellets were stored at −80 • c or directly used for further processing. during the purification procedure, samples and buffers were kept at 4 • c. the pelleted cells were resuspended in buffer a (20 mm tris ph 8.1, 150 mm nacl, 1 mm edta) and lysed by passing them twice through a 40 k french pressure cell (slm-aminco) at 1000 psi (6.9 mpa). subsequently, the lysate was centrifuged at 100,000 × g for 30 min, resulting in a supernatant (s100) and a pellet (p100) fraction. the s100 fraction was loaded onto an amylose affinity column (new england biolabs), which was washed with buffer a until the od 280 of the flowthrough dropped below 0.05. subsequently, the bound fusion protein was eluted from the column in 3-ml fractions using buffer a supplemented with 10 mm maltose. fractions containing the mbp-nsp4 fusion protein (as determined by sds-page analysis) were pooled and the concentration of the fusion protein was determined using the bradford protein assay (biorad). generally, 1 l of culture yielded about 50 mg of fusion protein. fusion protein-containing fractions were diluted to a concentration of 1 mg/ml and incubated for 16-18 h at 4 • c with 1 unit of thrombin (from human plasma; sigma) per mg of fusion protein. after dialysis against buffer b (20 mm tris ph 8.1, 10 mm nacl, 1 mm edta) at 4 • c, the cleaved fusion protein was loaded onto an anion exchange column (mono q, hr 5/5, pharmacia) and washed extensively with buffer b to remove traces of maltose and contaminating proteins. bound proteins were eluted from the column in 3-ml fractions using a 50 ml gradient of 10-500 mm nacl in 20 mm tris ph 8.1 and l mm edta. nsp4containing fractions were pooled, concentrated (10 mg/ml) and 1 ml fractions were loaded onto a sephadex g-75 gel filtration column (total bed volume 210 ml). fractions from this column containing nsp4 were repeatedly loaded onto an amylose affinity column, until all detectable traces of mbp, as judged from sds-page and coomassie brilliant blue r-250 staining, had been removed. nsp4 preparations were diluted in buffer b to a concentration of 1-5 mg/ml and stored at −80 • c. the purity and integrity of the nsp4 samples were confirmed by mass spectrometry. e. coli strain bl21(de3) was transformed with expression plasmid pet-nsp4his or pet-nsp4his-s120i and a single colony was incubated in 1 ml lba at 37 • c until the culture became turbid (approximately 3 h). subsequently, 50 l of the culture was plated on a lba agar plate and incubated overnight at 37 • c. the plate was sliced into small pieces and added to a 2-l flask containing 500 ml of lba. this culture was grown to an od 600 of 0.7 and subsequently protein expression was induced with 1 mm of iptg. bacteria were harvested 5 h after induction by centrifugation at 8000 × g for 15 min at 4 • c. cell pellets were either stored at −80 • c or immediately used for further processing. during the purification procedure, samples and buffers were kept at 4 • c. cells were resuspended in buffer c (50 mm phosphate ph 8.1, 500 mm nacl), lysed and centrifuged using the same method as described above for the purification of mbp-nsp4. the s100 fraction was loaded onto a talon metal affinity resin (clontech) column (total bed volume 10 ml) and the column was washed with buffer c until the od 280 of the flow-through dropped below 0.05. subsequently, the column was washed with 10 column volumes of buffer d (20 mm tris ph 8.1, 150 mm nacl), and 10 column volumes of buffer d supplemented with 10 mm imidazole, to wash off non-specifically bound proteins. nsp4his was eluted from the column with buffer e (5 mm tris ph 8.1, 25 mm nacl, 100 mm imidazole). nsp4his was more than 90% pure as judged from a coomassie brilliant blue r-250 stained protein gel and generally a yield of 15 mg nsp4his per 1l culture was obtained. nsp4his preparations were diluted with buffer g (5 mm tris ph 8.1, 25 mm nacl) to a concentration of 1-5 mg/ml and stored at −80 • c. using the tnt system (promega), plasmids plnsp6-7 and plnsp6-8, encoding eav nsp6-7 and nsp6-8, respectively, were in vitro transcribed and the resulting rna was translated in the presence of [ 35 s] methionine (amersham). typically, transcription/translation reactions with a volume of 25 l were carried out for 90 min at 30 • c. the reaction was stopped by adding 1/10 volume of 10× stopmix (20 mm methionine, 5 mg/ml cycloheximide (sigma), 1 g/ml rnase a) and 3 l of the reaction was incubated with 5 g of either purified nsp4his, mbp-nsp4, or non-purified cleaved mbp-nsp4 in 10 mm tris ph 8.1 containing 150 mm nacl, in a total volume of 15 l. samples of 3 l were taken at regular intervals up to 120 min after addition of the proteinase and reaction products were analyzed on 15% tricine gels. as a negative control, 5 g of purified nsp4his-s120i was used as enzyme in the reaction. peptide substrates were synthesized by solid-phase strategies on an automated multiple peptide synthesizer (syroii, multi-syntech, witten, germany). the purity of the peptides was determined by analytical reversed-phase hplc and proved to be at least 80%. the identity and homogeneity of the peptides was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and analytical reversed-phase chromatography. 16-mer peptides (representing the p9-p7 residues of a cleavage site) were synthesized by solid-phase chemistry (table 1) . each peptide was dissolved in 100% dmso at a concentration of 100 g/l and stored at −20 • c. the nsp4 activity assay was performed in a reaction volume of 100 l, using 25 l of nsp4 (0.8 g/l) and 0.2 l peptide (100 g/l) in the appropriate buffer to yield a final concentration of 0.2 g/l (∼10 m) nsp4 and 0.2 g/l (∼100 m) peptide in the assay. cleavage reactions were routinely incubated at 37 • c for 0.5-6 h. the reactions were terminated by the addition of 100 l of 10% trifluoroacetic acid and stored at −80 • c. prior to analysis on hplc, samples were centrifuged for 5 min at 14,000 × g to remove insoluble components. cleavage products were resolved using a 28 min, 5-90% linear gradient of acetonitrile in 0.1% trifluoroacetic acid. the absorbance was determined at 215 nm. peak areas were calculated by integration. the large-scale expression and purification of an mbp-nsp4 fusion protein was achieved using the protocol described in section 2. briefly, after induction of expression, large amounts of mbp-nsp4 fusion protein were recovered from the soluble s100 fraction ( fig. 2a) and purification of the protein using an amylose resin affinity column yielded a more than 90% pure fusion protein (as judged from coomassie brilliant blue r-250 stained protein gels). to prevent exposure of the nsp4 proteinase to relatively high temperatures, the release of nsp4 from the mbp moiety by cleavage with thrombin was not performed at the recommended temperature of 25 • c, but at 4 • c. an overnight digestion with 1 unit thrombin per mg of fusion protein was sufficient to cleave virtually all of the fusion protein ( fig. 2a) . nsp4 was further purified by anion exchange chromatography, gel filtration, and repeated loading of the nsp4-containing samples onto an amylose resin affinity column to remove all remaining traces of mbp. due to the cloning strategy, nsp4 lacked the c-terminal glu-204 residue (nsp4 numbering) and carried three additional amino acids at its extreme c-terminus (leu-ala-ser). after cleavage by thrombin, also the n-terminus of the proteinase remained attached to three foreign residues (gly-ser-met) (fig. 2c) . although mass spectrometry indicated that the purified nsp4 sample was homogeneous and of the expected molecular mass, there were two bands visible on the gel ( fig. 2a) , which were both recognized by a nsp4-specific antiserum (data not shown). it has been reported before that nsp4, with a predicted molecular mass of 21 kda, migrates as a 30 kda protein . presumably, nsp4 is prone to aberrant migration during sds-page. nevertheless, the recombinant protein obtained using this protocol was of sufficient purity and homogeneity to be successfully used for crystallogenesis and the subsequent nsp4 structure determination by x-ray crystallography (barrette-ng et al., 2002) . as alternative to the use of a mbp fusion protein, it was decided to employ the production of a c-terminally (his) 6 tagged protein as a second expression/purification strategy, a all peptides contained a protective b (amide) at their c-terminus. b c (cysteine) residues were replaced by o (2-aminobutyric acid). which was previously used to successfully purify many proteinases, including several from the 3c-like proteinase group (chisholm et al., 2001; someya et al., 2000; tibbles et al., 1999) . based on the specific affinity of histidine residues for cobalt ions, a convenient one-step purification could be performed using a chelating agarose column charged with cobalt chloride. we reasoned that the (his) 6 tag purification could be more efficient compared to the mbp-mediated purification, as it circumvents the potentially harmful thrombin treatment and does not include the laborious removal of mbp. the protocol described below essentially followed the original manufacturer's protocol for c-terminally his tagged proteins (described in section 2). however, some adjustments had to be made regarding the induction of protein expression. when an overnight liquid culture of pet-nsp4his-transformed e. coli bl21(de3) bacteria was used to inoculate a larger culture, induction of protein expression with iptg was, for unknown reasons, not successful. however, nsp4his synthesis could be induced in cultures grown for shorter periods of time or in colonies picked from freshly transformed plates. therefore, instead of using an overnight liquid culture as an inoculum, a droplet from a 3 h culture was plated on a lba plate, which was incubated overnight at 37 • c, sliced into small pieces, and then added to lba-containing flasks. with this method large cultures could be grown to the right density in a short period of time. after harvesting, lysis, and centrifugation of the cells, the s100 fraction was loaded on a column packed with talon metal affinity resin (clontech) charged with cobalt ions and the column was washed extensively with several buffers (see section 2) to remove contaminating proteins. after the elution of nsp4his from the column, the protein was more than 90% pure, as judged from a coomassie brilliant blue r-250 stained sds-page gel, and generally a yield of 15 mg of nsp4his protein per 1-l culture was obtained (fig. 2b) . active site mutant nsp4his-s120i was purified using the same protocols and yielded comparable quantities of protein. with this expression-and purification protocol, nsp4his could be purified within 8 h, whereas the mbp-nsp4 expression, cleavage and purification protocol would generally take 2 days. the differences between the amino acid sequences at the n-and c-termini of both expression products, and their comparison to the termini of native eav nsp4, are depicted in fig. 2c . to test whether active recombinant proteinases had been isolated, the proteolytic activity of purified mbp-nsp4 and nsp4his was tested in cleavage assays using in vitro synthesized substrates as described in section 2. substrates represented eav nsp6-7 and nsp6-8, which are naturally occurring processing intermediates of the minor processing pathway (wassenaar et al., 1997) . aliquots from in vitro translation reactions were incubated with purified recombinant nsp4his or nsp4his-s120i. samples were taken at 0, 15, 30, 60 and 120 min after addition of proteinase and analyzed on 15% tricine sds-page gels (fig. 3) . processing of the two substrates could be easily monitored since cleavage of the nsp6/7, nsp7/8 or both sites resulted fig. 3 . eav nsp4his activity in an in vitro cleavage assay. using the tnt system (promega), expression plasmids encoding eav nsp6-7 and nsp6-8 were in vitro transcribed and the resulting rna was in vitro translated in a rabbit reticulocyte lysate in the presence of [ 35 s] methionine. after a 90-min incubation at 30 • c, transcription and translation were stopped (for details see section 2). subsequently, from a typical 25 l reaction, 3-l aliquots were taken and incubated with 5 g of purified nsp4his or nsp4his-s120i (negative control) for up to 120 min in a total reaction volume of 15 l. samples were analyzed on a 15% tricine gel and labelled products detected by autoradiography using a phosphoimager (biorad). the in vitro assay yielded the expected processing products, as predicted from known nsp4 cleavage sites. in addition, a number of smaller cleavage products was detected (indicated with arrows), which may be derived from internal cleavage of nsp7 (see text). processing products nsp6 and nsp8 were not detected due to their small size of 2.3 and 5.5 kda, respectively. in a substrate size reduction of 2.3, 5.5, or 7.8 kda, respectively. fig. 3 illustrates that after a 15-min incubation with nsp4his about half of the nsp6-7 substrate had been processed, although the remaining substrate was processed much slower. similar results regarding the processing of the nsp6/7 site were obtained with the nsp6-8 substrate, but the second cleavage site in this substrate (the nsp7/8 site) was processed much faster. already after 15 min, almost no nsp6-8 or nsp7-8 could be detected, suggesting that the nsp7/8 site had been cleaved in all of the available substrates (fig. 3) . surprisingly, processing of each of the substrates produced several additional products (indicated with arrows in fig. 3 ). these were not observed after incubation with the inactive control enzyme nsp4his-s120i or when the reaction was performed without enzyme, suggesting that the generation of these products depended on the proteolytic activity of nsp4his. based on their sizes, these products may have been derived from internal cleavage of nsp7, a possibility that is currently being investigated in more detail. using an identical experimental set-up, partially purified cleaved and uncleaved mbp-nsp4 were also tested for proteinase activity. in these assays, no differences could be observed between these two proteinases, which both displayed activities similar to those of nsp4his. taken together, these results confirmed the activity of the eav nsp4his and mbp-nsp4 proteinases that had been purified from e. coli. the in vitro cleavage assay described in the previous paragraph is less useful for quantitative biochemical analysis of nsp4 catalytic activity, since the quantification of the substrate is not straightforward. consequently, an alternative assay based on the use of synthetic peptides as substrates was developed. using this approach other 3c-(like) proteinases, like those of the human coronavirus 229e, the feline infectious peritonitis virus, and several picornaviruses (cordingley et al., 1989; hammerle et al., 1991; hegyi et al., 2002; jewell et al., 1992; ziebuhr et al., 1997) , have been successfully characterized. a set of 16-mer peptides (table 1) , mimicking the p 9-p 7 positions of the six nsp4 cleavage sites downstream of nsp5 (ziebuhr et al., 2000) , was synthesized and the peptides were tested for their potential to be cleaved. in the initial experiment, 10 g of each peptide (final concentration approximately 100 m) was incubated at 25 • c with 37.5 g of either nsp4his or nsp4his-s120i (both at a concentration of 18 m), or without nsp4 proteinase, in a 100-l volume of a buffer containing 20 mm tris ph 8.1, 150 mm nacl, 1 mm edta. samples were taken directly after addition of nsp4 (t = 0 h) or after 3-and 6-h incubations and were analyzed by reversed-phase hplc. as an example, the results for peptide 222-36, representing the nsp7/8 cleavage site, are shown in fig. 4 . following incubation with nsp4his, the main peak representing the uncleaved peptide was reduced and the peptide was apparently converted into two new products. incubation with nsp4his-s120i or an assay without nsp4his did not yield these two new peaks, indicating that they were derived from nsp4his activity. when mass spectrometry was used to analyze the two newly formed products, it was found that the peaks with retention times of 8 and 9.7 min (fig. 4) were of the same mass as the c-terminal and n-terminal fragments of the peptide, respectively, assuming cleavage at the glu/gly bond representing the established eav nsp4 cleavage site in this substrate. all peptides could be cleaved under the selected conditions (approximately 25% hydrolysis after 6 h) without obvious differences between the peptides being detected (data not shown). the peptide representing the nsp7/8 cleavage site was selected to further optimize reaction conditions because of its slightly higher cleavage potential. although the purified eav nsp4his proteinase showed catalytic activity on all peptides, its activity was very weak compared to that of other 3c-like proteinases in similar experiments (cordingley et al., 1989; hammerle et al., 1991; hegyi et al., 2002; jewell et al., 1992; ziebuhr et al., 1997) . therefore, to investigate whether a differently purified nsp4 proteinase might perform better, the catalytic activity of nsp4his proteinase was compared to that of a partially purified mbp-nsp4 proteinase. the mbp-nsp4 protein was not further purified after the thrombin cleavage step, but instead the cleaved fusion protein sample was directly used in the peptide cleavage assay. a similar experimental set-up using hcv 229e 3clpro had shown that such a limited purification did not negatively affect the ability of the proteinase to cleave peptides (ziebuhr et al., 1995) . however, the enzymatic properties of the partially purified eav mbp-nsp4 were not different from those of the (his) 6 tagged nsp4 proteinase (data not shown), suggesting that the low catalytic activity had a different cause. the possibility of inhibition of the proteinase by contaminants or instability of the enzyme after storage was investigated by an additional wash on the column fig. 4 . eav nsp4his activity in an in vitro cleavage assay using synthetic peptide substrates. one hundred micromolars of peptide 222-36 was incubated with 10 m nsp4his according to the protocol described in section 2 and reaction products were analyzed by reversed phase hplc. the results are shown for a reaction without proteinase (panel a) and a reaction to which nsp4his had been added (panel b). by using mass spectrometry, the peaks with retention times of 8 and 9.7 min in the hplc profile of the sample incubated with nsp4his were identified as the c-terminal-and n-terminal fragments, respectively, of the 222-36 peptide substrate. with 60% isopropanol (franken et al., 2000) or by using the purified protein directly in the assay instead of after storage at 4 • c or −80 • c. however, the additional washing step did not increase catalytic activity and no differences were found between a freshly prepared sample and preparations stored at 4 • c for 3 days or stored at −80 • c for 2 weeks. this indicated that catalytic activity was not lost during storage, but was already low upon isolation of the enzyme, possibly due to the purification method and/or intrinsic properties of nsp4. alternatively, the parameters of the in vitro cleavage reaction might have been suboptimal. to address the latter possibility, the catalytic activity of nsp4his was compared at different temperatures (20, 25, 30 and 37 • c) using the starting buffer composition (20 mm tris ph 8.1, 150 mm nacl, 1 mm edta). although hydrolysis of the peptide turned out to be slightly faster at 37 • c, the differences in comparison to the other temperatures were marginal (data not shown). in an attempt to further optimize the conditions of the cleavage assay, 20 g of peptide 222-36 (final concentration 200 m) was incubated with 17.5 g of nsp4his (final concentration 8.3 m) in a volume of 100 l at 37 • c in different buffers that varied systematically in ph, nacl and glycerol concentration. the ph range tested included 6 (25 mm mes), 7 (25 mm mops), 8 (25 mm tris), and 9 (25 mm glycine) and these ph values were combined with nacl concentrations of 25, 500, and 1000 mm and glycerol concentrations of 10, 20, and 30%. after a 4-h incubation, the reaction was stopped by adding 100 l of 10% trichloroacitic acid and samples were analyzed using reversed-phase hplc. since the peak with a retention time of 8 min, representing the c-terminal part of the peptide, had a very low absorbance at 215 nm, only the peak with a retention time of 9.7 min (fig. 4) was quantified by integration. samples could not be quantified when less than 3% of the substrate was cleaved. although differences in catalytic activity were observed using the different buffers, no significant improvements were found compared to the starting reaction conditions in which 15% of the substrate was cleaved during a 4-h incubation at 37 • c. in the ph 6 buffer and at nacl concentrations of 500 and 1000 mm, the catalytic activity of nsp4 was even below detection limit. no significant differences were found between reactions performed at ph 7, 8, or 9, but catalytic activity seemed slightly enhanced by increasing the concentration of glycerol (data not shown). since high nacl concentrations inhibited nsp4his activity, cleavage conditions were further optimized by titrating the nacl concentration with 50-mm increments between 0 and 500 mm at a ph of 7.5 (25 mm mops) and a glycerol concentration of 10%. although the catalytic activity was slightly higher at glycerol concentrations of 30%, a lower concentration was chosen for technical reasons. however, increasing the nacl concentration above 25 mm only reduced the catalytic activity in the assay (data not shown). finally, the effect of the presence of 10 mm dtt and 2.5 mm edta on catalytic activity was investigated, but addition of these compounds did not make a detectable difference (data not shown). this report describes two protocols for the expression and purification of recombinant eav nsp4 and two approaches to assess the proteolytic activity of the purified enzyme in vitro. both purification protocols resulted in the isolation of highly pure nsp4 proteinase, a conclusion supported by the subsequent use of the nsp4 moiety of mbp-nsp4 crystallization and structural studies (barrette-ng et al., 2002) . the results from the assays in which we used substrates synthesized in rabbit reticulocyte lysates showed that the nsp4 proteinase was able to process its cognate cleavage sites in the nsp6-8 and nsp7-8 substrates, indicating that an active enzyme had been purified. although this was not a quantitative assay, the eav nsp4 proteinase displayed kinetics that were comparable to those of other 3c-like proteinases in similar experiments (grubman et al., 1995; thole and hull, 2002; tibbles et al., 1996 tibbles et al., , 1999 ziebuhr et al., 1995) . the unexpected small reaction products visible in fig. 3 , at first suggested aberrant nsp4 activity or additional cleavages by other proteinases in the system, possibly following activation by nsp4. however, re-examination of protein gels from several previous independent experiments, in which we used pp1a expression systems and eav-infected cells to investigate nsp4 activity confirmed the existence of minor bands of approximately the same size as the unidentified bands in fig. 3 , making it more plausible that these bands were derived from genuine nsp4 activity. a more elaborate analysis of the presence of internal cleavage sites for nsp4 in nsp7 will be presented elsewhere (van aken et al., manuscript in preparation) . to characterize nsp4 catalytic activity in more detail, assays using synthetic peptides as substrates were developed. for optimization of this cleavage assay, nsp4his was preferred over mbp-nsp4 because the purification of the latter was a more elaborate process. although it was reported that the addition of six his residues strongly inhibited the enzymatic activity of the human coronavirus 229e 3cl pro proteinase (ziebuhr et al., 1997) , in both our assays the catalytic activity of nsp4his was very comparable to that of partially purified uncleaved or cleaved mbp-nsp4. also, the in vivo trans-cleavage activity of nsp4his, as determined using the recombinant vaccinia virus/t7 expression system (data not shown), was comparable to that of untagged nsp4, suggesting that the presence of the (his) 6 tag did not affect nsp4 proteolytic activity. although the processing kinetics of in vitro synthesized substrates by nsp4his seemed comparable to that of, e.g. the distantly related human coronavirus 229e 3cl pro in an identical experimental set-up, this was not true for the synthetic peptide cleavage assays. despite our efforts to improve the performance of the enzyme by testing a variety of reaction conditions, nsp4 was unable to cleave more than 15% of its substrate during a 4-h incubation, whereas studies using the human coronavirus 229e and sars-coronavirus 3cl pro enzymes reported complete conversion of a comparable amount of substrate in less than 2 h, using a 10-fold lower enzyme concentration compared to that used in our nsp4 experiments (shi et al., 2004; ziebuhr et al., 1997) . a possible explanation for this discrepancy is the lack of nsp2 in our assay, which was suggested to act as nsp4 cofactor in the polyprotein processing via the major pathway in vivo (wassenaar et al., 1997) . in the flaviviridae family, the ns4a cofactor (hepatitis c virus) and the ns2b cofactor (dengue virus type 2) have been shown to enhance in vitro ns3 proteinase activity up to 100-fold (steinkuhler et al., 1996; yusof et al., 2000) . yet another explanation may be found in the observations made during the crystallographic studies of nsp4 of barrette-ng et al. (2002) , who reported that three out of four copies of nsp4 in the unit cell had a collapsed oxyanion hole, which would render the proteinase practically inactive. if the majority of the purified nsp4his and mbp-nsp4 molecules would have had such a collapsed oxyanion hole, this might explain the low activity of both proteinases. possibly this is a consequence of nsp4 expression in e. coli, since both forms of the proteinase (mbp-nsp4 and nsp4his) displayed low proteolytic activity. expression in a different system (e.g. insect cells or yeast) might give better results. also, we cannot rule out that the foreign amino acid residues present at either the n-or c-terminus of the recombinant nsp4 might have inhibited its proteolytic activity. in coronavirus main proteinases, the n-terminus was found to be of critical importance to the geometry of in particular the s1 subsite, suggesting that changes (or extensions) in this part of the protein may adversely influence proteolytic activity (anand et al., 2002) . if this property is shared with the distantly related arterivirus main proteinase, this might explain the finding of the collapsed oxyanion hole (barrette-ng et al., 2002) in the majority of the molecules. although it seemed that either only a small part of the purified nsp4 was proteolytically active or that the catalytic activity of the enzyme was low, the cleavage of cognate sites in both assays confirmed the specificity of the e. coli-derived nsp4 proteinase. thus, the work described in this study may lay the groundwork for the more extensive biochemical characterization of the eav main proteinase, including studies aiming to identify inhibitors of proteolytic activity. structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain structure of arterivirus nsp4-the smallest chymotrypsin-like proteinase with an alpha/beta c-terminal extension and alternate conformations of the oxyanion hole expression and partial purification of recombinant tomato ringspot nepovirus 3c-like proteinase: comparison of the activity of the mature proteinase and the vpgproteinase precursor cleavage of small peptides in vitro by human rhinovirus 14 3c protease expressed in escherichia coli cleavage-site preferences of sindbis virus polyproteins containing the non-structural proteinase. evidence for temporal regulation of polyprotein processing in vivo all subgenomic mrnas of equine arteritis virus contain a common leader sequence equine arteritis virus is not a togavirus but belongs to the coronavirus like superfamily expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent putative papain-related thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha-and coronaviruses viral cysteine proteases identification of the active-site residues of the 3c proteinase of foot-and-mouth disease virus site-directed mutagenesis of the putative catalytic triad of poliovirus 3c proteinase mutational analysis of the active centre of coronavirus 3c-like proteases hepatitis a virus 3c proteinase substrate specificity poliovirus protein 3cd is the active protease for processing of the precursor protein p 1 in vitro viral proteinases polypeptide requirements for assembly of functional sindbis virus replication complexes: a model for the temporal regulation of minus-and plus-strand rna synthesis on the size of the active site in proteases dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors arteriviruses topley and wilson's microbiology and microbial infections the 5 end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease the arterivirus nsp2 protease. an unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases the arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3c-like serine proteases proteolytic processing of the replicase orfla protein of equine arteritis virus complete nucleotide sequence of the chiba virus genome and functional expression of the 3c-like protease in escherichia coli nidovirales polyprotein processing as a strategy for gene expression in rna viruses activity of purified hepatitis c virus protease ns3 on peptide substrates characterization of a protein from rice tungro spherical virus with serine proteinase-like activity characterization in vitro of an autocatalytic processing activity associated with the predicted 3c-like proteinase domain of the coronavirus avian infectious bronchitis virus activity of a purified his-tagged 3c-like proteinase from the coronavirus infectious bronchitis virus proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication processing of the equine arteritis virus replicase orflb protein: identification of cleavage products containing the putative viral polymerase and helicase domains alternative proteolytic processing of the arterivirus replicase orf1a polyprotein: evidence that nsp2 acts as a cofactor for the nsp4 serine protease protein 3cd is the major poliovirus proteinase responsible for cleavage of the p 1 capsid precursor purified ns2b/ns3 serine protease of dengue virus type 2 exhibits cofactor ns2b dependence for cleavage of substrates with dibasic amino acids in vitro characterization of a human coronavirus (strain 229e) 3c-like proteinase activity biosynthesis, purification, and characterization of the human coronavirus 229e 3c-like proteinase virus-encoded proteinases and proteolytic processing in the nidovirales we thank anton poliakov (university of alabama, usa) and willy spaan (leiden university medical center) for helpful discussions and support. d.v.a. was supported by a grant from the leiden university stimuleringsfonds. key: cord-272458-72dybi7t authors: desforges, marc; miletti, tina c.; gagnon, mylène; talbot, pierre j. title: activation of human monocytes after infection by human coronavirus 229e date: 2007-07-31 journal: virus res doi: 10.1016/j.virusres.2007.06.016 sha: doc_id: 272458 cord_uid: 72dybi7t human coronaviruses (hcov) are recognized respiratory pathogens that may be involved in other pathologies such as central nervous system (cns) diseases. to investigate whether leukocytes could participate in respiratory pathologies and serve as vector for viral spread towards other tissues, the susceptibility of human leukocytic cell lines and peripheral blood mononuclear cells (pbmc) to hcov-229e and hcov-oc43 infection was investigated. human primary monocytes/macrophages were susceptible to hcov-229e infection, but strongly restricted hcov-oc43 replication. moreover, productive hcov-229e infection of primary monocytes and of the thp-1 monocytic cell line led to their activation, as indicated by the production of pro-inflammatory mediators, including tnf-α, ccl5, cxcl10 and cxcl11 and mmp-9. moreover, an in vitro chemotaxis assay showed that motility towards chemokines of thp-1 cells and primary monocytes was increased following an acute or persistent hcov-229e infection. taken together, these results suggest that infected monocytes could serve as a reservoir for hcov-229e, become activated, participate in the exacerbation of pulmonary pathologies, as well as serve as potential vectors for viral dissemination to host tissues, where it could be associated with other pathologies. human coronaviruses (hcov) are ubiquitous enveloped positive-stranded rna viruses, represented by strains 229e, oc43, nl63, hku1, and possibly sars-cov. except for sars-cov, the etiological agent of severe acute respiratory syndrome (sars; ksiazek et al., 2003) , hcov have primarily been associated with mild upper and lower respiratory tract diseases, with the common cold representing the typical hcovinduced pathology (myint, 1995) . however, over the years, hcov have regularly been linked to other pathologies, such as severe respiratory distress syndrome in newborns (sizun et al., 1995; gagneur et al., 2002) , and as important triggers of exacerbations of acute asthma (nicholson et al., 1993; el-sahly et al., 2000) . more recently, both previously (229e and oc43) and newly described (nl63 and hku1) hcov strains were associated with more severe acute lower respiratory tract pathologies, including pneumonia, in both infants and immunocompromised * corresponding author. tel.: +1 450 687 5010x4300; fax: +1 450 686 5566. e-mail address: pierre.talbot@iaf.inrs.ca (p.j. talbot). patients (woo et al., 2005; gerna et al., 2006) . even though it is recognized that hcov primarily infect airway epithelial cells and cause diseases of the respiratory tract in humans, viral material was reported in other cells and tissues. for example, hcov-229e infection of human monocytic cell lines and of primary monocytes/macrophages was reported (collins, 2002; desforges et al., 2006) . infection of peritoneal macrophages (patterson and macnaughton, 1982) and murine dendritic cells expressing human aminopeptidase n (apn; wentworth et al., 2005) by hcov-229e suggests that this virus may use myeloid cells to disseminate to other tissues, where it could provoke other types of pathologies. other viruses, such as human immunodeficiency virus type 1 (hiv-1; nottet et al., 1996; persidsky et al., 1997) , human cytomegalovirus (hcmv) (smith et al., 2004) and its murine counterpart (mcmv) in immunocompromised mice (reuter et al., 2004) , are well-characterized examples of viruses that reach host tissues via infected leukocytes. indeed, as previously suggested for hcmv (smith et al., 2004) , blood-borne monocytes and their differentiated counterparts, macrophages, could be involved in hematogenous dissemination of hcov-229e to host tissues, including the cns, as we previously showed this virus to be neuroinvasive (arbour et al., 2000) . in these examples of viral spread, pro-inflammatory molecules, such as cytokines, matrix metalloproteinases (mmp) and chemokines and their receptors, were shown to play a crucial role in leukocyte activation and their dissemination from blood to various tissue. in the current study, susceptibility of the thp-1 human monocytic cell line and of peripheral blood mononuclear cells (pbmc) to infection by hcov-229e and hcov-oc43 was investigated to verify the possibility that these infectious agents may on the one hand activate monocytic cells that could participate in lung injury, and on the other hand use a hematogenous route to disseminate into host tissues. we show that human monocytic cells were susceptible to infection and that cell death was induced in a portion of the cells, in correlation with the initial moi for the 229e strain. the thp-1 cell line presented a pattern of infection very similar to that of primary monocytes. both cell types were activated following infection, as shown by the production of pro-inflammatory mediators. moreover, infection by hcov-229e increased cell motility and chemokine-driven cell migration. furthermore, the establishment of a persistent infection in monocytes/macrophages is consistent with the possibility that these cells serve as a reservoir and potential vector for virus dissemination to host tissue. hcov strains (229e and oc43) were originally obtained from the american type culture collection (atcc). hcov-229e was produced in the human l132 cell line (atcc ccl5) and hcov-oc43 was produced in human hrt-18 cells (gift from dr. david brian, tennessee university, knoxville, tn, usa). both cell lines were grown in minimum essential medium (mem) supplemented with 10% (v/v) fbs (wysent) and nonessential amino acid (invitrogen). the human monocytic cell line thp-1 (gift from dr. daniel oth, inrs-institut armand-frappier, laval, quebec, canada) was cultured in rpmi 1640 (invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (fbs) (wysent), and 2-mercaptoethanol 2 × 10 −5 m (invitrogen) at 37 • c in a humidified atmosphere containing 5% (v/v) co 2 . mouse igg1 mabs 5-11h.6 and 1-10c.3, directed against the s protein of hcov-229e and hcov-oc43, respectively, and anti-hev 4-e11.3, specific for the n protein of hcov-oc43 (gift from the late dr. serge dea, inrs-institut armand-frappier) were used in immunoperoxidase assays, immunofluorescence and flow cytometry. peripheral blood was obtained from healthy volunteers. leukocytes were separated by gradient centrifugation through ficoll-hypaque (amersham biosciences). pbmc were washed twice and viable cells brought to a final concentration of 3 × 10 6 ml −1 . monocytes were enriched by adsorption into 24or 6-well plastic plates (corning) for 2 h at 37 • c in rpmi 1640 supplemented with 10 mm hepes, 1 mm pyruvate sodium, non-essential amino acids, 100 u/ml penicillin, 100 g/ml streptomycin (invitrogen). cultures were then washed three times with medium to remove unattached lymphocytes, while adherent monocytes were fed new medium. a portion of these monocytes were cultured with 2% (v/v) autologous serum (decomplemented and filtered) and infected the next day. the remaining cells were induced to differentiate into macrophages for 7 days, with a change of medium after 4 days, in 10% (v/v) autologous serum in rpmi, before infection. lymphocytes were cultured in rpmi supplemented with 10% (v/v) serum and 2 × 10 −5 m 2-mercaptoethanol. for the chemotaxis assay and evaluation of the cytokine/chemokine profile, monocytes were isolated from pbmc by a negative immunoselection procedure, using the monocytes isolation kit ii (miltenyi biotec) and an automacs apparatus. isolated monocytes were then resuspended in sfm macrophage medium (invitrogen). the thp-1 human cell line was infected in triplicate at a multiplicity of infection (moi) of 0.01, 0.1 and 1 and incubated 4 h at 37 • c, washed twice with serum-free rpmi 1640. infections proceeded for up to 3 days in rpmi 1640 supplemented with 5% (v/v) fbs (except for evaluation of mmp-9, where the cells were incubated in rpmi without serum). uninfected controls were prepared by incubating cells with medium alone and 4 • c controls were obtained by incubating cells with virus on ice, to measure bound virus that presumably did not penetrate the cells. undifferentiated non-adherent monocytic thp-1 cells (1.5 × 10 6 cells in each well of a 6-well plate) were differentiated by incubating the cultures for 48 h with phorbol myristate acetate (pma) (sigma) at a final concentration of 100 nm. after this incubation period, the cells were adherent and considered to be differentiated macrophage-like cells. primary lymphocytes, monocytes and macrophages were infected separately in complete rpmi 1640 supplemented with 1% (v/v) serum. infection was then performed in triplicate with both hcov strains at an moi of 1.0 (hcov-229e infections at a moi of 0.01 and 0.1 were also performed for primary monocytes) and incubated 4 h at 37 • c. cells were then washed twice in rpmi 1640 and infection proceeded in the same medium supplemented with 2% (v/v) for monocytes and 10% (v/v) fbs for macrophages and lymphocytes in 6-well plates (corning) at 37 • c for up to 7 days. a kinetics of infection was performed for two donors where samples of supernatant were harvested at 6, 24, 48 and 72 hpi for viral titration and cells were fixed with pbs supplemented with 4% (w/v) paraformaldehyde (biopharm-sigma) for 20 min and kept in pbs at 4 • c until processed for detection of viral antigens. the metabolic activity of the infected thp-1 cells or primary cells was evaluated using the mts/pms tetrazolium assay (cory et al., 1991) , as this represents an evaluation of cell survival after infection. mts (promega) was added to the culture medium (final concentration: 0.6 mm) with pms (sigma-aldrich) (final concentration: 0.014 mm). reduction of mts, a measure of mitochondrial respiration, and therefore a direct indication of cell viability, was monitored by optical density reading at 540 nm. for evaluation of cytokine/chemokine and mmp-9 production, serum-free medium for monocytes/macrophages (sfm, invitrogen) was used for the culture of primary monocytes. the ipa was performed on l-132 cells (hcov-229e) or hrt-18 cells (hcov-oc43) as described previously (sizun et al., 1998) . primary antibody was antiviral mab, 5-11h.6 (anti-hcov-229e) or 1-10c.3 (anti-hcov-oc43) and secondary antibody was horseradish peroxidase-conjugated goat antimouse immunoglobulin (kpl). bound antibodies were detected by incubation with 0.025% (w/v) 3,3 -diaminobenzidine tetrahydrochloride (bio-rad) and 0.01% hydrogen peroxide in pbs and infectious virus titers were calculated by the karber method. paraformaldehyde-fixed primary monocytes and macrophages (in 24-well plates) were permeabilized for 5 min with methanol at −20 • c. thp-1 cells were allowed to adhere to poly-d-lysine-coated glass slides for 45 min and were fixed the same way. primary antibodies, mabs 5-11h.6, 1-10.c3 and 4-e11.3 (section 2.2), were added to plates for 1 h at room temperature. the 5-11h.6 antibody served as isotypic control for 4-e11.3 and 1-10.c3 and the latter served as isotypic control for 5-11h.6. following three washes with pbs, the alexa-fluor-488 mouse-specific goat antibody was added (invitrogen) for 1 h at room temperature. after three washes with pbs, cells were incubated for 5 min with dapi (sigma-aldrich) at 1 g/ml in order to stain the dna and the plates were washed once and kept in pbs at 4 • c until further analysis using a fluorescence microscope. analysis on lymphocytes was performed by flow cytometry using the undifferentiated thp-1 cell line as positive control of infection. total rna was extracted from primary cells using trizol tm (invitrogen) according to the manufacturer's instructions. five micrograms of total rna were reverse transcribed at 37 • c for 90 min using 0.5 g of oligo dt (invitrogen), 50 u of expand tm momulv reverse transcriptase, 60 u of rnaguard tm , 0.4 mm of dntps and rt buffer (roche diagnostics). pcr primers used for detection of the n gene of hcov-229e were e1 (nucleotides 498-521; 5 -aggcgcaagaattcagaaccagag-3 ) and e3 (nucleotides 806-783; 5 -agcaggactctgattacga-gaagg-3 ) (schreiber et al., 1989) . primers o1 (nucleotides 215-238; 5 -cccaagcaaactgctacctctcag-3 ) and o3 (nucleotides 497-520; 5 -gtagactccgtcaatatc-ggtgcc-3 ) were used for detection of hcov-oc43 (stewart et al., 1992) . twenty percent of the rt was used in the pcr and incubated with the o1 and o3 primers or e1 and e3 primers, pcr buffer (roche diagnostics) and 0.4 mm dntps. after addition of expand tm high-fidelity pcr system dna polymerase (roche diagnostics), pcr consisted of 30 cycles of 1 min at 94 • c, 1 min at 60 • c and 1 min at 72 • c. amplicons were separated by electrophoresis in 1.5% (w/v) agarose. to get a more complete cytokine/chemokine profile following hcov-229e infection of human monocytic cells, the proteome profiler human cytokine array (r&d systems), which evaluates the production of 36 inflammation-related molecules, was used according to the manufacturer's instructions. to quantitate production of the tnf-␣ cytokine by infected thp-1 cells and primary monocytes, elisa was performed using quantikine tm (r&d systems) according to the manufacturer's indications. medium was recovered at different times post-infection and was centrifuged immediately. supernatants were harvested and kept at −80 • c until further analysis. a gelatin-based zymography, modified from heussen and dowdle (1980) , was used to detect the secretion and gelatinase activity of mmp-2 and mmp-9, as previously described (edwards et al., 2000) . cells were cultured in serum-free conditions and either treated with 100 ng/ml pma, mock-infected or infected with hcov-229e. supernatants were collected and concentrated 10-fold by ultrafiltration using centricon ® ym-50 centrifugal filter devices (amicon, millipore corporation). ten microliters of samples were mixed with non-reducing buffer and loaded onto 9% (w/v) polyacrylamide gels containing 0.4% (w/v) gelatin. after electrophoresis, gels were incubated twice in 2.5% (v/v) triton x-100, rinsed with distilled water and incubated in enzyme activation buffer (100 mm tris-hcl, ph 7.4, 15 mm cacl 2 ) overnight at 37 • c. after staining with coomassie blue (0.1% (w/v) in acetic acid:methanol (1:3)), and destaining (acetic acid:methanol (1:3)), mmp activity was detected as clear bands of gelatin degradation. the specificity of the observed bands was confirmed by incubating the gel in activation buffer containing 10 mm edta, a specific inhibitor of ca 2+ -dependent gelatinases/type iv collagenases (data not shown). mmp-9 activity was also measured in serum-free medium (invitrogen) following hcov-229e infection using the fluorokine e human mmp-9 activity assay kit (r&d systems) according to the manufacturer's instruction. human recombinant chemokines ccl2, ccl5 and cxcl12 (r&d systems) were diluted in rpmi 1640 supplemented with 1% (v/v) fcs. cell migration was evaluated using transwell inserts with a 5-m pore size fitted in 24-well plates (corning costar). briefly, 100 l of rpmi 1640 medium supplemented with 1% (v/v) fcs, containing 5 × 10 5 thp-1 cells or primary monocytes were loaded into the insert above the well containing 600 l of the same medium containing either no chemokine or either one of the ccl2, ccl5 and cxcl12 chemokines, each at 100 ng/ml. after 4 h at 37 • c, cells that migrated across the insert towards the lower chamber were harvested and counted using a hemacytometer. an aliquot of these cells was allowed to adhere for 45 min to poly-d-lysine-coated glass slides and were processed as in section 2.6, to detect viral antigens by immunofluorescence. infection of primary monocytes, macrophages and lymphocytes was performed at an moi of 1 and virus replication was monitored for up to 7 days post-infection (dpi). hcov-oc43 infectious viral particles were never detected in any of the pbmc cell components of 10 healthy donors. similarly, hcov-229e infectious virus could never be detected in lymphocytes. however, infectious hcov-229e was detected at 1 dpi in monocytes and macrophages for most donors and production was low, with a maximum of 10 5 tcid 50 /ml for macrophages and 10 4 tcid 50 /ml for monocytes, and was below the detection level as early as 3 dpi (data not shown). a kinetics of infectious virus production over the first 48 h post-infection (hpi) confirmed that primary monocytes and macrophages were susceptible to a weak productive hcov-229e infection at an moi of 1, which reached a maximum titer of 10 5 tcid 50 /ml at 18 hpi, followed by a decrease at 24 and 48 hpi (fig. 1a) . in comparison, infectious hcov-oc43 particles were below detection level as early as 24 hpi (fig. 1a) and no hcov-oc43 antigens could ever be detected (data not shown), indicating restriction of viral replication. nevertheless, rt-pcr showed that hcov-oc43 rna remained present up to 48 hpi, even when the infection was performed at 4 • c, suggesting that it came from the inoculum that remained attached to cells (data not shown). a kinetics of infectious virus production confirmed that lymphocytes were not susceptible to infection, since neither in panels a and b, the limit of detection is 0.5 tcid 50 /ml. (c) evaluation of cell morphology and of the relative metabolic mitochondrial activity, indicative of cell viability, at 3 dpi in primary monocytes and macrophages, as well as in the undifferentiated and pma-differentiated thp-1 cell line. all infections were performed at an moi of 1, which induced cell death in a significant portion of the cell culture. syncytia-like structures are easily observed in primary macrophages and pma-differentiated thp-1 cells. infectious virus nor viral antigens were ever detected (data not shown). nevertheless, small amounts of viral rna from both hcov strains were detected in primary lymphocytes (data not shown). human monocytic thp-1 cells are widely used as an in vitro model to study interactions between viruses and monocytes (nordoy et al., 2003; ponzetto et al., 2004; cooper et al., 2005; yokota et al., 2005; yen et al., 2006) , because they display several characteristics of monocytes, and they can be induced to differentiate with pma to more mature macrophage-like cells (tsuchiya et al., 1982) . the kinetics of hcov-229e infection of thp-1 cells (pmadifferentiated or not) at an moi of 1 showed a similar pattern to primary monocytes and macrophages (fig. 1b) . moreover, like primary monocytes and macrophages, the thp-1 cells were highly susceptible to hcov-229e-induced cell death, regardless of the state of differentiation, as mitochondrial metabolic activity dropped significantly at 72 hpi ( fig. 1c) , suggesting that the decrease in infectious titers may in part be due to a lower number of viable cells. indeed, microscopic observation of infected cells revealed an important cytopathic effect (cpe) and the formation of syncytia-like structures in primary macrophages and in pmadifferentiated thp-1 cells (fig. 1c) . also, similarly to primary monocytes and macrophages, the pma-differentiated thp-1 cells restricted hcov-oc43 replication, with no detection of production of infectious virus (fig. 1b ) or viral antigens (data not shown). however, the non-differentiated thp-1 cells were susceptible to a productive hcov-oc43 infection (fig. 1b) , with no cpe, infected cells remaining as viable as mock-infected cells at 72 hpi (data not shown). to correlate infectious virus production and cell viability, infections were performed at a moi of 0.01, 0.1 and 1. as shown in fig. 2 , infectious hcov-229e production ( fig. 2a) and cell survival (fig. 2b) were very similar and dependent on the initial moi for both primary monocytes and thp-1 cells. indeed, infectious virus production was delayed in infections at lower moi (0.01 and 0.1), but reached the same level at 48 hpi as with an moi of 1 ( fig. 2a) . as expected, cell viability was higher when infection was performed at these lower moi (fig. 2b) . when hcov-229e infection of human primary monocytes/macrophages was performed at a moi of 1, infectious virus was under the detection limit at 3 dpi but viral antigens were still easily detected within the infected cells until at least 5 dpi (data not shown). even though primary macrophages may be kept in culture for a long period of time, primary monocytes are very difficult to keep in culture for more than a few days. therefore, primary cultures of monocytes did not survive long enough to characterize the establishment of a long-term infection. the thp-1 cells allowed us to investigate this possibility. when hcov-229e infection of both primary monocytes and thp-1 cells was performed at a moi of 0.1, the kinetics of infection was similar, as shown by infectious virus production ( fig. 2a) and detection of viral antigens (fig. 3a) . a long-term hcov-229e infection of thp-1 cells was established for at least 10 cell passages, over a 2-month period. indeed, a carrier-state type of persistent infection was observed: only a small proportion (1-2%) of the culture appeared to produce viral antigens, yielding a low infectious virus titer (fig. 3b) . to investigate consequences of hcov infection of thp-1 cells and of primary monocytes on their state of activation, we investigated the profile of cytokine/chemokine production following infection at a moi of 0.1, the highest moi that allowed survival of the majority of cells (fig. 2b ) and the establishment of a persistent infection of thp-1 cells, at 48 hpi, which corresponded to the peak of infectious virus production for all moi tested for both thp-1 cells and primary monocytes. results are shown in fig. 4 . even though the use of the cytokine array is not meant to be a precise quantitative measure of the amount of molecules produced, a reproducible increase in production of some cytokines/chemokines was observed following hcov-229e infection in two independent experiments. indeed, even though there was a constitutive production of low amounts of the proinflammatory cytokine tnf-␣ and of the chemokine cxcl10 in primary monocytes, of the chemokine cxcl11 in thp-1 cells and of the chemokine ccl5 (rantes) by both types of cells, the level of production of these molecules was clearly increased after infection (fig. 4) . a slight but reproducible increase in il-16 and cxcl12 production was also observed following infection of the thp-1 cells and primary monocytes, respectively (fig. 4) . several other inflammatory factors (see complete list in fig. 4) were produced by both cell types, without significant modulation after infection, including high levels of il-1␤, il-1ra, cxcl1, mif, ccl3, il-6 and cxcl8. strikingly, even though the steady-state profile of cytokine/chemokine was not exactly similar in thp-1 and primary monocytes, the production of the same four cytokines/chemokines, tnf-␣, ccl5, cxcl10, and cxcl11, was reproducibly modulated the same way in either type of cells following hcov-229e infection. since the production of the pro-inflammatory cytokine tnf-␣ was increased after hcov-229e infection (fig. 4) , the kinetics of its production was quantitated after infection. hcov-229einfected thp-1 cells and primary monocytes produced an amount of tnf-␣ in direct correlation with the moi, over a period of 72 h (fig. 5a) , confirming the result obtained with the cytokine/chemokine profiles (fig. 4) . a t-test analysis revealed that the production of tnf-␣ began to be significantly modulated at 48 hpi. furthermore, the production of tnf-␣ was increased even more after hcov-229e infection of pmadifferentiated macrophage-like thp-1 cells and uv-inactivated hcov-229e also induced the production of a small amount of tnf-␣ that remained stable up until 96 hpi when compared to mock-infected thp-1 cells (data not shown). matrix metalloproteinases (mmp) can also be linked to monocyte activation. therefore, the modulation of mmp-2 and -9 production and activity (type iv collagenases) was investigated. two bands of gelatinolysis were identified as pro-mmp-9 (92 kda) and pro-mmp-2 (72 kda), based on their molecular weight in polyacrylamide gels and their inhibition in presence of edta. while pro-mmp-9 appeared to be constitutively expressed in the thp-1 cell line and primary monocytes, an increased expression was observed after 72 hpi with hcov-229e, when compared to mock-infected cells (fig. 5b) . pma was used as a positive control (data not shown). even though pma induced an increase in the expression of pro-mmp-2 and mmp-9 in thp-1 cells, no modulation of mmp-2 was detected upon infection of either cell type (data not shown). quantitation of active mmp-9 by fluorescence elisa revealed that the amount of mmp-9 secreted in the medium started to increase at 48 hpi and that the differences in mmp-9 secretion were statistically significant (t-test) between mock-and infected-cells (fig. 5c) . since monocytic cells were activated by hcov-229e infection, their potential to respond to chemokines was evaluated by a chemotaxis assay. our results indicate that an acute or persistent infection induced an increase in motility of the thp-1 cells and primary monocytes, and in the capacity of these cells to respond to ccl5 and cxcl12-driven migration in vitro (fig. 6) . even though hcov-229e-infected thp-1 cells were more responsive to the ccl2-driven migration at 24 hpi compared to mock-infected thp-1 cells, this increase was only transient, since no difference in migration was observed at 48 hpi ( fig. 6a and b) . persistently infected thp-1 cells were also slightly more responsive to the ccl2-driven migration. on the other hand, hcov-229e and mock-infected primary monocytes were equally responsive to the ccl2-driven migration ( fig. 6a and b) . immunofluorescence analysis revealed that only a portion of the migrating cells were producing viral antigens (fig. 6c) . hcov are recognized respiratory pathogens, infecting mostly airway epithelial cells (wang et al., 2000) , but viral material was detected in host tissue other than the respiratory tract, including the cns (arbour et al., 2000) . since they have access to lung tissue and express cd13 (apn), the hcov-229e receptor (yeager et al., 1992) , on their cell surface (ashmun and look, 1990) , infected activated leukocytes could participate in initiating an overwhelming immune response that could lead to more serious respiratory pathologies, as well as represent vehicles for viral spread to other tissues, like it was shown for hiv-1 (nottet et al., 1996; persidsky et al., 1997) and human cytomegalovirus (hcmv) (smith et al., 2004) . therefore, the present study was designed to evaluate this possible mechanism for hcov-related pulmonary diseases, such as asthma exacerbations and respiratory distress syndrome in newborns, and in dissemination to host tissues such as the cns. the oc43 strain of hcov (hcov-oc43) was reported to be capable of productively infecting primary monocytes and macrophages (collins, 1998 (collins, , 2002 . however, in our study, only viral rna was detected (data not shown). this could be explained by virus attachment to cell surface lectin-like molecules, as shown for sars-cov and other enveloped viruses such as hiv, dengue and hepatitis c virus (altmeyer, 2004) . alternatively, it may be related to the differentiation state of monocytes and its correlation with susceptibility to hcov-oc43 infection. indeed, promonocytic thp-1 cells, which were susceptible to a weak productive hcov-oc43 infection, represent less differentiated cells than primary monocytes/macrophages. upon pma-induced differentiation, they lost susceptibility to infection, which is consistent with monocytes being only susceptible to infection during a brief period during their differentiation. this was also suggested for hiv-1 (schuitemaker et al., 1992; pauza et al., 1988) , hcmv (weinshenker et al., 1988) and measles virus (helin et al., 1999) . the detection of hcov-oc43 rna in primary monocytes/macrophages (data not shown) is consistent with these cells participating in virus dissemination, as suggested for sars-cov (yilla et al., 2005) . however, infectious hcov-oc43 particles were not detected as early as 24 hpi postinfection, suggesting that viral rna detected at 48 hpi represents non-infectious virus, still cell-associated. on the other hand, primary monocytes and macrophages were susceptible to hcov-229e infection (fig. 1a) , in contrast with a report of an abortive hcov-229e infection of peritoneal macrophages (patterson and macnaughton, 1982) . our results with primary monocyte-derived human macrophages not only confirm susceptibility to hcov-229e, but demonstrate their production and release of small amounts of infectious virus into the culture medium. moreover, detection of hcov-229e antigens up to 5 dpi (data not shown) suggests that virions still remain in cells for a while even after a short burst of infectious virus producfig. 6 . thp-1 cell and primary monocyte motility and chemokine-driven migration are increased after hcov-229e infection. infections were performed at a moi of 0.1. (a) chemotaxis assay showed that hcov-229e infection of thp-1 cells by itself induced a significant increase in cell motility and migration across transwell inserts. ccl5 and cxcl12-driven migration was also significantly increased following infection but ccl2-driven migration was only significantly modified at 24 hpi and in persistently infected cells. (b) a comparative chemotaxis assay showed that both thp-1 cell and primary human monocyte motility and chemokine-driven migration across transwell inserts was significantly increased. ccl5-driven migration was already significantly increased at 24 hpi and remained at that level until 48 hpi and cxcl12-driven migration was significantly increased at 48 hpi. ccl2-driven migration was only significantly increased at 24 hpi for the thp-1 cells but not significantly modified for primary monocytes. (c) a representative field of thp-1 cells and primary monocytes that have migrated across transwell inserts and were allowed to adhere to poly-d-lysine-coated glass slides. detection of hcov-229e s protein (green cells with white arrows) was performed using the mab 5-11h.6. cell nuclei were stained with dapi (blue). results are representative of three independent experiments. mock-infected thp-1 cells (thp-mock) at 24, 48 hpi and at 60 dpi (persist: persistently infected kept in culture for 2 months, representing 10 passages) were compared to hcov-229e-infected thp-1 cells (thp-229e-0.1) at 24, 48 hpi and 60 dpi and mock-infected primary monocytes (primarymono-mock) at 24 and 48 hpi were compared to hcov-229e-infected primary monocytes (primarymono-229e-0.1) at 24 and 48 hpi. the levels of statistical significance between mock-and hcov-infected cells were evaluated for each condition. the p values were calculated by a t-test and are represented by * where the difference between mock and infected cells was significant. *p < 0.05; **p < 0.01; ***p < 0.001. tion. kinetics of monocyte infection (fig. 1a) suggests that it is productive, as previously reported (collins, 2002) . however, in contrast to the latter report, our results show a transient productive infection, with titers starting to drop between 24 and 48 hpi when an initial moi of 1 is used. we also show that infection led to cell death in a portion of the cell culture in a moi-dependent manner (fig. 1c) , which may involve apoptosis (collins, 2002) . our results clearly indicate that susceptibility of thp-1 cells to hcov-229e infection correlates very well with results on primary cells. a weakly productive long-term hcov-229e infection of thp-1 cells at lower initial moi was also previously reported for hiv-1 (mikovits et al., 1990) and cells remained responsive to pma-induced differentiation into macrophagelike cells and produced more infectious virus (konopka et al., 1993) . moreover, even though monocytes have a life span of only a few days in circulation (cline et al., 1978) , a long-term infection in thp-1 monocytic cells is highly relevant, since during their migration towards tissue, monocytes differentiate into macrophages, which are long-lived cells that can harbor virus for a long period of time. persistently infected thp-1 cells had an increased motility and were still responsive to chemokines (fig. 6a) , which strongly suggests that monocytes/macrophages could be a reservoir and potential vector for hcov-229e spread into host tissue, as suggested for hcmv (söderberg-naucler et al., 1994; sinclair and sisson, 1996) . moreover, we show that hcov-229e infection of thp-1 cells and primary monocytes leads to their activation, as indicated by the production of several inflammation-related mediators and responsiveness to chemokine gradients. the faint increase in il-16 production by infected thp-1 cells (fig. 4) could be caused by apoptosis of a small portion of the culture, as reported for primary monocytes (elssner et al., 2004) . however, the increase in il-16 was not seen in primary monocytes, as these cells produced it constitutively (fig. 4) . others have reported a faint increase in cxcl8 production and no production of tnf-␣ following hcov-229e infection of thp-1 cells (yen et al., 2006) . this apparent discrepancy may be due to their use of thp-1 cells manipulated to overexpress dc-sign, which could have altered their capacity to produce tnf-␣, or to the fact that they measured tnf-␣ production at 1 dpi, whereas we analyzed its production over a 3-day period. the same may apply to the apparent discrepancy in the production of cxcl8 in hcov-229e infected thp-1 cells (yen et al., 2006) . constitutive production of high levels of cytokines/chemokines by primary monocytes (fig. 4) is not surprising: for instance cxcl8, il-1␤, il-6 and tnf-␣ were shown to be constitutively produced by human monocytes (naldini et al., 2006) . the pro-inflammatory cytokine tnf-␣ was constitutively produced at very low levels in thp-1 and primary cells (fig. 5a) , consistent with reports for thp-1 cells (heidinger et al., 2006) and primary monocytes (nguyen et al., 2006) . interestingly, we show that tnf-␣ production was modulated by viral replication in a moi-dependent manner in both cell types (fig. 5a ). this cytokine was linked to some asthmatic complications and in acute respiratory distress syndrome (ards; mukhopadhyay et al., 2006) , two pathologies associated with sporadic hcov infection (nicholson et al., 1993; sizun et al., 1995; el-sahly et al., 2000; gagneur et al., 2002) . on the other hand, tnf-␣ was shown to increase icam-1 expression on endothelial cells (weirtheimer et al., 1992) including those of the bbb (defazio et al., 1998) , and icam-1 is known to facilitate the passage of leukocytes (including monocytes) through the bbb (dietrich, 2002) . therefore, considering the neuroinvasive properties of hcov-229e, the production of tnf-␣ by infected monocytes could lead to an increase spread of these cells towards other tissues, including the cns. the strong production of the cxcl10 and cxcl11 chemokines after hcov-229e infection is very interesting since these chemokines have a high chemotactic potential towards t cells (qin et al., 1998) and they were upregulated and provoked an increased recruitment of t cells after hiv-1 infection of monocyte-derived macrophages (foley et al., 2005) . the increased production of ccl5 in hcov-229e-infected cells is also of interest as this chemokine has a chemoattracting potential for memory and activated t cells (schall et al., 1990) , nk cells (loetscher et al., 1996) , dendritic cells (lin et al., 1998) , and monocytes (fine et al., 2001) . therefore, migration of hcov-229e-infected monocytes into the respiratory tract could lead to recruitment of additional immune cells that may eventually cause an uncontrolled upregulation of inflammatory molecules, as is the case in ards. indeed, an increased level of expression of matrix metalloproteinases (mmp) was seen in patients with ards (corbel et al., 2002) , an interesting observation considering that hcov-229e was associated with severe respiratory distress syndrome in newborns (gagneur et al., 2002) . moreover, it was recently shown that the in vitro recruitment of human peripheral blood leukocytes exposed to supernatants of sars-cov and hcov-229e-infected thp-1 dc-sign cells (yen et al., 2006) was significantly higher compared to supernatant from uninfected cells. in the particular case of the cns, a role for ccl5 in the pathogenesis of mhv-induced neurologic disease was suggested, which correlated with macrophage infiltration and demyelination (lane et al., 2000) . also, expression of ccl5 and its ccr5 receptor was strongly upregulated during cns infection by west nile virus, which was associated with an important infiltration of immune cells including macrophages (glass et al., 2005) . as hcov-229e is also neuroinvasive (arbour et al., 2000) , migration of hcov-229e-infected monocytes into the cns is plausible and the increased production of mmp-9 by monocytes following hcov-229e infection was also previously described for hiv-1-infected monocytes (dhawan et al., 1995) . given that mmp-9 is implicated in promoting leukocyte trafficking across the bbb (gidday et al., 2005; bar-or et al., 2003) , the upregulation of mmp-9 in thp-1 cells and primary monocytes following hcov-229e infection is highly relevant. a release of cytokines, such as tnf-␣, during infection may also be involved in the upregulation of mmp in monocytes/macrophages (welgus et al., 1992) . the significant induction of tnf-␣ production by monocytes after hcov-229e infection (fig. 5a) suggests a synergistic effect between mmp-9 and tnf-␣ to facilitate the migration of monocytes into tissues, including the cns. the mmp-9 production of monocytes was enhanced in the presence of chemokines such as ccl5 and cxcl12 (webster and crowe, 2006) . a role for neutrophilderived mmp-9 (zhou et al., 2002) was also suggested in the breakdown and integrity loss of the bbb (zhou et al., 2003) during mhv infection of the cns. the increased motility of hcov-229e-infected monocytes (fig. 6) is consistent with the hypothesis that infected monocytes could serve as vectors for viral spread to tissues, as suggested for hcmv (smith et al., 2004) . furthermore, the increased chemokine-driven migration of infected cells suggests that migration of hcov-229e-infected monocytes towards tissue could be facilitated during an inflammatory response in which the production of ccl2 and ccl5 would be increased within the inflamed tissue. the production of ccl5 appear more important than that of ccl2 in facilitating migration of hcov-229e infected cells towards tissue, since the increased migratory capacities of infected cells were more evident towards ccl5 (fig. 6) . the increased responsiveness to cxcl12 (fig. 6) is also very interesting since this chemokine is constitutively produced in tissues such as the cns (cartier et al., 2005) . this latter chemokine was shown to facilitate the passage of lymphocytes and monocytes through endothelial cells in vitro (wu et al., 2000) and was implicated in the baseline traffic of t cells and monocytes in the cns (calderon et al., 2006) . monocytes can be divided into two main subsets, a shortlived "inflammatory subset" that homes to inflamed tissue to trigger an immune response, and a "resident" subset, with a longer life span, that homes to noninflamed tissues, including the lungs where they serve to replenish either the alveolar macrophages or the dendritic cell populations (landsman et al., 2007) , and the brain, where they serve as putative microglia precursors (geissmann et al., 2003) . either type of monocytes may propagate a viral infection (reuter et al., 2004) . therefore, virus-induced monocyte activation could facilitate their passage through endothelial cells towards host tissue, especially the cns in immunocompromised individuals, as reported for murine cytomegalovirus (mcmv) (reuter et al., 2004) . the fact that hcov-229e could only infect partially immunocompromised transgenic mice (lassnig et al., 2005) suggests that hcov-229e could also take advantage of immunosuppression to gain access to tissue other than the respiratory tract, including the cns in susceptible individuals. altogether, our results indicate that monocytes are susceptible to hcov-229e infection and that they become activated shortly after being infected, as shown by their increased capacity to produce pro-inflammatory mediators and chemokines. this could have important consequences on the development of more serious respiratory tract diseases and even in the capacity of the virus to invade other tissues. even though hcov-229e is a respiratory pathogen, our previous studies did demonstrate that it has neuroinvasive properties. since monocytes have access to cns, our observation that monocytes/macrophages support coronavirus replication is consistent with the possibility that monocytes may contribute to hcov-229e spread to the cns. moreover, the long-term infection of the thp-1 monocytic cell line strongly suggests the possibility of a viral reservoir within monocyte subsets and is consistent with human monocytes harboring hcov and participating in virus spread towards tissue in general and particularly the cns. virus attachment and entry offer numerous targets for antiviral therapy neuroinvasion by human respiratory coronaviruses metalloprotease activity of cd13/ aminopeptidase n on the surface of myeloid cells analyses of all matrix metalloproteinase members in leukocytes emphasize monocytes as major inflammatory mediators in multiple sclerosis a role for cxcl12 (sdf-1␣) in the pathogenesis of multiple sclerosis: regulation of cxcl12 expression in astrocytes by soluble myelin basic protein chemokine receptors in the central nervous system: 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migration are induced by distinct chemokines and are regulated on maturation activation of nk cells by cc chemokines negative regulation of human immune deficiency virus replication in monocytes: distinction between restricted and latent expression in thp-1 cells role of tnf-a in pulmonary pathophysiology human coronavirus infections cutting edge: il-1␤ mediates the proangiogenic activity of osteopontin-activated human monocytes protein tyrosine kinase and p38 map kinase pathways are involved in stimulation of matrix metalloproteinase-9 by tnf-␣ in human monocytes respiratory viruses and exacerbations of asthma in adults cytomegalovirus infection induces production of human interleukin-10 in macrophages mechanisms for the transendothelial migration of hiv-1-infected monocytes into brain replication of human respiratory coronavirus strain 229e in human macrophages human immunodeficiency virus infection of monoblastoid cells: cellular differentiation determines the pattern of virus replication a model for monocyte migration through the blood-brain barrier during hiv-1 encephalitis effect of hcv infection on thp-1 monocytoid cells the chemokine receptors cxcr3 and ccr5 mark subsets of t cells associated with certain inflammatory reactions systemic immune deficiency necessary for cytomegalovirus invasion of the mature brain selective attraction of monocytes and t lymphocytes of the memory phenotype by cytokine rantes sequence analysis of the nucleocapsid protein of human coronavirus 229e differential tropism of clinical hiv-1 isolates for primary monocytes and promonocytic cell lines latent and persistent infections of monocytes and macrophages neonatal nosocomial respiratory infection with coronavirus: a prospective study in a neonatal intensive care unit comparison of immunofluorescence with monoclonal antibodies and rt-pcr for the detection of human coronaviruses 229e and oc43 in cell culture human cytomegalovirus induces monocyte differentiation and migration as a strategy for dissemination and persistence reactivation of latent human cytomegalovirus in cd14+ monocytes is differentiation dependent human coronavirus gene expression in the brains of multiple sclerosis patients induction of maturation in cultured human monocytic leukemia cells by a phorbol diester human coronavirus 229e infects polarized airway epithelia from the apical surface matrix metalloproteinases, their production by monocytes and macrophages, and their potential role in hiv-related diseases phorbol ester-induced differentiation permits productive human cytomegalovirus infection in a monocytic cell line intercellular adhesion molecule-1 gene expression in human endothelial cells. differential regulation by tumor necrosis factor-alpha and phorbol myristate acetate neutral proteinase expression by human mononuclear phagocytes: a prominent role of cellular differentiation cells of human aminopeptidase n (cd13) transgenic mice are infected by human coronavirus-229e in vitro, but not in vivo characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia mechanisms of leukocytes trafficking into the cns human aminopeptidase n is a receptor for human coronavirus 229e modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro sars-coronavirus replication in human peripheral monocytes/macrophages induction of suppressor of cytokine signaling-3 by herpes simplex virus type 1 confers efficient viral replication matrix metalloproteinase expression correlates with virulence following neurotropic mouse hepatitis virus infection neutrophils promote mononuclear cell infiltration during viral-induced encephalitis we gratefully thank francine lambert and marcel desrosiers for excellent technical assistance. we also thank dr. alain lamarre, inrs-institut armand-frappier, for providing macsisolated leukocytes. this work was supported by grant mt-9203 from the institute of infection and immunity, canadian institutes of health research (cihr) to pierre j. talbot. mylène gagnon and tina miletti are grateful to the fondation armand-frappier for studentship support. 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 coronavirus, cv777 the pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, cv777 detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa propagation of the virus of porcine epidemic diarrhea in cell culture spike glycoprotein cleavage recognition site analysis of infectious bronchitis virus pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus isolation of porcine epidemic diarrhea virus (pedv) in korea derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate proteomic analysis of up-regulated proteins in human promonocyte cells expressing severe acute respiratory syndrome coronavirus 3c-like protease heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in 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-284479-75zgljet authors: garcía-serradilla, moisés; risco, cristina; pacheco, beatriz title: drug repurposing for new, efficient, broad spectrum antivirals date: 2019-04-15 journal: virus res doi: 10.1016/j.virusres.2019.02.011 sha: doc_id: 284479 cord_uid: 75zgljet emerging viruses are a major threat to human health. recent outbreaks have emphasized the urgent need for new antiviral treatments. for several pathogenic viruses, considerable efforts have focused on vaccine development. however, during epidemics infected individuals need to be treated urgently. high-throughput screening of clinically tested compounds provides a rapid means to identify undiscovered, antiviral functions for well-characterized therapeutics. repurposed drugs can bypass part of the early cost and time needed for validation and authorization. in this review we describe recent efforts to find broad spectrum antivirals through drug repurposing. we have chosen several candidates and propose strategies to understand their mechanism of action and to determine how resistance to antivirals develops in infected cells. viruses are a constant threat to humans, animals and plants. there are hundreds of viruses that can cause disease in humans but there is no treatment for most of them. emerging or reemerging viruses are a public health concern (howard and fletcher, 2012) . some of these pathogens are the rift valley fever virus (rvfv), dengue virus (denv), west nile virus (wnv), yellow fever virus (yfv), ebola virus (ebov), sars-and mers-cov, zika virus (zikv), crimean congo hemorrhagic fever virus (cchfv), severe fever with thrombocytopenia syndrome virus (sftsv), chikungunya virus (chikv) and influenza a virus (iav). recent outbreaks of these viruses are summarized in table 1 . international travel and migrations, globalization of commerce, technology and industry, agriculture development or climate changes are favoring the emergence and reemergence of viruses that could easily spread and potentially become a pandemic (jones et al., 2008) . a clear example of the potential of an emerging virus to become a pandemic is the emergence of human immunodeficiency virus (hiv) in the 1980s (http:// aidsinfo.unaids.org/) (sharp and hahn, 2011) . there is no cure for hiv, although antiretroviral treatment can control the virus ( barre-sinoussi et al., 2013) . in the case of influenza virus there are both vaccines and antiviral drugs. however, these viruses constantly change and antiviral resistance emerges . in 2013, human infections with a new avian influenza virus in china caused considerable concern due to the pandemic potential of this virus (su et al., 2015) . the experts consider that the virus could gain the ability to spread among people, triggering a global epidemic (su et al., 2015; tanner et al., 2015) . due to all these reasons, we urgently need new, efficient, broadspectrum antivirals (howard and fletcher, 2012; schor and einav, 2018) . the traditional strategy in drug development of one virus one drug, based in the development of molecules that specifically target a viral protein, has been very successful for certain viruses like hiv or hepatitis c virus (hcv) (barre-sinoussi et al., 2013; li and de clercq, 2017) . in fact, most approved antiviral treatments target a viral protein, and thus are usually quite virus specific. however, direct acting antivirals (daa) have some drawbacks. the development of new therapeutic compounds is time and resources consuming (dimasi et al., 2016) . the whole process from the discovery of a new drug that can potentially treat a specific disease or condition to its approval to be used in humans can take several years (on average 12 to 16 years) (nosengo, 2016) . the first stage, drug discovery, can take between 3 to 6 years. then preclinical studies, usually in animal models, are carried out. these studies that are aimed to test the efficiency and safety of a molecule in a suitable model before it is tested in humans can go on for about 3 years. if a molecule has shown promising results up to here, then clinical trials in humans are started. these trials, from phase i to phase iii, usually last about 5 years. finally, if a molecule successfully passes all the process it needs to get the approval from the appropriate agency. at the end it is estimated that only about 5% of the candidate molecules successfully pass the whole process. adding to this the cost of drug development, about 1-2 billion dollars or more in some cases (dimasi et al., 2016) , the one virus one drug strategy is not the most cost efficient approach. in addition, the use of daas can easily lead to the appearance of drug resistant viruses due to the elevated mutation rate of viruses (iyidogan and anderson, 2014; menendez-arias et al., 2014; perales et al., 2015) . to circumvent these limitations other strategies aim to develop molecules that target host cellular factors that are needed for viral replication. although viruses can also find ways to use alternative routes to replicate in a host, emergence of resistant viruses is less likely (lin and gallay, 2013; zeisel et al., 2013) . in addition these approaches focused in host cellular pathways needed for viral replication can favor the development of broad spectrum antivirals. there are families of viruses that share certain cellular pathways. thus, drugs that inhibit a specific cellular target could work for several viruses. as a drawback drugs that inhibit a host cell target have higher chances of producing undesirable side effects in the patient compared to drugs that target a viral protein. drug repurposing or repositioning is an alternative approach that can lead to less costly and faster approval of new treatments for viral infections (mercorelli et al., 2018) . a clear advantage of drug repositioning over traditional drug development is that since the repositioned drug has already passed a significant number of tests, its safety is known and the risk of failure is reduced. although repositioned drugs can most likely skip phase i clinical trials, they still need to go through phase ii and iii trials to test their efficacy for the new condition (nosengo, 2016) . in some instances these drugs might have lost their patents, as is the case of generic medicines. in these cases, when a new formulation or a new medical use exists for an "old" drug, second medical use patents can be obtained for them. on the other hand, increasing implication of practicing physicians and researchers at academic centers in clinical trials can also help in the repurposing of these drugs (nosengo, 2016) . additionally, there are thousands of drugs that have already passed clinical trials that confirm their safe use in humans. however, for some reasons, they lack or have poor efficiency to treat the condition under scrutiny and have not reached the market. these collections of drugs are another source of potential antivirals. several drugs in the market have been tested for their potential use as broad spectrum antiviral treatments ( table 2 ). many of these compounds have shown promising results in preclinical studies. below we summarize the main results and antiviral mechanisms of selected candidates for repositioning as broad spectrum antiviral drugs. digoxin is a cardiac glycoside or cardiotonic steroid that has been used for treating certain heart conditions including heart failure, atrial fibrillation, atrial flutter, and paroxysmal atrial tachycardia (gheorghiade et al., 2006) . cardiac glycosides comprise a large family of natural compounds that share a common steroid structure. digoxin was first isolated from foxglove plant, digitalis lanata, in 1930, although foxglove plant extracts have been used from ancient times to treat heart conditions (gheorghiade et al., 2006) . digoxin has been shown to block the na + /k + atpase, raising intracellular na + levels. the increase in intracellular na + leads to an increment in intracellular ca2+ due to inhibition of na + /ca 2+ exchanger. elevated intracellular ca2+ ultimately leads to an increased contractibility of the cardiac muscle (smith, 1988) . inhibition of the na + /k + atpase has been shown to have an effect in multiple signaling cascades through modulation of neighboring tyrosine kinases, including src, ultimately affecting gene expression (nesher et al., 2007) . thus, cardiac glycosides are in the spotlight for drug repurposing. some studies have shown the potential use of digoxin and other cardiac glycosides to treat cancer (prassas and diamandis, 2008) . cardiac glycosides can induce apoptosis in diverse cancer cells by multiple mechanisms including inhibition of na + /k + atpase, suppression of nuclear factor-kappab and inhibition of topoisomerase ii (ishida et al., 2016; vaklavas et al., 2011) . by performing a chemical screen with an insect cell-based reporter system, the cardiac glycoside digoxin was identified as a specific inhibitor of the retinoic acid receptor-related orphan nuclear receptor (rorγt) transcriptional activity (huh et al., 2011) , which is required for induction of il-17 transcription and for the manifestation of t h 17-dependent autoimmune disease in mice. this study indicated that digoxin and its derivatives could be used as therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease. recent studies have shown that digoxin and other cardiac glycosides can have a potential use as antivirals. wong and colleagues reported the suppression of hiv-1 replication by digoxin (wong et al., 2013) . digoxin was shown to accomplish its antiretroviral effects by two mechanisms: 1) inducing oversplicing of hiv-1 rna which reduces the accumulation of viral rnas encoding for structural proteins needed for new virion assembly, and 2) selectively inhibiting the expression of hiv-1 regulatory protein rev. these studies showed that digoxin exerted its anti-hiv effect by altering the function of a subset of sr proteins, a conserved family of serine and arginine-rich proteins involved in rna splicing. a screening of a drug library for compounds inhibiting late steps of hiv-1 replication cycle also identified several members of the cardiac glycoside family (laird et al., 2014) . the cardiac glycosides digoxin and ouabain have also been shown to impair replication of human cytomegalovirus (hcmv) and herpes simplex virus (hsv) that belong to the family of dsdna viruses herpesviridae (dodson et al., 2007; hartley et al., 2006; kapoor et al., 2012) . inhibition happens at an early post-entry step and produces a (grosso et al., 2017) . although the precise mechanism of action is not fully clear, these drugs altered viral mrna processing, blocking replication before viral dna synthesis. some cardiac glycosides like procillaridin a, bufallin, covallatoxin and digitoxin are able to inhibit hepatitis b virus (hbv) in cell cultures (okuyama-dobashi et al., 2015) . in this study, however, digoxin did not show an anti-hbv effect. digoxin also inhibits alphaviruses (singlestrand positive-sense rna viruses) like chikungunya, ross river virus and sindbis virus, as well as the unrelated mammalian orthoreovirus (polysegmented double-strand rna virus) and vesicular stomatitis virus from the rhabdoviridae family (negative-sense rna virus) (ashbrook et al., 2016) . in this study digoxin was shown to impair chikungunya infection at an early post-entry step. some coronaviruses like feline infectious peritonitis virus, murine hepatitis virus, and mers-cov are inhibited by ouabain and bufalin (burkard et al., 2015) . the antiviral effect of these cardiac glycosides was observed only when the drug was added prior to infection. infection was not affected when the drugs were added 2 h post-infection, suggesting that for these viruses the drugs were acting during the entry step. sunitinib is a small molecule that inhibits multiple tyrosine kinase receptors like vascular endothelial growth factor receptor vegfr, platelet-derived growth factor receptors pdgfrα and pdgfrβ, fibroblast growth factor receptor 1, and epidermal growth factor receptor (mendel et al., 2003; sun et al., 2003) . sunitinib has been approved by the fda for the treatment of some cancers (atkins et al., 2006) . recent studies have shown that sunitinib may also have broad spectrum antiviral activity. intracellular trafficking of viruses relies on the endocytic and exocytic cellular pathways. these processes usually require signal transduction, suggesting that kinase inhibitors may have antiviral activity by blocking the endocytic or exocytic pathways. binding of the hcv core protein to adaptor protein 2 (ap-2) μ2 subunit (ap2m1) is essential for hcv assembly. phosphorylation of ap2 by adaptor associated kinase (akk1) and cycling g-associated kinase (gak) regulate this interaction (neveu et al., 2012 . it has also been shown that ap1 and ap2 co-traffic with hcv viral particles within the cell (bekerman et al., 2017) . these studies showed that sunitinib prevents hcv entry and assembly, apparently through an inhibitory effect on aak1 and gak, with no effect on hcv rna replication. erlotinib, another anticancer drug, also inhibits hcv entry and assembly. similarly, sunitinib and/or erlotinib can restrict infection by denv and ebov in vitro and in a murine animal model and have potent in vitro antiviral activity against zikv, wnv, chikv, junin virus (junv) and respiratory syncytial virus (rsv) (bekerman et al., 2017; pu et al., 2018) . sunitinib has also been shown to inhibit hcmv infection in cell culture (cai et al., 2014) and hiv-1 infection of resting cd4 t cells (guo et al., 2013) . although these studies showed the potential antiviral activity of sunitinib, this drug can also inhibit protein kinase r (pkr) and 2′5′oligoadenylate synthetase (oas)/rnase l system that act as antiviral effectors in response to type i interferons (jha et al., 2013) . the capacity of sunitinib to block these two innate immunity pathways could hamper its potential use as antiviral. the development of more selective gak inhibitors could bypass this hurdle. in this sense, a screening of a library based on a bicyclic, heteroaromatic flat scaffold designed to discover novel ligands of gak, led to the identification of a hit compound based on a isothiazolo[4,3-b]pyridine scaffold (kovackova et al., 2015) . some isothiazolo[4,3-b]pyridine derivatives with low nanomolar affinity for gak exhibited inhibitory activity against hcv in cell culture, acting at the level of viral entry and assembly (kovackova et al., 2015) . chloroquine (cq) is a weak base 4-aminoquinolone derivative that can easily enter the cells and accumulate in acidic compartments like endosomes, lysosomes or trans-golgi network vesicles raising their ph (thome et al., 2013) . in the clinic cq has been used for the treatment of non-resistant malaria (slater, 1993; white, 1996) . additionally cq has been proven to have anti-inflammatory properties, and has been approved for the clinical management of some autoimmune diseases such as rheumatoid arthritis and lupus erythematosus (rainsford et al., 2015) . many viruses use acidic organelles at some point of their replication cycle. this has prompted the study of cq and its hydroxyl analogue hydroxychloroquine (hcq) as potential antiviral drugs. cq and hcq have been tested and proved to have in vitro, and in some instances in vivo, antiviral activity against several viruses, as described below. two main mechanisms of action have been described for the antiviral activity of cq (al-bari, 2017). on the one hand, many viruses use acidic endocytic vesicles, like endosomes or lysosomes, to enter the host cell. the acidification of these vesicles triggers conformational changes in the envelope proteins of the virus that induce the fusion of the viral and cellular membranes and the delivery of the virion content into the cell. cq raises the ph of these vesicles impeding the conformational changes needed for viral entry or uncoating and blocking the infection. cq has also been shown to impair maturation of some viral proteins in the golgi network by blocking the action of ph-dependent enzymes, like proteases or glycosyltransferases, which are needed for the maturation process. the antiretroviral effect of cq in cell cultures has been shown to occur through the inhibition of the glycosylation of hiv viral particles (savarino et al., 2004) , while the antiviral activity of cq against sars-cov seems to be related to the poor glycosylation of the sars-cov receptor ace2 (vincent et al., 2005) . cq has been reported to inhibit denv replication by blocking the cleavage of the prm protein that occurs in acidic compartments (boonyasuppayakorn et al., 2014; randolph et al., 1990) . cq and some of its derivatives have been reported to have strong antiviral activity against sars-cov and mers-cov in different cell lines with ic 50 ranging from 3.0 to 8.8 μm (de wilde et al., 2014; dyall et al., 2014; keyaerts et al., 2004; vincent et al., 2005) . however, in a sars-cov mouse model cq treatment did not reduce virus titer in the lungs (barnard et al., 2006) some studies have reported inhibitory effect of cq against influenza a viruses (iav) h3n2 and h1n1 in vitro at concentrations similar to doses used for malaria treatment (ooi et al., 2006) . however, the susceptibility of iav to cq seems to depend on the ph requirements of the viruses and the electrostatic potential of hemagglutinin 2 (di trani et al., 2007) . in a mouse model of infection with iav, cq treatment dramatically increased the survival of infected mice when administered therapeutically but not when administered as a prophylactic treatment (yan et al., 2013) . furthermore, a randomized, double-blinded placebo controlled clinical trial did not protect against influenza infection (paton et al., 2011) . dengue virus and wnv replication has also been reported to be blocked by cq in cell culture (boonyasuppayakorn et al., 2014; farias et al., 2014) . a recent study has demonstrated the efficacy of cq in the prophylactic and therapeutic treatment of aotus monkeys inoculated with denv (farias et al., 2015) . however, a double-blinded, placebocontrolled clinical trial in vietnam failed to show a beneficial impact of cq treatment in the outcome of the infection except for a modest reduction in the percentage of patients that developed dengue hemorrhagic fever in the cq-treated arm (tricou et al., 2010) . chloroquine also exhibits antiviral activity against zikv, another member of the flaviviridae family, in different cell lines with an ic 50 around 9.8-14.2 μm (delvecchio et al., 2016) . additionally, some studies have demonstrated that chloroquine can protect mice against zikv infection shiryaev et al., 2017) . cq has also been reported to inhibit chikv in cell cultures in a dose dependent manner when added at an early point during infection (sourisseau et al., 2007) . although in this study the therapeutic index of the drug in cell cultures was low, the results pointed at the potential use of cq for the treatment of chikv infection. however, a clinical trial in the french reunion island during an outbreak of chikv to test the efficacy and safety of cq treatment did not show evidence of a significant antiviral effect in cq treated patients compared to placebo treated patients (de lamballerie et al., 2008) . this lack of antiviral effect could be due to the high viral load in the serum of patients during the acute phase of the infection. the rather narrow therapeutic index of cq leaves little room for a dose adjustment that could allow the use of cq as a therapeutic treatment in acute chikungunya infection. nonetheless, in cell cultures, where the viral titer is lower, cq strongly inhibits chikv infection at concentrations achieved in plasma of individuals on cq treatment for malaria, which suggest that cq could be used as a prophylactic treatment during chikungunya outbreaks to prevent transmission (khan et al., 2010) . however, a recent study in non-human primates showed that chloroquine treatment exacerbated the disease and/or suppressed the antiviral immunity in the chloroquine treated macaques compared to the placebo group (roques et al., 2018) . cq and hcq were both shown to inhibit hiv replication in monocytic and t cell lines as well as in monocytes and primary t cells at a post-transcriptional step (sperber et al., 1993; tsai et al., 1990) . cq can also inhibit other viruses like cchfv (ferraris et al., 2015) , hepatitis a virus (hav) (bishop, 1998) and ebov (madrid et al., 2013) in cell lines. additionally, cq has been shown to protect mice against a challenge of a deadly dose of ebov (madrid et al., 2013) . in cell culture cq has shown potent antiviral activity against a broad range of viruses and in some cases efficacy in animal model of infection, as discussed above. however, clinical trials aimed to test the efficacy of cq as antiviral has shown very limited antiviral effect. the rather narrow therapeutic index of chloroquine might be behind the limited antiviral effect of chloroquine in clinical trials. the development of chloroquine derivatives with lower toxicity could improve their effectiveness for the treatment of viral infections. the immunomodulatory activity of cq can also contribute to the control of viral diseases reducing the excessive release of cytokines and other proinflammatory mediators. cyclophilin a (cypa) is a peptidylprolyl isomerase that is expressed in the cytosol (schmid, 1995) . cyclosporin a (csa) is a cyclic undecapeptide with immune suppressive activity that mainly targets cellular cyclophilins (cyps) (handschumacher et al., 1984; schreiber, 1991) . the cyp-csa complex inhibits the phosphatase activity of calcineurin needed for the nuclear translocation of the nuclear factor of activated t cells (nfat), which eventually leads to the block of transcription of cytokines and inhibition of t cell activation (matsuda and koyasu, 2000) . csa is one of the drugs approved by the fda for immunosuppressive therapy to avoid rejection in organ transplants (hartono et al., 2013) . csa has been reported to have antiviral activity against a wide range of viruses including human papilloma virus (hpv) (bienkowska-haba et al., 2009 ), vesicular stomatitis virus (vsv) (bose et al., 2003) , vaccinia virus (vv) (damaso and moussatche, 1998) , hiv-1 (franke et al., 1994; thali et al., 1994; wainberg et al., 1988) , and hcv (kaul et al., 2009; nakagawa et al., 2004; yang et al., 2008) . although the immunosuppressive activity of csa does not make it a good candidate for antiviral treatment, some csa derivatives, like alisporivir, nim811 and scy-635, with reduced immunosuppressive properties that retain the ability to bind cyclophilin have been developed and proved to conserve the antiviral activity (flisiak et al., 2008; ma et al., 2016; paeshuyse et al., 2006; watashi et al., 2014) . the replication of certain viruses like hiv, hcv, hpv, vsv, vv or influenza virus relies on their interaction with cyclophilins at certain steps of their replication cycle. thus, the antiviral activity of cyclosporine a (csa) and some of its nonimmunosuppressive analogs against these viruses has been shown to be related to its ability to bind cellular cyclophilins and inhibiting the interaction with the viral proteins (bienkowska-haba et al., 2009; bose et al., 2003; damaso and moussatche, 1998; franke et al., 1994; kaul et al., 2009; nakagawa et al., 2004; thali et al., 1994; wainberg et al., 1988; yang et al., 2008) . csa also blocks the replication of diverse coronaviruses (de wilde et al., 2018 (de wilde et al., , 2011 pfefferle et al., 2011) . the nucleocapsid protein of sars-cov specifically binds cypa (luo et al., 2004) , and cypa has been found to be incorporated into the sars-cov particle (neuman et al., 2008) . however, sirna knock-down of cellular cypa and cypb, the main targets of csa, did not have an effect in the infectivity of this virus, suggesting that either these cyclophilins are not required for viral replication or that the remaining levels of cellular cyclophilins are enough to support normal replication (de wilde et al., 2011) . additionally csa and some csa analogs have been shown to inhibit hbv entry into cells (watashi et al., 2014) . however, this inhibitory activity was independent on the binding to cypa or calcineurin, and correlated to the ability of csa to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (ntcp), a membrane protein that has been proposed to be the hbv receptor. cypa has been reported to interact with influenza a virus matrix protein m1 suppressing viral replication in cell cultures and animal models liu et al., 2009) . the role of cypa in the viral cycle is unclear. although it seems to act at several steps of the viral life cycle, the isomerase activity of cypa does not seem to be implicated (liu et al., 2009) . overexpression of cypa inhibits m1 translocation into the nucleus (liu et al., 2009 ) while depletion of cypa accelerated the replication of the virus (liu et al., 2012b) . additionally cypa has been shown to increase degradation of m1 though the ubiquitin proteasome system (liu et al., 2012b) . the effect of csa on influenza a virus infection has also been investigated. csa-treated mice were administered a dose of influenza a virus that would be lethal for untreated mice, but they survived (schiltknecht and ada, 1985) . csa has also been reported to inhibit the propagation of several strains of influenza a virus in cell cultures blocking a late step of the replication cycle by mechanisms that might implicate cypa-dependent and -independent pathways (hamamoto et al., 2013; liu et al., 2012a; ma et al., 2016) . globally these results point at non-immunosuppressive csa analogs as promising broad-spectrum antivirals. the most advanced csa analogue is alisporivir or debio 025 that has been evaluated in phase ii and phase iii clinical trials with hcv infected patients (clinicaltrials.gov identifier: nct01215643 and nct02753699) with promising results on their safety and efficacy. further studies to evaluate the efficacy of csa analogs with reduced immunosuppressive activity as antivirals in clinical trials are assured. the antibacterial effect of silver metal has been known from ancient times (barillo and marx, 2014) . recent advances in nanotechnology have allowed the introduction of silver nanoparticles (agnps) for biomedical applications (burdusel et al., 2018) . agnps can be prepared by several techniques, including physical, chemical and biological methods. depending on the size and the technique used to prepare the nanoparticles their properties and toxicity can vary (thorley and tetley, 2013; ullah khan et al., 2018) . agnps have been proven to have broad antimicrobial activity against gram positive and gram negative bacteria (kim et al., 2007; morones et al., 2005) . in addition, some studies have shown that agnps also have antiviral activity against a broad range of viruses including herpes simplex virus (baram-pinto et al., 2009) , influenza virus (papp et al., 2010; xiang et al., 2013 ), hepatitis b virus (lu et al., 2008 , hiv-1 lara et al., 2010a, b; sun et al., 2005) , rift valley fever virus (borrego et al., 2016) , dengue virus (murugan et al., 2016; sujitha et al., 2015) and tacaribe virus (speshock et al., 2010) . the antiviral mechanism of action of agnps is not well defined. agnps are thought to interfere with virus-cell attachment and entry into the target cell or by directly inducing structural changes in the virion turning it non-infective xiang et al., 2013) . additionally, agnps can enter the cell and exert their antiviral activity by interfering with cellular proteins needed for viral replication or by direct interaction with viral proteins. silver ions (ag + ) can react with thiols and phosphate groups present on proteins and nucleic acids inhibiting different steps of the replication cycle (lara et al., 2010a) . agnps formulated as topical vaginal gels could be used to prevent the transmission of sexually transmitted infections like hiv and hsv. studies in an in vitro system using human cervical tissue cultures that simulate in vivo conditions have shown encouraging results inhibiting hiv transmission (lara et al., 2010b) . although the use of agnps for biomedical applications seems promising, further studies are needed to better understand their potential fig. 1 . experimental strategy to study the mechanism of action of antiviral drugs in single cells and to investigate the origin of antiviral drug resistance. toxicity and long term effects on human health and in the environment. the toxicity of agnps is highly related to the release of ag + , which directly depends on the size of the nanoparticles. the smaller the size the higher the amount of ag + released. ag + has been reported to alter mitochondrial function (chappell and greville, 1954; kone et al., 1988) . moreover, exposure of cells to agnps induce the production of high levels of reactive oxygen species (ros) and jnk and p53 activation leading to mitochondria-dependent cell apoptosis (hsin et al., 2008) . therefore, further studies are needed to better understand the mode of action of agnps, their cell specificity and toxicological issues in order to generate new and more effective compounds as well as the use in combination with other drugs in the treatment of different viral diseases. high content imaging (hci) is a popular and basic tool of early drug discovery in multiple disease research areas (carpenter, 2007) . hci was developed with the contribution of major advances in robotics, imaging and automated image processing (moffat et al., 2017) . hci is already a fundamental technology for antiviral screening and validation (bernatchez et al., 2018; hoenen, 2017; lowen et al., 2018; mudhasani et al., 2014; panchal et al., 2010; tan et al., 2014) . moreover, recent developments in light and electron microscopy are having a significant impact in virology. in particular, live-cell microscopy, super-resolution microscopy, correlative light and electron microscopy (clem) and 3d imaging are now fundamental tools for studying virus-cell interactions (bykov et al., 2016; fernandez de castro et al., 2017; francis and melikyan, 2018; risco et al., 2014; witte et al., 2018) . microscopy is also a powerful tool for studying antivirals. with clem, scientists can study events at the level of single cells in culture (fernandez de castro et al., 2014; hellstrom et al., 2015; tenorio et al., 2018) and infected animals (lowen et al., 2018) . clem is starting to show its potential for testing antivirals because changes in viral structures assembled in cells where the treatment was effective and in those where the antiviral drug failed can be now studied in detail (berger et al., 2014; lowen et al., 2018; martinez et al., 2014) . cell sorting and clem in combination with proteomics, transcriptomics and lipidomics, will be exceptionally informative in identifying new targets for antiviral drugs, to characterize the mechanism of action of old and new antiviral compounds and to understand how resistance to antivirals is developed in cells (fig. 1) . the strategy for these studies can be as follows: duplicated cell cultures infected with a fluorescent virus and treated in the presence or absence of an antiviral drug are processed either by clem or cell sorting. fluorescence microscopy will reveal interesting features in single cells that are then processed for ultrastructural analysis. electron microscopy will show how viral structures are affected by the antiviral drug. in parallel, key cell factors involved in cell resistance to infection or in viral escape to the antiviral drug will be identified by comparing fluorescent and nonfluorescent cells separated by cell sorting and studied by proteomics, transcriptomics and lipidomics. for these studies, cells that differentiate into tissues and 3d organoids (liu et al., 2013; takebe et al., 2015; walters et al., 2013) will provide more physiologically relevant cellular systems to study virus infection and to test antivirals. emerging viruses are a major threat to human health and the rate at which new pathogenic viruses are emerging has accelerated in the past fifty years. the frequent outbreaks have highlighted the urgent need for new antiviral treatments. generally, antiviral drug development has focused on targeting viral components to block virus entry, replication, morphogenesis and propagation, or on the modulation of the host immune response (zhu et al., 2015) . drug repurposing is an important alternative. in this review we have chosen several repurposed drugs with proven antiviral activity. with different mechanisms of action, digoxin, sunitinib, chloroquine, cyclosporine a and silver nanoparticles are promising candidates for broad spectrum antivirals that could be used in a combined antiviral therapy. drug repurposing is now facilitated by a number of resources (pollastri and campbell, 2011) , such as pathogen target bioinformatics resources, public data repositories of screening data, structural biology resources and compound collections, such as the library of small molecules from the national institutes of health (http://www.nihclinicalcollection.com) (ashbrook et al., 2016; cao et al., 2015) or the canadian drugbank (https://www.drugbank. ca/), a large drug database commonly used by computational drug repositioning methods. drugbank contains fda approved drugs and those in clinical trials and is frequently updated with new information. with new and powerful screening assays and prediction tools, in silico structure-based screening of these large chemical libraries is identifying potential inhibitors of viral infections including repurposed compounds that target a variety of viral proteins and host factors (abu bakar and ng, 2018; barrows et al., 2016; dowall et al., 2016; martinez et al., 2015; yuan et al., 2017) . another strategy that will surely reveal more promising candidates is based on comparing data from databases of pathogens (brown and patel, 2017; sharma et al., 2015) with databases of drugs, e.g., drug information from the national library of medicine (https://www.nlm.nih.gov/learn-about-drugs.html). imaging studies combined with functional assays will show the localization and intracellular effects of these new inhibitors (panchal et al., 2010) . combination therapies with more than one drug against more than one target will be necessary to minimize problems of resistance to repurposed antiviral drugs. the authors declare no conflict of interest. nonstructural proteins of alphavirus-potential targets for drug development targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases antagonism of the sodium-potassium atpase impairs chikungunya virus infection sunitinib maleate inhibition of herpes simplex virus type 1 infection by silver nanoparticles capped with mercaptoethane sulfonate silver in medicine: a brief history bc 335 to present evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice past, present and future: 30 years of hiv research a screen of fda-approved drugs for inhibitors of zika virus infection anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects daclatasvir-like inhibitors of ns5a block early biogenesis of hepatitis c virus-induced membranous replication factories, independent of rna replication development and validation of a phenotypic high-content imaging assay for assessing the antiviral activity of small-molecule inhibitors targeting zika virus target cell cyclophilins facilitate human papillomavirus type 16 infection examination of potential inhibitors of hepatitis a virus uncoating amodiaquine, an antimalarial drug, inhibits dengue virus type 2 replication and infectivity potential application of silver nanoparticles to control the infectivity of rift valley fever virus in vitro and in vivo requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype a standard database for drug repositioning biomedical applications of silver nanoparticles: an up-to-date overview atp1a1-mediated src signaling inhibits coronavirus entry into host cells correlative light and electron microscopy methods for the study of virus-cell interactions in vitro combination of anti-cytomegalovirus compounds acting through different targets: role of the slope parameter and insights into mechanisms of action a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs image-based chemical screening effect of silver ions on mitochondrial adenosine triphosphatase effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo inhibition of vaccinia virus replication by cyclosporin a analogues correlates with their affinity for cellular cyclophilins on chikungunya acute infection and chloroquine treatment. vector borne zoonotic dis cyclosporin a inhibits the replication of diverse coronaviruses screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture cyclophilins and cyclophilin inhibitors in nidovirus replication chloroquine, an endocytosis blocking agent, inhibits zika virus infection in different cell models different ph requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza a viruses mycophenolic acid inhibits dengue virus infection by preventing replication of viral rna innovation in the pharmaceutical industry: new estimates of r&d costs inhibitors of the sodium potassium atpase that impair herpes simplex virus replication identified via a chemical screening approach repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection interaction of silver nanoparticles with hiv-1 chloroquine interferes with dengue-2 virus replication in u937 cells antiviral activity of chloroquine against dengue virus type 2 replication in aotus monkeys metallothioneins for correlative light and electron microscopy threedimensional imaging of the intracellular assembly of a functional viral rna replicase complex evaluation of crimean-congo hemorrhagic fever virus in vitro inhibition by chloroquine and chlorpromazine, two fda approved molecules the cyclophilin inhibitor debio-025 shows potent anti-hepatitis c effect in patients coinfected with hepatitis c and human immunodeficiency virus live-cell imaging of early steps of single hiv-1. infection specific incorporation of cyclophilin a into hiv-1 virions contemporary use of digoxin in the management of cardiovascular disorders suppression of adenovirus replication by cardiotonic steroids genistein interferes with sdf-1-and hiv-mediated actin dynamics and inhibits hiv infection of resting cd4 t cells cyclosporin a inhibits the propagation of influenza virus by interfering with a late event in the virus life cycle cyclophilin: a specific cytosolic binding protein for cyclosporin a ionic contra-viral therapy (icvt); a new approach to the treatment of dna virus infections immunosuppressive drug therapy. cold spring harb correlative light and electron microscopy enables viral replication studies at the ultrastructural level mycophenolic acid inhibits hepatitis c virus replication and acts in synergy with cyclosporin a and interferon-alpha luciferase-expressing ebolaviruses as tools for screening of antivirals emerging virus diseases: can we ever expect the unexpected? the apoptotic effect of nanosilver is mediated by a ros-and jnk-dependent mechanism involving the mitochondrial pathway in nih3t3 cells digoxin and 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reversal by thiol reagents selective inhibitors of cyclin g associated kinase (gak) as anti-hepatitis c agents minocycline suppresses dengue virus replication by down-regulation of macrophage migration inhibitory factor-induced autophagy a novel cell-based high-throughput screen for inhibitors of hiv-1 gene expression and budding identifies the cardiac glycosides mode of antiviral action of silver nanoparticles against hiv-1 pvpcoated silver nanoparticles block the transmission of cell-free and cell-associated hiv-1 in human cervical culture drug repurposing of minocycline against dengue virus infection protective activity of tetracycline analogs against the cytopathic effect of the human immunodeficiency viruses in cem cells current therapy for chronic hepatitis c: the role of directacting antivirals cyclophilin a protects mice against infection by influenza a virus chloroquine, a fda-approved drug, prevents zika virus infection and its associated congenital microcephaly in mice curing a viral infection by targeting the host: the example of cyclophilin inhibitors cyclophilin a interacts with influenza a virus m1 protein and impairs the early stage of the viral replication cyclosporin a inhibits the influenza virus replication through cyclophilin a-dependent and -independent pathways cyclophilin a restricts influenza a virus replication through degradation of the m1 protein generation of functional organs from stem cells countering zika virus: the usamriid response silver nanoparticles inhibit hepatitis b virus replication nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a discovery of cyclosporine a and its analogs as broad-spectrum anti-influenza drugs with a high in vitro genetic barrier of drug resistance a systematic screen of fda-approved drugs for inhibitors of biological threat agents abacavir and mycophenolic acid, an inhibitor of inosine monophosphate dehydrogenase, have profound and synergistic anti-hiv activity imaging the 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use characterization of reemerging chikungunya virus inhibition of human immunodeficiency virus type 1 replication by hydroxychloroquine in t cells and monocytes interaction of silver nanoparticles with tacaribe virus epidemiology, evolution, and recent outbreaks of avian influenza virus in china green-synthesized silver nanoparticles as a novel control tool against dengue virus (den-2) and its primary vector aedes aegypti discovery of 5-[5-fluoro-2-oxo-1,2-dihydroindol-(3z)-ylidenemethyl]-2,4-dimethyl-1h-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase silver nanoparticles fabricated in hepes buffer exhibit cytoprotective activities toward hiv-1 infected cells minocycline attenuates hiv infection and reactivation by suppressing cellular activation in human cd4+ t cells vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven 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of susceptible cells by human immunodeficiency virus type 1 generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity cyclosporin a and its analogs inhibit hepatitis b virus entry into cultured hepatocytes through targeting a membrane transporter the treatment of malaria concepts in light microscopy of viruses digoxin suppresses hiv-1 replication by altering viral rna processing inhibitory effects of silver nanoparticles on h1n1 influenza a virus in vitro inhibition of a/human/hubei/3/2005 (h3n2) influenza virus infection by silver nanoparticles in vitro and in vivo anti-malaria drug chloroquine is highly effective in treating avian influenza a h5n1 virus infection in an animal model cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro structure-based discovery of clinically approved drugs as zika virus ns2b-ns3 protease inhibitors that potently inhibit zika virus infection in vitro and in vivo host-targeting agents for prevention and treatment of chronic hepatitis c -perspectives and challenges broad-spectrum antiviral agents neuroprotective and anti-human immunodeficiency virus activity of minocycline this work was supported by a research grant from the spanish ministry of economy, industry and competitiveness (bio2015-68758-r, mineco-feder to cr). bp was recipient of a post-doctoral contract from the severo ochoa center of excellence program. key: cord-266453-v1hbust8 authors: sztuba-solińska, joanna; fanning, sean w.; horn, james r.; bujarski, jozef j. title: mutations in the coat protein-binding cis-acting rna motifs debilitate rna recombination of brome mosaic virus date: 2012-10-16 journal: virus res doi: 10.1016/j.virusres.2012.10.001 sha: doc_id: 266453 cord_uid: v1hbust8 we have previously described the efficient homologous recombination system between 5′ subgenomic rna3a (sgrna3a) and genomic rna3 of brome mosaic virus (bmv) in barley protoplasts (sztuba-solińska et al., 2011a). here, we demonstrated that sequence alterations in the coat protein (cp)-binding cis-acting rna motifs, the bbox region (in the intercistronic rna3 sequence) and the rna3 packaging element (pe, in the movement protein orf), reduced crossover frequencies in protoplasts. additionally, the modification of bbox-like element in the 5′ utr region strongly debilitated crossovers. along the lines of these observations, rna3 mutants not expressing cp or expressing mutated cps also reduced recombination. a series of reciprocal transfections demonstrated a functional crosstalk between the bbox and pe elements. altogether, our data imply the role of cp in sgrna3a-directed recombination by either facilitating the interaction of the rna substrates and/or by creating roadblocks for the viral replicase. genetic rna-rna recombination is the process of joining noncontiguous rna fragments and is one of the dominant forces shaping the architecture of rna viral genomes (sztuba-solińska et al., 2011b) . the process of replicase-driven template switch is the most widely accepted mechanism of recombination (ranjith-kumar et al., 2002; shapka and nagy, 2004; hu et al., 2003) . highly structured cis-acting replication elements stall the progressing rna-dependent rna polymerase (rdrp) complex, which subsequently forces the dissociation of the replicase from the original template and promotes its re-initiation at the acceptor site on another template (suzuki et al., 2003; dedepsidis et al., 2010; draghici and varrelmann, 2010; sztuba-solińska et al., 2011b) . studies with brome mosaic virus (bmv) demonstrated that the homologous crossovers occur near the secondary structures, e.g., within a 3 trna-like structure (tls), which appear to serve as roadblocks for the progression of rdrp. interestingly, the effects were more evident during (+)-strand synthesis (olsthoorn et al., 2002) . moreover, viral proteins have been shown to affect the distribution of crossover sites (figlerowicz et al., 1997 (figlerowicz et al., , 1998 panaviene and nagy, 2003) or even to mediate recombination. for example, hiv-1 nucleocapsid protein (nc), an rna chaperone, not only enhanced homologous strand transfer during reverse transcription but also facilitated the annealing of complementary strands (negroni and bus, 2000; roda et al., 2003) . in addition, coronavirus nucleocapsid protein (n) enhanced the frequency of template switching (zúñiga et al., 2010) , and the close association between the protein n and structural protein nsp3 (a component of replicase complex) implied a role in the replicase-driven process (hurst et al., 2010) . bmv is a tripartite rna virus, where rna components 1 and 2 (rna1 and rna2) encode, respectively, replicase proteins 1a and 2a, while rna3 encodes a movement protein (3a) and a coat protein (cp) (fig. 1) . the cp is expressed from subgenomic (sg) rna4. the multipartite composition of bmv genome and the production of subgenomic rnas make it a useful model for studies on rna recombination. both inter-or intra-segmental crossovers in bmv rnas were observed at the 3 tls (nagy and bujarski, 1994 , 1996 . moreover, the rna3 subgenomic promoter (sgp) was mapped as an active recombination hot-spot (wierzchoslawski et al., 2004) , additionally engaged in the formation of nonreplicating 5 subgenomic rna3a (sgrna3a) (wierzchoslawski et al., 2006) . binding of bmv cp molecules to the rna3 elements. this schematic summarizes the secondary elements on bmv rnas known to serve as binding sites to either cp or rna-dependent rna-polymerase (rdrp) proteins. bmv genomic rnas are represented as thick black lines with the 5 cap structure and 3 trna-like element shown at the ends. cp is indicated with a purple asterisk, while rdrp is represented as a red circle. the names for each binding site are shown below with exact nucleotide positions. the predicted secondary structures (with mfold) are represented on the top of the diagram. sequences altered in the rna mutants tested are indicated by solid boxes. the question marks above the 5 utr bbox-like elements indicate the potential, unconfirmed cp/rdrp binding(s). recently, we reported the efficient homologous recombination between bmv sgrna3a and genomic rna3 in barley protoplasts (sztuba-solińska et al., 2011a) . both in vitro and in vivo data suggested that the exposed 3 terminal polya tail of sgrna3a can prime the extension of (+) strand rna synthesis on the (−) sense rna3 templates. in addition, more upstream regions also supported homologous crossover events, most likely by internal strand transfers during (−) sense rna synthesis. among the cis-acting motifs known to bind bmv cp ( fig. 1) , the bbox element is located within the intercistronic region of the rna3 (+) strand as part of the intergenic replication enhancer (ire) (pogue et al., 1992; baumstrak and ahlquist, 2001) . another motif is a highly structured position-dependent rna3 packaging element pe that is localized between nucleotides (nts) 550 and 820 and is considered as a selective domain for cp interaction facilitating specific packaging of bmv rna3 (choi and rao, 2003) . since sgrna3a represents an exact copy of the 5 half of (+) strand rna3, it also carries these two cis-acting elements. in this work, we show that sequence alterations within both motifs, either on rna3 and/or on the sgrna3a components, diminished the recombination frequency in protoplasts. a series of reciprocal transfections revealed putative interactions between these motifs in establishing the crossover locations. moreover, the transfection of protoplasts with the rna mutants incapable of cp expression or that expressed the cp variants with altered domains recognizing the cp-binding motifs also reduced the recombination frequency. our study offers novel insight into bmv rna recombination and indicates that associations of cp with the rna cis-acting motifs can mediate rna recombination process. full-length cdna clones, corresponding to wt bmv rnas 1, 2, and 3 (russian strain) were obtained from p. ahlquist laboratory as plasmids pb1tp3, pb2tp5, and pb3tp7, respectively (janda et al., 1987) . the plasmids were used as templates to synthesize the capped full-length transcripts in vitro using the megascript t7 kit (ambion, austin, tx). moloney murine leukemia virus (mmlv) reverse transcriptase, pfu dna polymerase (agilent technologies), restriction enzymes, and dntps were obtained from the promega corporation or new england biolabs, inc. all rna3 variants were pcr-amplified from plasmid pb3tp7 using the primers listed in supplementary table 1 . primer 1 was used to introduce the 5 a to u substitution at nt position 2, which was shown to increase both the (−) strand rna3 production and the frequency of rna3-sgrna3a crossovers (sztuba-solińska et al., 2011a) . the bbox-rna3 derivative carrying uu to aa substitutions at nts 1102-1103 was generated using fusion pcr. the 5 segment was generated with primers 1 and 2, whereas the 3 segment was generated with primers 3 and 4 followed by pcr amplification using both fragments and primers 1 and 4. similarly, the pe-rna3 variant was generated using fusion pcr with flanking primers 1 and 4 of the 5 fragment (synthesized using primers 1 and 5), carrying the pe deletion (nt 660-762) and the 3 fragment (synthesized with primers 6 and 4). the 5 box-rna3 construct carried u to a substitutions at nts 23-24 and nt 29, which were generated using single-step pcr with primers 7 and 4. the cp0-rna3 construct carried a g to c substitution at position 1242 and an a to c substitution at position 1239, which were both and generated using primers 8 and 9. subsequently, the pcr products were purified using the qiaquick ® pcr purification kit (qiagen), digested with pflmi (nt 816) and sali (nt 1254) enzymes and re-ligated between the pflmi and sali sites into pb3tp7. the resulting product was used as template for the next round of pcr with primers 1 and 4. the cp-bbox rna3 construct expressing the cp derivative carrying alanine substitutions within the aa residues t145, d148, n151, y155, y157, s159, v162, p163, and k165, was created using fusion pcr with primers 1 and 10 for the 5 fragment (carrying the desired mutations plus the 5 a to u substitution at nt position 2) and primers 11 and 4 for the 3 fragment. subsequently, the overlapping segments were mixed and co-amplified in the next round of pcr using primers 1 and 4. likewise, the cp-slc construct carrying alanine substitutions within the cp aa residues v27, p29, v30, v32, p34, q39, and k41 was created using fusion pcr with flanking primers 1 and 4 and two fragments: the 5 fragment (generated with primer 1 and 12; the latter carrying the desired mutations plus the 5 a to u substitution at nt position 2) and the 3 segment (obtained with primers 13 and 4). the previously published pjs-22 plasmid (sztuba-solińska et al., 2011a) was used as a template to generate sgrna3a derivatives: sg, bbox-sg, pe-sg, 5 box-sg using fusion pcr with primers specific for each corresponding rna3 derivative (indicated above). for each construct, the obtained pcr product was digested with bglii to transcribe the full-length sgrna3a. these sgrna3a-derived transcripts carried the polya tail plus four extra bases (gauc) at the 3 end. the cdna templates comprising either the bbox (fr bbox) or the pe (fr pe) fragment rna3 sequences were synthesized using pcr amplification of the above corresponding full-length rna3 cdna clones (wt or mutated) with the following oligonucleotides (supplementary table 1 ): primers 27 and 28 for the bbox fr (nts 1070-1140) and primers 29 and 30 for the pe fr (nts 633-817). the cdna templates were subsequently transcribed in vitro in the presence of alphap 32 -ctp, and the unincorporated radioactivity was removed on a sephadex g-25 spin minicolumn. radioactive rna3 probes were used for the rna-cp filter binding assays (below). the cdna templates for the synthesis of wt and mutated variants in the cis-acting rna motifs were generated from the corresponding rna3 cdna derivatives using one-step pcr with primers 21 and 22 for the intergenic bbox motif, 23 and 24 for the packaging element (pe), and 25 and 26 for the 5 bbox related region. the final cdna templates were sequenced to confirm their correct assembly. subsequently, the rnas were synthesized in vitro using the megascript t7 kit (ambion, austin, tx) and purified using microspin tm g-50 columns (ge healthcare). barley mesophyll protoplasts were isolated from the five-dayold barley seedlings according to the protocol of rao (2007) with modification performed in our laboratory (sztuba-solińska et al., 2011a) . on average, one million protoplast cells were inoculated with 1 g of each desired combination of the in vitro-capped transcripts using a peg-mediated transfection protocol, as described previously (sztuba-solińska et al., 2011a). total rna extracted from protoplast samples was separated in denaturing 1.2% agarose gels (sambrook and russell, 2001) . the rna3-size material was cut out from the gel and purified using spin columns (ambion, cat# 605 am10065) followed by chloroform extraction and ethanol precipitation. the final rna preparation was subjected to rt-pcr with primers 17 and 18 (supplementary table 1 ) and the products were purified with the qiaquick ® pcr purification kit (qiagen) and cloned into the pgem-t easy vectorsystem (promega). the resulting clones were analyzed by restriction digestion. the accumulation of bmv rnas in protoplasts was detected using northern blotting (kroner et al., 1989) . the blots of the total rna extracts (see above protocol) were hybridized with a probe specific to the 200 nts at the 3 end of the (+)strand rna as described previously (wierzchoslawski et al., 2004) . the hybridization signals were quantified on scanned x-ray films using imagequant 5.0 software from amersham biosciences. to verify that the recombinant rnas emerged during rna replication rather than during rt-pcr (cocquet et al., 2006) , protoplasts were co-transfected with a mixture of wt bmv rna3 and sg construct, but without wt rnas 1 and 2. another control involved the rt-pcr amplification of the rna3 sequences from a mixture of wt bmv rnas and sg construct, omitting the protoplast transfection. the rna transcripts used in these experiments were pre-treated with an excess of rnase-free dnase (ambion) to remove the plasmid dna template and prevent the occurrence of dna-amplified pcr products. subsequent analyses of cdna clones did not detect rna3 recombinants (not shown). in yet another control, total rna was extracted from protoplasts that were transfected with wt (virion) bmv rna. no false positive recombinants were identified. the wild type and mutated cp sequences were amplified from pb3tp7 plasmid or from the corresponding fusion pcr products: cp-bbox and cp-slc (described above) using primers 19 and 20 that carried, respectively, the bamhi and xhoi restriction sites. the pcr products were digested with restriction enzymes and re-ligated into the corresponding sites on the pet21a(+) expression vector (emd biosciences). the resulting constructs were transformed in escherichia coli bl21 (de3) cells. following a 5 ml overnight incubation at 37 • c, a 50 ml lb/amp subculture was inoculated with 1 ml of the overnight culture. once at mid-log phase growth (o.d. 600 = 0.6), the subculture was used to inoculate a 1 l lb/amp culture. the expression of the bmv-cps was induced with 0.1 mm iptg once the cells reached mid-log phase. after 3 h incubation at 37 • c, the cells were harvested by centrifugation at 8000 × g for 15 min. the pellet was resuspended in 10 mm tris/ph 8 and sonicated for 4 one min on/off cycles with a model 60 sonic dismembrator (fisher scientific) at an output power of 21 w. the lysed cells were centrifuged at 22,700 × g for 15 min, and the supernatant fraction was loaded onto a histrap hp column (ge healthcare), and the column was washed with 20 mm imidazole, 50 mm phosphate and 500 mm nacl ph 7.4. the protein was eluted with 500 mm imidazole, 50 mm phosphate and 500 mm nacl ph 7.4, and further purified on a hiload 26/60 superdex 75 prep grade fplc column (10 mm tris, 150 mm nacl, ph 8.0). finally, the protein was dialyzed in a buffer containing 50 mm tris-hcl, ph 7.5, 300 mm nacl and 1 mm dtt to keep the molecules in a monomeric stage (yi et al., 2009b) . purified wt or mutant cp preparations (200 ng) were incubated with serial dilutions of 1 nm of radiolabeled rnas (ratios 1:0; 1:10; 1:100 and 1:1000) in the rna binding buffer (50 mm tris-hcl, ph 7.5, 50 mm nacl and 4 mm mgcl 2 , as described in yi et al., 2009a) , in a total volume of 20 l. two microliters of each reaction mixture were applied on the nitrocellulose membrane, irradiated with uv at 1000 mj for 2 min, dried, washed with the binding buffer, dried again and the retained radioactivity was visualized using autoradiography on an x-ray film. the previously described barley protoplast recombination system (sztuba-solińska et al., 2011a) involved wt bmv rnas 1 and 2, and wt or modified rna3 and sgrna3a. recombination events were tracked based on the inheritance of three restriction marker mutations (bamhiii, hindiii and psti) located within the 3a orf and near the 5 and 3 sides in all the sgrna3a constructs (fig. 2) . the marker mutations were translationally silent, stable, and they did not affect the accumulation of progeny rna3 recombinants in protoplasts (sztuba-solińska et al., 2011a) . moreover, all of the rna3 constructs that were used contained a 5 a to u substitution at nt 2, which was shown previously to decrease the accumulation of (+) rna3 strands without affecting (−) strands . in our previous study, this particular substitution was fig. 2 . the distribution of crossover sites between recombining rna3 and sgrna3a derivatives in co-transfected barley protoplasts. the rna3/sgrna3a inocula are indicated on the left of each transfection experiment panel (for further description of the rna constructs, see section 2). all rna3 derivatives bear a point mutation (a to u) at nt position 2 near the 5 end (black square) to lower the production of (+) sense rna3. the positions of marker restriction sites are indicated below each construct. the column on the right shows the number of recombinants bearing the indicated restriction marker sites identified among 100 insert-bearing cdna clones; the rest of cdna clones represented the wt input rna3 sequence. these numbers are repeated inside the shaded rectangles (for recombinants with single markers) or above the brackets (for recombinants with multiple markers). the numbers summarize the results from two independent transfection experiments per each combination of the inoculated rna substrates. to calculate total recombination frequency, the number of all recombinant-bearing clones should be summarized and divided by 100. total protoplast rna was extracted after 48 h post inoculation, separated on a 1.2% denaturing agarose gel, blotted and probed with the 3 rna probe (see section 2). lanes 1 and 10, mock-inoculated protoplasts as the negative control; lanes 2 and 11, protoplasts transfected with wt bmv rnas 1, 2 and 3 as positive controls; lanes 3-9, and 11-15, protoplasts co-transfected with different combinations of rna3 and sgrna3a variants (shown on top). below, digital bands were analyzed with imagequant (ge-healthcare), and the measured areas were defined as absolute intensity. these numbers were used to calculate the relative intensity of each band. the mean from multiple experiments was used to plot the graph. error bars represent the calculated standard errors. also shown to increase rna3-sgrna3a crossovers in protoplasts, as well as it kept the (+) rna3 strand synthesis at lower level (mut-rna3) (sztuba-solińska et al., 2011a). since mut-rna3 recombined with sgrna3a (sg) at elevated frequency (42%), we hypothesized that sgrna3a primed rna recombination on (−) sense rna3 templates. the pe-rna3 construct, in addition to the a to u mutation, it also carried pe deletion (nts 660-762), whereas the pe-sg sgrna3a construct, besides containing the same deletion, it contained three previously described silent restriction marker mutations (bamhi, hindiii and psti, see fig. 2 ). these marker mutations allowed mapping of the recombination sites within the 5 region of rna3 and were present in all the sgrna3-derived constructs used in this work (sztuba-solińska et al., 2011a) . the pe-rna3 and pe-sg constructs were co-transfected (together with wt rna1 and 2) into barley protoplasts ( fig. 2a) . only three recombinant clones were identified among 100 rt-pcr-generated cdna clones, thus the recombination frequency (3%) was greatly reduced as compared to the previously published results for mut-rna3 and sg co-transfection (42%) (sztuba-solińska et al., 2011a). the control amplifications did not show rt-pcr generated recombinants (see section 2). the three characterized clones all caried the 5 -most bamhi marker mutation which implied that the residual rna3-sgrna3a crossovers occurred within the regions 5 upstream to pe element. in yet another experiment, the pe-rna3 derivative was co-transfected with sg construct ( fig. 2a) . similarly, the recombination frequency approached only 3%, with three recombinant rna3 clones containing the 5 bamhi marker site, implying internal strand switching. again, the level of rna3 (fig. 3 , lane 6) was alike that of mut-rna3, reflecting an equal availability of rna3 substrate for crossovers. overall, when compared with the 42% frequency between mut-rna3 and sg, the lack of functional pe in rna3 effectively reduced the recombination frequency to a marginal value, demonstrating the importance of the pe motif. however, the presence of the pe region in the sgrna3a donor did not seem to be critical because both sg and pe-sg recombined at comparably low rate. a structured region near the 3 sgrna3a polya tail, referred to as the intergenic bbox motif, participates in the assembly of the bmv replicase complex on rna3 via interactions with protein 1a (baumstrak and ahlquist, 2001) , and it binds cp molecules via a specific peptide domains, as mapped by yi et al. (2009b) . to determine whether bbox structure contributes to rna3-sgrna3a crossovers, uu to aa substitutions previously found to lower the affinity for bmv cp (yi et al., 2009a) were introduced within the bbox hairpin loop (fig. 1) . co-transfection with a mixture of bbox-rna3 and bbox-sg generated only three rna3 recombinants (per 100 cdna clones analyzed): two clones contained all three marker sites, whereas one clone contained only the 5 bamhi site. in a separate experiment, the bbox-rna3 was co-transfected with sg construct to determine whether the presence of the wt bbox motif in sgrna3a could rescue the previously reported high recombination frequency for unmutated rnas. indeed, among 100 cdna clones, there were 42 recombinants (fig. 2b) , of which the majority carried all three marker restriction sites (74%, 32 clones), while few had either single (bamhi -four clones) or double (bamhi/hindiii -three clones, bamhi/psti -one clone, hindiii/psti -two clones) restriction sites. this distribution of crossovers suggested that the recombination was mainly governed via the incorporation of sgrna3a (referred to as primer extension). here, the unchanged 3 end motifs of sgrna3a, the 3 polya tail and bbox, would efficiently prime the (+) sense rna3 synthesis. next, we examined the potential interplay between the pe and bbox motifs. the co-transfection of protoplasts with the pe-rna3 and bbox-sg constructs resulted in only two recombinants: one containing the central hindiii site and one with the 3 psti site (fig. 2c) . thus, the absence of pe in rna3 along with the bbox mutation in sgrna3a minimized the crossover frequency. however, the reverse arrangement (bbox-rna3 and pe-sg) supported a 13% recombination frequency (fig. 2c ). in this case, all recombinants contained three marker mutations. these results suggested that the disrupted interaction of cp with the rna3 bbox lowers the frequency of internal crossovers, while the presence of unchanged 3 end motifs, in particular the bbox in sgrna3a supports the incorporation of full-length sgrna3a during synthesis of progeny (+) sense rna3 molecules. previous studies implied that the 5 utr region was another possible rna3-sgrna3a recombination hotspot (sztuba-solińska et al., 2011a). the 5 bbox-like motif was mutated by replacing u with a residues at nt positions 23-24 and at nt position 29 in order to change its 7-nt hairpin-loop sequence (fig. 1, top) . the resulting 5 box-rna3 and 5 box-sg constructs (fig. 2d ) did not generate any recombinants in protoplasts. likewise, the 5 box-rna3 × sg (fig. 2d ) produced only one recombinant per 100 clones analyzed, and it carried the internal hindiii site. thus, the intact 5 utr of rna3 seems to be required for efficient crossovers. we conclude that the 5 utr must participate in the recombination activity of the rna3 template. the bmv cp was shown previously to specifically bind not only to bbox and pe elements (rao, 2006; yi et al., 2009a,b) but also to stem-loop c (slc) of the 3 tls in rna3 (fig. 1) . the cp domains responsible for the binding to bbox and slc motifs were mapped (yi et al., 2009b) . to address whether the absence of cp affects sgrna3a-rna3 recombination, cp0-rna3 was created. cp0-rna3 did not transcribe sgrna4 due to mutations introduced within the sgrna4 promoter region (+1 g to c and −2 a to c), as previously reported (sivakumaran et al., 2004) , and thus in the above system the expression of cp is debilitated. the results of disrupted sgrna4 transcription along with an unaffected level of rna3 accumulation in protoplasts are shown in fig. 3 (compare lanes 12 and 13) . among four identified recombinants (per 100 analyzed cdna clones), three acquired a complete set of sg-derived restriction markers, while one clone inherited only the central hindiii and the 3 psti sites (fig. 2e ). this suggested that the remnant recombination activity could occur via extension of sgrna3a serving as a primer during copying of progeny rna3. to test this idea further, two previously mapped specific rna binding domains on cp (yi et al., 2009b) were mutated, including critical amino acid regions 143-165 (bbox motif binding domain) and 27-41 (the 3 slc stem-loop binding domain). since the pe binding domain was not mapped on bmv cp molecule, the corresponding amino acids could not be mutated. two cp derivatives were generated: the cp-bbox mutant carrying alanine substitutions for residues t145, d148, n151, y155, y157, s159, v162, p163 and k165, and the cp-slc variant carrying alanine substitutions for residues v27, p29, v30, v32, p34, q39 and k41. to examine recombination frequency, both rna3 cp mutants were co-transfected separately with sg in barley protoplasts. the cp-bbox rna3 generated the following recombinants: three carrying all sgrna3a marker restriction sites (likely reflecting sgrna3a extension on progeny rna3), one carrying the 5 bamhi and the 3 psti sites, and one carrying only the 5 bamhi site (fig. 2f ). in the case of cp-slc rna3, only one recombinant emerged, containing the inner hindiii site (fig. 2g ). taken together, these results revealed that either the elimination of cp expression or the disturbed cp binding to cis-acting motifs on the rna substrates, or both, inhibited the rna3-sgrna3a recombination. to determine if sequence modifications affected the accumulation of bmv rna3 and/or sgrna3a substrates in host cells, barley protoplasts were co-transfected with transcripts of rna3 and sgrna3a (1:1 molar ratio) along with wt rnas 1 and 2. the subsequent northern blot analysis of total rna extracts confirmed previously reported observations ; sztuba-solińska et al., 2011a); a noticeable reduction in the production of (+) strands was observed in all mutated rna3 variants that carried the a to u substitution in their 5 noncoding regions (lanes 3-9 and 12-15 in fig. 3) , with all rna3 and sgrna3a derivatives accumulating to levels comparable with the control inoculum (fig. 3, lanes 3 and 12) . moreover, the addition of full-length sgrna3a (sg construct) (fig. 3, lanes 4, 6, 8, 12 , 13, 14, and 15) did not affect the ratio of accumulating bmv rna components versus the control inoculum (fig. 3, lane 3 and 12) , and the ratios of bmv rnas were not affected by mutations introduced within the cp orf (fig. 3, lanes 13, 14, 15 ) as compared to the control transfection with bmv rna1 and 2, mut-rna3 and full-length sgrna3a (sg construct) (fig. 3 , lane 3 and 12). furthermore, the cp0-rna3 mutant did not produce sgrna4 as expected (fig. 3, lane 13) , and the addition of the sg construct did not markedly influence the accumulation of the remaining bmv rna components. overall, we concluded that the observed differences in the recombination frequencies and in the distribution of crossover sites were not due to the altered stability or distorted accumulation of the tested rna derivatives. to determine whether the altered stability of transfected rna substrates affected the observed crossover frequencies in protoplasts, stability assays were performed as previously described (sztuba-solińska et al., 2011a) . various combinations of the transcribed genomic rna3 and sgrna3a radioactive variants were co-transfected into barley protoplasts along with unlabeled rna1 and 2. the protoplasts were thoroughly washed to remove the untransfected radioactive material, and the total rna was extracted at three time points. the stabilities of the rna3 and sgrna3a variants were assessed in comparison with the previously tested wt rna3 and wt sgrna3a transcripts. all of the transcript variants were stable to a similar degree within the 75 min assay time (fig. 4) , indicating that the observed frequencies during the protoplast assays reflected recombination rates rather than altered rna stability. to determine whether the lack of recombination activity correlated with diminished cp-rna3 binding, both the wt and two bmv cp mutants were expressed in e. coli and purified as described in methods section. a single band corresponding to cp was identified after sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) (shown in fig. 1 supplement) . the affinity of these cp mutants to the rna3 templates was analyzed with filter-binding assays. equimolar concentrations of serially diluted (1; 1:10; 1:100; 1:1000) radioactive wt or mutated rna3 probes (comprising the regions of individual cis-acting sequences, as shown on top of fig. 5 ), were incubated with equimolar wt or mutated cp preparations. the complexes were transferred onto nitrocellulose filters, washed, and the bound radioactivity was determined via autoradiography. as shown in fig. 5 , the wt cp bound efficiently to wt and mutated full-length rna3 probes (top section), as well as to wt pe (wt pe fr) and to wt bbox (wt bbox fr) rna3 fragments (fig. 5, second section, lines 1 and 2) , because the interaction was at the detectable level even with 1:1000 diluted probe. this reflected the fact that more than one cp peptides were found to be cross-linked to the bmv rnas by using the reversible cross-linking and peptide fingerprinting (rcap) assays (yi et al., 2009b) . however, the deleted pe ( pe fr) and mutated bbox (mut bbox fr) rna3 fragments interacted at least 100 fold less efficiently with wt cp (fig. 5, second section, lines 3 and 4 , respectively) as the 1:10 diluted probes barely generated detectable signal. likewise, the cp variant with mutated peptide recognizing bbox motif (cp-bbox) also bound inefficiently to both pe fr and mut bbox fr probes (fig. 5, third section, lines 3 and 4, respectively) and to an increased (three-fold) amount of wt bbox fr probe (line 1), yet binding to wt pe fr was reasonably high (fig. 5, line 2) . this analysis revealed separate pe and bbox binding sites on the cp molecule, which is consistent with the previously published data of yi et al. (2009b) . the cp-slc mutant bound with relative efficiency to both wt pe and bbox fr (fig. 5 , bottom section, lines 1 and 2) but it bound with much less efficiency to the pe and bbox mutated counterparts (fig. 5, lines 3 and 4) . collectively, our data suggest that all three rna3 motifs interact with separate regions on the bmv cp molecule, and confirm that the inhibition of cp binding parallels the diminished rna3-sgrna3a recombination frequency. both rna secondary structures and protein factors participate during rna recombination in bmv (bujarski and dzianott, 1991; nagy and bujarski, 1993; figlerowicz, 2000; olsthoorn et al., 2002; panaviene and nagy, 2003) and in other rna viruses (suzuki et al., 2003; dedepsidis et al., 2010; draghici and varrelmann, 2010; zúñiga et al., 2010) . here, we report about coat protein-binding cisacting rna motifs as essential factors supporting rna3-sgrna3a homologous recombination in bmv system. the starting point of our analysis was the recombination frequency between mut-rna3 and sg construct (wt sgrna3a sequence carrying three additional silent marker mutations) that previously reported to be at high 42% level (sztuba-solińska et al., 2011a) . the mut-rna3 × sg experiment generated only six recombinants with more random distribution of three markers; thirteen carrying bamhi-psti double markers; one and two carrying (respectively) bamhi-hindiii or hindiii-psti double markers; and eleven, one, and eight carrying (respectively) psti, hindiii, or bamhi single markers. this suggested that crossovers happened near the 3 end of sgrna3a or more internally, likely near the pe encapsidation region. based on prior and current studies we speculated that recombination occurred by two mechanisms (fig. 6a and b) : (1) sgrna3a-directed primer extension on (−) strand rna3, where the exposed 3 terminal regions of sgrna3a would promote the template switch during (+) strand rna synthesis, or by (2) strand switching between (+) strands [during (−) strand synthesis] which could engage internal secondary structures of rna3 and sgrna3a. here, the sgrna3a would act as a "docking partner" that anneals to the newly synthesized (−) sense rna3. both, the bmv rdrp capability to participate in primer extension with sgrna3a on (−) rna3 templates as well as its potency to switch among rna templates were demonstrated previously in vitro (sztuba-solińska et al., 2011a) . we focused our current study on analyzing the potential role of specific cis-acting rna motifs in rna3-sgrna3a crossovers. our recent data (sztuba-solińska et al., 2011a) suggested that the rna3 position-dependent packaging element (pe) (choi and rao, 2003; annamalai and rao, 2006) focuses the rna3-sgrna3a crossovers. it was shown that removal of pe did not inhibit rna3 accumulation in barley protoplasts (choi and rao, 2003) . here, we confirm that the deletion of a large portion of pe did not affect replication of rna3 in protoplasts (fig. 3) but it strongly debilitated recombination (transfection pe-rna3 × sg supported only 3% recombination frequency; fig. 2a) . similarly, the pe-rna3 × pe-sg transfection, in which both constructs were missing the pe motif, generated only three recombinants, all carrying the 5 bamhi marker mutation. in contrast, when the same pe motif was deleted only in sgrna3a (transfection bbox-rna3 × pe-sg), this alteration was much less debilitating for recombination (reduction to 13%), likely as a combined effect with the mutated bbox element (fig. 2c ) that was shown to increase recombination. the observed effects could be due to disrupted interactions between the pe-rnas and the cp, suggesting a link between recombination and encapsidation (annamalai and rao, 2006; annamalai et al., 2008) . previously, it was shown that partial pe deletion eliminated rna3 packaging (choi and rao, 2003) . since pe is functional only in (+) strands we speculate that the lack of cp binding to this motif might disrupt the initial interaction between the (+) strands of rna3 and sgrna3a, which in normal conditions would be supported via cp-cp dimerization/oligomerization. bmv cp ability to bind small rna motifs together with rdrp was previously proposed to regulate the timing of bmv rna synthesis and viral encapsidation (zhu et al., 2007) . additionally, bmv cp was shown to co-purify with bmv replicase (bujarski et al., 1982) , and both bmv cp and viral rnas were found to co-localize at viral "replication factories" (bamunusinghe et al., 2011; schwartz et al., 2004; diaz et al., 2012; seo et al., 2012) . the bound cp might then support rna crossovers during replication inside the factories. in addition, the bound cp might create obstacles for the replicase complex and promote strand switching. opposite effects were observed after mutating another cpbinding element, the bbox hairpin loop (baumstrak and ahlquist, 2001) . this structured motif participates in the assembly of the bmv replicase complex (via interaction with protein 1a) as well as it binds cp molecules (baumstrak and ahlquist, 2001; yi et al., 2009b) . however, mutations within the cp-bbox binding region did not repress bmv rna accumulation in vivo (yi et al., 2009a) . additionally, its proximity to the rna3 polya region, previously shown to concentrate the crossovers (wierzchoslawski et al., 2004) , is suggestive of its involvement in rna3-sgrna3a recombination. in our studies, the bbox mutations debilitated recombination, but while occurring in sgrna3a rather than in rna3 (fig. 2b) . for the latter (bbox-rna3 × sg) the frequency maintained the wt level (42%) (sztuba-solińska et al., 2011a) with the majority of recombinants carrying all three marker mutations (32 out of 42 recombinants). this effect suggested the prevalence of sgrna3a-directed primer extension (fig. 6a) . mechanistically, we speculate that the bbox-bound cp can expose the 3 end in sgrna3a, facilitating its annealing to another (−) sense rna3 template (template switch) followed by the sgrna3a-directed extension into the progeny (+) sense rna3 (fig. 6a ). in addition, the sgrna3a-bound cp might interact with the replicase complex that is already present at the subgenomic promoter, which would also facilitate primer extension. at the same time, cp binding at the internal rna3 bbox motif would create an obstacle for progressing rdrp supporting the inner strand switching process. in addition, the presence of the polya tail in sgrna3a primer might be essential. we have demonstrated that bmv rdrp can initiate at the polya tail of sgrna3a in vitro (sztuba-solińska et al., 2011a) . the results from reciprocal protoplast inoculations support these observations. the transfection with pe-rna3 × bbox-sg practically eliminated recombination (fig. 2c ) which might be due to missing pe in rna3. along these lines, a reverse transfection with bbox-rna3 × pe-sg generated recombinants that carried all three sgrna3a markers; again reflecting the sgrna3a-directed primer extension on (−) templates. since the bbox-rna3 × pe-sg experiment supported the crossovers near the 3 end of sgrna3a we conclude that the unmodified sgrna3a bbox structure supported primer extension due to binding to both bmv cp and bmv replicase proteins. additionally, the lack of cp binding to the modified bbox in rna3 likely removes the road block for bmv replicase, and thus prevents the rdrp from inner strand switching. the results from the bbox-rna3 × sg co-transfection experiment (76% recombinants with all three marker sites) agree with the lines of this hypothesis. previously, yi et al. (2009a) reported that the bbox motif in the 5 utrs of bmv rnas 1 and 2 mediates cp binding. analogous motif was also reported at the 5 utr of rna3 (pogue et al., 1992) ; however, its cp-binding activity was not reported. here we show that mutations in the bbox like element did not affect the level of rna3 (+) strands in protoplasts (fig. 3) . similar observations were reported by pogue et al. (1992) after deleting the 5 bbox like sequence from bmv rna3. apparently, this region does not participate in the initiation of rna3 (+) strands. however, two cotransfections involving rna3 and/or sgrna3a 5 box-like mutants (5 box-rna3 × 5 box-sg and 5 box-rna3 × sg) dramatically debilitated recombination (none and one recombinant, respectively, per 100 clones; fig. 2d ). these results complement our previously published low recombination rates with the 3 -nested sgrna3a derivatives that were lacking the 5 bbox-like motif (sztuba-solińska et al., 2011a) . the effect of the disruption of the 5 bbox-like sequence on sgrna3a-rna3 recombination might be due to changes in the entire topology (structure and/or communication with other motifs) of the recombining rnas, making them less prone to crossovers. these possibilities will be a subject of further experimentation. to substantiate our conjectures, an rna3 mutant that cannot transcribe sgrna4 and thus did not synthesize cp (cp0-rna3) was tested. here as well, the accumulation of bmv rnas in protoplasts was not detectably affected. this agrees with earlier reports that blocking sgrna synthesis had little effect on bmv rna3 accumulation in barley protoplasts (grdzelishvili et al., 2005) . the corresponding recombination frequency was at very low level (fig. 2e) . thus, the lack of cp per se diminished recombination between rna3 and sgrna3a even if sequences of the cis-acting cp binding elements remained intact. therefore, we conclude that cp plays an important role during homologous recombination between rna3 and sgrna3a. to address further the role of rna-cp interactions from the protein side, two cp domains mapped to bind rna3 were altered by replacing codons of several amino acids with those for alanine, and then tested for recombination activity. one domain was the previously mapped bbox binding region on cp (yi et al., 2009a,b) . indeed, the amino acid substitutions diminished the interaction in vitro of the corresponding mutant cp preparation (cp-bbox mutant) with the bbox rna probe (fig. 5) , and the recombination frequency in protoplasts was minimal (5%). yet another cp mutant (named cp-slc) carried multiple amino acid replacements (again with the alanine residues) within a separate domain that was mapped to bind to the 3 slc hairpin (yi et al., 2009b) , the rna3 (−) strand replication signal (chapman and kao, 1999) . similar to cp-bbox, the recombination frequency with cp-slc was very low after co-transfecting with wt sgrna3a (fig. 2g) . collectively, these observations revealed that either the lack of cp or the disrupted binding of cp molecules to their cognate rna binding signals debilitated significantly the recombination activity. one can speculate that the 3 slc-bound cp increases the interaction between sgrna3a and rna3 molecules via long distance oligomerization (dashed arrow in fig. 6b ). under this scenario, the strand switching during (−) strand synthesis could be facilitated. alternatively, the 3 slc-bound cp could boost the (−) strand synthesis (chapman and kao, 1999) , which would increase the frequency of strand switching. filter binding assays revealed significant inhibition of cp-rna binding for mutated sequences, as compared to the unchanged rna or cp regions (fig. 5) . this was observed for point mutations within the bbox element, for a large deletion within the pe region, as well as for cp amino acid substitutions within binding domains to bbox and 3 -slc elements. these observations confirm previous reports of yi et al. (2009b) and choi and rao (2003) . most importantly, the strength of cp binding directly correlates with the recombination frequency, which further supports the predictions on the important contribution the cp plays during homologous recombination. in conclusion, our data reveal important role of cp in homologous bmv rna recombination and both mechanisms, the sgrna3a-mediated primer extension and internal strand switching. as regarding the biological meaning of homologous crossovers with subgenomic components, these mechanisms can serve as an efficient strategy that rna viruses may use to diversify their rna genomes and/or to guard the integrity of viral genome. it allows them to not only shuffle their genes so to adjust to ever-changing environment, but also to exploit subgenomic components as potential backup in case of extensive damage of their replication-competent genomic rnas. the fact that bmv utilizes its multifunctional cp to orchestrate recombination proves once more the economical nature of virus life cycle under the limited resources. it is possible that cp-mediated recombination might be a common process among (+) stranded rna viruses. the accumulating evidence demonstrates close proximity of viral rna replication and cp translation inside the host cell (annamalai et al., 2008; seo et al., 2012) . thus, it is tempting to assume that rna recombination processes are linked not only spatially but also they share protein factors. yet, further studies are necessary to define more details of these mechanisms. packaging of brome mosaic virus subgenomic rna is functionally coupled to replication-dependent transcription and translation of coat protein replication-coupled packaging mechanism in positive-strand rna viruses: synchronized coexpression of functional multigenome rna components of an animal and a plant virus in nicotiana benthamiana cells by agroinfiltration subcellular localization and rearrangement of endoplasmic reticulum by brome mosaic virus 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predicted stem-loop structures and an internal subgenomic promoter-like motif recombination of 5 subgenomic rna3a with genomic rna3 of brome mosaic bromovirus in vitro and in vivo rna-rna recombination in plant virus replication and evolution dissecting the requirement for subgenomic promoter sequences by rna recombination of brome mosaic virus in vivo: evidence for functional separation of transcription and recombination characterization of a novel 5 subgenomic rna3a derived from rna3 of brome mosaic bromovirus brome mosaic virus capsid protein regulates accumulation of viral replication proteins by binding to the replicase assembly rna element rna binding by the brome mosaic virus capsid protein and the regulation of viral rna accumulation rna-binding proteins that inhibit rna virus infection coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription we would like to thank stuart hill for comments and discussions and barbara ball for assistance with figures. jjb was supported through a grant from the national science foundation (mcb-0920617) and through the plant molecular biology center at northern illinois university. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.virusres.2012.10.001. key: cord-257715-pbcr81qm authors: pignatelli, j.; jimenez, m.; luque, j.; rejas, m.t.; lavazza, a.; rodriguez, d. title: molecular characterization of a new ptov strain. evolutionary implications date: 2009-03-20 journal: virus res doi: 10.1016/j.virusres.2009.02.019 sha: doc_id: 257715 cord_uid: pbcr81qm toroviruses are emergent viruses, belonging to the nidovirales order, that remain mostly ignored, despite they are able to infect different species of domestic animals and humans, causing enteric diseases and diarrhea. thus far, only five variants of porcine torovirus (ptov) have been identified. in this report we describe the identification and partial characterization of a new strain of porcine torovirus (ptov-bres) that was detected by rt-pcr in a swine faecal specimen from a farm in brescia (italy). the complete genes coding for the nucleocapsid (n), hemagglutinin-esterase (he) and membrane (m) proteins were amplified, and sequence analysis showed that ptov-bres is a new ptov strain that, based on the he gene sequence, is phylogenetically related to p4 strain, that was up to now the only member of a distinct ptov lineage. the nucleocapsid protein from ptov-bres was expressed in insect cells as a his-tagged protein, purified by affinity chromatography and used to develop an elisa method to detect antibodies against ptov. this assay was evaluated using a serum collection including 45 samples from three commercial farms from spain. high antibody prevalence against ptov was observed in the three farms, both in adult animals and in piglets, which could suggest that ptov might be endemic in spanish porcine population. the elisa method developed in this work could be useful in future epidemiological surveys about toroviruses. toroviruses have been described as potential gastroenteritis causing agents in horses, cows, pigs and humans. they were classified as a new genus within the family coronaviridae, from the order nidovirales (cavanagh et al., 1994) , although the possibility of establishing two subfamilies, coronavirinae and torovirinae within the family coronaviridae has been proposed and is currently under consideration (gonzalez et al., 2003; coronaviridae study group, 2008) . torovirus genome consists of a single rna molecule of about 25-30 kb. genome organization is similar to that of coronavirus: the 5 two-thirds contain two large and overlapping open reading frames, orf1a and orf1b, that code for the replication machinery. the last third of the genome contains four open reading frames, orfs 2-5, coding respectively for the structural proteins: spike (s), membrane (m), hemagglutinin-esterase (he) and nucleocapsid (n). torovirus particles have a characteristic torus-shape nucleocapsid formed by the n protein interacting with the viral rna. the nucleocapsid is surrounded by an envelope that contains the triple spanning m protein, and the s and he proteins that conform the large and short spikes, respectively. the first torovirus was identified in 1972 after examining a diarrheic faecal sample from a horse in berne (switzerland), and thus it was named berne virus or bev (weiss et al., 1983) . a morphologically related virus was later found in a cattle farm from breda (iowa), and this bovine torovirus (btov) was designated brv (woode et al., 1982) . over the years, there have been a few reports describing the presence of toroviral particles in faecal samples from humans (htov) (beards et al., 1986; duckmanton et al., 1997; koopmans et al., 1997; krishnan and naik, 1997; jamieson et al., 1998; uziel et al., 1999; lodha et al., 2005) and pigs (ptov) (scott et al., 1987; woode, 1987; durham et al., 1989; penrith and gerdes, 1992; lavazza et al., 1996) . ptov has later been detected by rt-pcr in swine faecal specimens from farms in the netherlands, belgium, hungary and italy (kroneman et al., 1998; matiz et al., 2002) . moreover, a partial genomic characterization of five european ptov isolates has been reported (smits et al., 2003) . epidemiological surveys about toroviruses have been largely concentrated on btov, and they showed that this virus is distributed worldwide, having been detected in united states, japan, south korea, india, and in different european countries like united kingdom, germany, belgium, france, switzerland, and italy (liebler et al., 1992; koopmans et al., 1991; ito et al., 2007; park et al., 2007; van kruiningen et al., 1992; krishnan and naik, 1997; koopmans et al., 1989; weiss et al., 1984; lavazza, 1989) . moreover, high seroprevalences against btov have been reported in cattle from united kingdom (brown et al., 1987) , the netherlands and germany (koopmans et al., 1989) . although there are few reports about ptov epidemiology, high seroprevalences, similar to those of btov, have also been reported in swine populations from switzerland (weiss et al., 1984) and the netherlands (kroneman et al., 1998) . despite this extensive geographical distribution and the broad host range, these viruses have attracted little attention. this is likely due in part to the fact that torovirus infection has not been associated with disease causing important losses in livestock, but also to the lack of an "in vitro" system to work with most of these viruses, that has precluded the development of specific tools for their diagnosis. for a long time only the equine isolate bev could be grown in cell cultures (weiss et al., 1983) , and, it has only been very recently reported the ability of a btov variant isolated in japan to grow in cells derived from a human rectal adenocarcinoma (kuwabara et al., 2007) . in addition, btov can be propagated in experimentally infected gnobiotic calves (woode et al., 1982) . thus, there have been a few reports where indirect elisa using partially purified btov or bev particles were used for torovirus serodiagnosis, but purification procedures are not affordable by all laboratories and, in addition, this assay would also provide low sensitivity for detection of antibodies to human and porcine toroviruses (brown et al., 1987) . torovirus serodiagnosis has also been performed by analyzing the ability of clinical serum samples to neutralize bev infectivity (weiss et al., 1984) . the virus neutralization test is "a priori" a very sensitive assay that provides also information about the existence of a protective humoral immune response in the animal under study, however, since bev, a non-homologous virus, would be used to search for antibodies to torovirus in cattle, human or swine sera, the sensitivity of the assay would be reduced. in addition seroneutralization test is a time-consuming and difficult assay that cannot be used to perform extensive studies. thus, alternative strategies have to be undertaken to generate torovirus specific antigens for immune detection. a frequently used alternative is the expression of immunologicaly relevant viral proteins in recombinant expression systems (chirnside et al., 1995; denac et al., 1997; pelosi et al., 1999; farkas et al., 2005; bogdanova et al., 2007; hou et al., 2007) . for this purpose the nucleocapsid proteins from many viruses have proven to be very useful. specifically, the n proteins of different coronaviruses including sars have been expressed either in bacteria or through the baculovirus expression system and have been used for serodiagnosis (timani et al., 2004; woo et al., 2004; wang et al., 2005) . moreover, the n protein is the most abundant protein in torovirus particles (horzinek et al., 1985) , and strong responses against it have been reported from experimental infections of gnobiotic calves with btov . therefore, n protein from ptov appears as an interesting viral antigen to develop diagnostic methods. in addition, since the nucleocapsid proteins are usually highly conserved, serodiagnosis tests based on the use of ptov n protein may prove useful for detection of antibodies against other toroviruses. the aim of this work was to characterize a new ptov strain that was detected in a faecal sample from italy, which was previously considered as torovirus positive by electron microscopy diagnosis, and to develop an elisa diagnostic system to detect antibodies to torovirus. the elisa was evaluated by analyzing pig serum samples from three different spanish farms. our results, although obtained with a reduced number of animals, indicate a high ptov seroprevalence suggesting that ptov might be endemic in spanish farms. equine dermal (e. derm) (atcc ® ccl-57 tm ) cells were kindly provided by r.j. de groot (utrecht university. utrecht. the nether-lands). they were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 15% foetal calf serum (fcs), 100 units/ml of penicillin, and 100 mg/ml of streptomycin. high five tm (highfive) insect cells were cultured at 27 • c in tc100 medium supplemented with antibiotics and 10% fcs. the equine torovirus strain berne (p138/72) (bev) was propagated in e. derm cells as described previously (weiss and horzinek, 1986) , and bev particles were purified by sucrose gradient centrifugation. porcine respiratory and reproductive virus (prrsv) and transmissible gastroenteritis virus (tgev), both provided to us by l. enjuanes (cnb-csic, spain), were propagated in monkey-derived ma104 (atcc ® crl-2378.1 tm ) cells and in swine testis fibroblasts (st), respectively. both viruses were partially purified by centrifugation over a 50% sucrose cushion. three porcine faecal specimens (513/02, 970/02, 1318/02), taken in brescia (italy) in 2002, and one bovine faecal sample (1812/03) taken at the same location in 2003, were described to contain torovirus-like particles when examined by electron microscopy, but, with the exception of 1318/02, all samples were also shown to contain other viral particles that could correspond to coronavirus, enterovirus and circovirus. swine blood samples were collected from three commercial farms located in different regions in the northern part of spain. ten blood samples were collected from sows in a farm located in navarra, where diarrhea and gastroenteritis symptoms in piglets had been reported previously, but they were not observed at the time of serum collection. twenty sow blood samples were collected from a farm located in aragon where most newborn piglets were suffering from diarrhea at the moment of collection. fifteen blood samples were collected from 6 to 8 week-old healthy piglets from a farm with high sanitary conditions located in galicia. blood samples were centrifuged at 1500 × g during 10 min at 4 • c, and the obtained sera were stored at −80 • c until their use. a commercial porcine serum obtained from healthy pigs was purchased from abd serotec and has been named as cps throughout the paper. a serum specimen from a piglet infected under field conditions with ptov, previously described (antiserum 233/90; lavazza et al., 1996) was used as ptov positive serum control, and was referred to as ␣bres. an anti-bev hyperimmune polyclonal serum (␣-bev) was generated in our laboratory (garzon et al., unpublished results). s. van gucht (gent university, belgium) provided us three serum samples from caesarean-derived, colostrum-deprived (cd/cd) pigs kept under germ free conditions (spf) that were used as negative control, and sera from two other pigs that were reared under these same conditions, but that had subsequently been inoculated with either porcine respiratory coronavirus (prcv) (␣prcv) or prrsv (␣prrsv), and that were used as cross-reactivity controls. in addition, 69 serum samples from cd/cd piglets taken at 0-36 days of age, were provided by j. segales (cresa. barcelona, spain) and were also used as negative controls. a rabbit hyperimmune serum against the n protein from ptov-bres was raised by immunizing a white new zeeland rabbit with chromatography purified n chimerical protein fused to a histidine tag, and expressed in insect cells by a baculovirus recombinant (see below). the faecal samples containing toroviral particles were diluted 1:5 in sterile phosphate buffered saline (pbs), homogenized by vortexing and centrifuged at 3000 × g for 10 min at 4 • c. for toroviral particle aggregation the supernatants were mixed (v:v) with a convalescent serum sample from a torovirus infected pig (␣bres), previously diluted 1:40 in pbs. after a 30 min incubation period at 37 • c, viral particles were adsorbed to collodion-carbon coated copper grids by ultacentrifugation in a beckman airfuge, at 21 psi for 15 min as previously described (lavazza et al., 1996) . for detection of bound antibodies, particles adsorbed to the surface of the grids were incubated with protein-a conjugated with 10 nm colloidal gold particles as previously described (rodriguez et al., 1996) . after washing in distilled water, samples were negatively stained with 2% sodium phosphotungstate (pta) in water for 1 min, allowed to dry, and observed in a jem1010 electron microscope (jeol, japan). pictures were taken with a bioscan digital camera (gatan). the faecal samples containing toroviral particles were diluted 1:1 in pbs, homogenized by vortexing and centrifuged at 3000 × g for 10 min at 4 • c. the supernatant was aliquoted and kept at −80 • c. 200 l of each supernatant were used for viral rna extraction using a commercial kit (high pure rna isolation kit, roche applied science) following the manufacturer's recommendations. the rna was recovered in 50 l of dnase, rnase free water, aliquoted and maintained at −80 • c. all specimens were extracted in a dedicated class 2 laminar flow hood using dedicated pipettes and aerosol resistant tips. reverse transcription (rt) reaction was performed using superscript ii reverse transcriptase (invitrogen, corp.) following the manufacturer's instructions with minor modifications. briefly, 8 l of the rna sample were mixed with a 1 mm dntps mix (roche applied science) and 1 mm random hexamer primer (roche applied science) and incubated for 10 min at 65 • c and 1 min on ice. reverse transcription reaction was completed by adding 9 l of reaction mix containing 1x rt buffer, 10 mm dtt, 8 mm mgcl 2 , 40 u ribonuclease inhibitor (fermentas) and 200 u of superscript ii reverse transcriptase and incubating for 10 min at 25 • c, followed by 45 min at 45 • c and finally 15 min at 65 • c. to table 1 oligonucleotide primers: location within torovirus genome and sequences. . primer sequence a polarity location minimize cross-contamination reverse transcription reaction was carried out in an independent room using dedicated pipettes with aerosol resistant pipette tips. the cdna preparation was aliquoted and conserved at −80 • c until use. sequences from etov, berne strain (bev), btov, strains brv-1, brv-2, b145, b150, b155, b6 and ptov, strains markelo, p4, p10, p9 and p78 were retrieved from genbank database (http://www.ncbi.nlm.nih.gov) and aligned using megalign program (dnastar, inc.). primers tovm5 and tovm3 were designed based on a conserved region from the m gene (table 1) . besides, primers 593 and 620 previously described (kroneman et al., 1998) were used to amplify a ptov specific fragment from the n gene. to amplify the full coding sequences for the structural proteins m, n and he from ptov, sets of primers were designed using ptov conserved sequences at both 5 and 3 regions from each gene (table 1) . tails containing recognition sites for bamhi and xbai restriction enzymes (underlined in the table) were added to each forward and reverse primer, respectively. as described below, to amplify the entire he gene a new primer, ptov-bres-he5 , had to be designed. diagnostic pcr reactions were carried out using 2 l of cdna and a reaction mix containing 1× pcr reaction buffer, 0.2 mm of each dntp, 2.5 mm mgcl 2 , 0.2 m of each primer, tov-m5 and tov-m3 or 593 and 620, and 1.25 u of taq platinum (invitrogen, corp.) in a final volume of 25 l. the mixture was subjected to an initial denaturation step of 2 min at 92 • c, followed by 30 cycles of 45 s at 92 • c, 40 s at 45 • c and 1 min at 62 • c. a final extension step of 5 min at 62 • c was added at the end. to amplify sequences of m and n genes using the primer pairs ptov-m5 -m3 and ptov-n5 -n3 , respectively, we used the same thermal cycling as described above. in the case of the he gene we could not obtain amplification from the cdna using the designed primer pair, ptov-he5 -he3 . however, using primers tov-m5 and ptov-he3 a 1700 bp fragment of ptov genome comprising the 3 -end of m gene and the complete he gene was obtained. this fragment was amplified using a high fidelity polymerase (triplemaster polymerase mix from eppendorf), and was sequenced to determine the 5 end region of he gene from ptov-bres, and thus, the primer ptov-bres-he5 was designed accordingly. to amplify just the complete he gene, a second amplification reaction was performed using ptov-bres-he5 and ptov-he3 primers and cdna as template. briefly, 2 l of ptov-bres cdna were added to a reaction mix containing 1× high fidelity buffer, 200 m of each dntp, 200 nm of each primer and 0.71 u/l of triplemaster polymerase mix. thermal cycling conditions for both reactions were as follows: 1 cycle of 94 • c; 30 cycles of denaturation at 94 • c for 20 s, annealing at 50 • c for 20 s and elongation at 72 • c for 2 min. all pcr reactions were performed in physically separated rooms and dedicated pipettes and filter tips were used. several negative controls of distilled water for each step of the procedure, rna isolation, reverse transcription and pcr were included in each assay. pcr reaction products were visualized in 0.8-1% agarose gels and extracted from agarose using the commercial gel extraction kit (qiagen) following instructions provided by the manufacturer. for sequencing analysis, all rt-pcr products corresponding to the n, m and he genes were cloned in the commercial vector pgem-t ® -easy (promega) and the resulting plasmids, pgt-ptov-m, pgt-ptov-n and pgt-ptov-he, respectively, were used to transform e. coli dh5␣ competent bacteria. at least 5 independent clones were used for sequencing in the sequencing facility of the national centre of biotechnology (madrid, spain). obtained sequences were analyzed using seqman program (dnastar, inc.) and aligned using clustalx method with torovirus sequences available from ncbi database using megalign software (dnastar, inc.). the rt-pcr amplified products and the corresponding sequences from genbank were compared using the program megaling (dnastar, inc.) employing clustalw method. this program was used to carry out phylogenetic analysis from gene sequences. phylogenetic analyses were performed using the neighbour-joining method, and statistical parameters of phylogenetic trees were determined by bootstrap analysis using 1000 replicates. the nucleotide sequences described in this paper have been submitted to the ddbj nucleotide sequences database and are retrievable from genbank. the accession numbers for the complete n, m, and he genes of ptov-bres are fj232068, fj232069, and fj232070, respectively. a baculovirus recombinant that contains the nucleotide coding sequence for ptov n protein fused at its 5 -end to a sequence encoding a 6-histidine tag was generated by using the bac-to-bac system (invitrogen, corp.) . for this, the pgt-ptov-n vector clone containing the consensus sequence for the n gene from ptov-bres isolate was used to subclone this gene into the baculovirus transfer vector pfastbac tm b (invitrogen, corp.). the n gene was extracted from pgt-ptov-n by enzymatic restriction with bamhi and xbai and cloned into pfastbac tm b, previously digested with the same restriction enzymes, giving rise to pfb-ptov-n vector. competent e. coli dh10bac cells, containing a bacmid (baculovirus shuttle vector plasmid) and a helper plasmid, were used to generate recombinant bacmids according to the manufacturer's instructions. the obtained recombinant bacmid, bac-ptov-n, was transfected into monolayers of highfive insect cells using lipofectin (invitrogen, corp.). culture medium containing recombinant baculovirus rbac-ptov-n was collected at 72 h post-transfection, clarified by centrifugation and stored at 4 • c as virus stock. transfected cells were collected and recombinant ptov-n protein expression was analyzed by western blot using a commercial polyclonal antibody against the histidine tag (␣his) and the ␣bres serum. the recombinant n protein fused to a histidine tag at its n terminus was purified using bd talon tm imac resin (bd biosciences clontech). highfive cells were infected with rbac-ptov-n at a multiplicity of infection (moi) of 5 plaque forming units per cell (pfu/cell) and maintained for 48 h at 28 • c. infected cells were harvested, pelleted by centrifugation at 1000 × g 10 min and resuspended in lysis buffer (50 mm h 2 napo 4 , ph 8.0, 300 mm nacl, 0.1% np-40) containing protease inhibitors (complete mini tablets from roche applied science). cell suspensions were then incubated on ice for 30 min and sonicated. the insoluble fraction, which contains the n protein, was recovered by centrifugation at 3000 × g 5 min and solubilized with denaturing buffer containing 50 mm h 2 napo 4 , ph 8.0, 300 mm nacl, and 6 m guanidine. after a second centrifugation at 3000 × g for 5 min, the supernatant was taken and mixed with the talon tm resin equilibrated with denaturing buffer, and incubated for 1 h at 4 • c in a rotating shaker. unbound proteins were removed by centrifugation at 500 × g for 5 min and resin was washed with 10 volumes of denaturing buffer three times, and two times with a buffer containing 50 mm h 2 napo 4 , ph 8.0, 300 mm nacl, 8 m urea. the n protein was eluted from the resin with successive elution steps with equal resin volume of elution buffer (50 mm h 2 napo 4 , ph 8.0, 300 mm nacl, 8 m urea and 1 m imidazol). concentration and purity of ptov-n protein was analyzed by sds-page and western blot. protein concentration was determined according to bca assay (pierce) using a 1:5 dilution of the eluted protein in distilled water and bovine serum albumin (sigma) as standard prepared similarly. aliquots of the purified recombinant n protein were electrophoresed on sds-page gels (400 ng/lane). the protein was transferred to a nitrocellulose membrane that was cut into strips corresponding to each gel track. western blots were carried out with porcine serum samples diluted 1:100. first, non-specific binding of antibodies to the nitrocellulose membrane was blocked by overnight incubation at 4 • c in pbs containing 0.05% tween 20 (pbst) and 5% skim milk. the membranes were then incubated with porcine serum samples diluted in pbst with 5% skim milk. after extensive washings, the membranes were incubated with secondary goat anti-porcine igg antibodies coupled to horseradish peroxidase. western blots were developed by incubating the nitrocellulose membranes with a solution containing 0.5 mg/ml 4chloro-1-naphtol (sigma) in pbs and 0.015% h 2 o 2 , during 20 min at room temperature, and then overnight at 4 • c, in distilled water. the presence of neutralizing antibodies against torovirus in swine sera was analyzed by plaque reduction test using bev virus. this method, that is a conventional procedure to determine neutralizing antibody titers for many viruses, has not been previously used for bev. the reason being that bev virus titers have always been determined by establishing the 50% tissue culture infectious dose (tcid 50 ) and neutralization assays were performed accordingly. instead, we titrated bev stocks by determining the number of pfu/ml (garzon et al, unpublished results). thus, bev neutralization assay was performed as follows: 25 l of heat inactivated diluted serum (two-fold serial dilutions in dmem, from 1:4 to 1:4096) were incubated with an equal volume of a viral suspension containing 100 pfu of bev for 1 h at 37 • c. virus-serum mixtures were added to confluent monolayers of e. derm cells grown in 12-well plates. after 1 h of virus adsorption at 37 • c, the infection medium was removed and 2 ml per well of 2× dmem medium mixed (v/v) with 1.9% agar and containing 0.05 mg/ml of deae-dextrane and 2% fcs was added, and the cells were maintained at 37 • c for 3 days. after removing the agar overlay, cells were stained with crystal violet and lysis plaques were counted. the virus-neutralizing titer (nd 50 ) of each serum sample corresponds to the reciprocal of the highest serum dilution giving 50% reduction in the number of plaques with respect to that obtained with the virus without serum. serum samples were tested by duplicate and negative (spf) and positive (cps) control sera were included in each test. neutralization titers were calculated using spearman-kärber formula. serum samples with neutralizing antibody titers equal to or higher than 4 (or 0.6 that is the equivalent value in logarithmic scale) were considered as positive. an indirect elisa was established in our laboratory to test for the presence of antibodies against porcine torovirus in pig serum samples using ptov-n protein as antigen. optimal concentration of reagents was established by checkerboard titration. paired rows of a 96-well microtiter plate (maxisorp, nunc) were coated with 400 ng/well of purified n protein diluted in 50 mm carbonatebicarbonate buffer (ph 9.6) by overnight incubation at 4 • c. after coating and at each washing step, plates were rinsed 3 times with 150 l/well of pbst. subsequently, plates were blocked for 2 h at 37 • c with 150 l/well of pbst-3% bsa. then 50 l of each serum sample diluted 1:100 in pbst-1% bsa was added in paired wells and the plates were incubated 1 h at 37 • c. next, commercial goat anti-pig igg antibodies conjugated to horseradish peroxidase (sigma) diluted in pbst-1% bsa was added and the plates were incubated 1 h at 37 • c. the enzymatic reaction was developed using o-phenylenediamine dihydrochloride (opd fast tm , sigma). after 10 min at room temperature the reaction was stopped by adding 50 l/well of 2n sulphuric acid, and absorbance at 492 nm was measured with a multichannel spectrophotometer (titertek multiscan mcc/340). torovirus-like particles were observed by electron microscopy analysis in three porcine (513/02, 970/02, 1318/02) and one bovine (1812/03) faecal samples, in the course of a diagnostic examination of animals suffering diarrhea in brescia, italy. in some of these samples (513/02, 970/02 and 1812/03), other viral particles were also observed that could correspond to coronavirus, circovirus and enterovirus (data not shown). moreover, in two of the porcine samples (513/02 and 970/02), the torovirus-like particles were not well preserved and were difficult to detect. to enhance the efficacy and specificity of the electron microscopy method for toroviral particle detection, faecal suspensions were incubated with convaslescent serum from a torovirus infected pig (␣-bres) facilitating particle aggregation, as previously described (lavazza et al., 1996) . samples were then ultracentrifuged onto a grid, and adsorbed viral particles were incubated with protein a-gold for antibody detection, and negatively stained with 2% pta. fig. 1 shows immunogold-labeled toroviral particles in one of the porcine faecal samples (1318/02), which will later be identified as ptov-bres. the results obtained with the other three samples were not conclusive. to detect toroviral genomic rna in these samples we designed a new primer set based on a region from the m gene that is conserved in all available torovirus sequences (tovm5 and tovm3 described in table 1 ). optimal rt-pcr conditions were established using serial dilutions of a titrated bev stock and using primers tovm5 and tovm3 we were able to detect up to 10 3 pfu/ml (data not shown). these primers were then used for rt-pcr detection of ptov and btov in the faecal samples from three pigs and one calf described above. the expected 410 bp fragment was amplified from one of the porcine specimens (1318/02) ( fig. 2a, lane 1) and also from the bovine stool sample (fig. 2b, lane 1) . sequence of the ptov fragment showed a 95% similarity with respect to other available ptov sequences and 80% with respect to btov sequences. conversely, the fragment amplified from btov showed a 98% similarity with respect to other btov isolates, but only an 80% identity with respect to ptov isolates. both fragments showed a 79-80% similarity with respect to bev. a second diagnostic pcr was carried out to amplify specifically a fragment from ptov n gene, using ptov specific primers 593 and 620 (table 1) . a 180 bp fragment was obtained only from the 1318/02 sample ( fig. 2a, lane 2) , but not from the other two porcine samples (not shown), nor from the bovine one. the amplified fragment was of the expected size and sequence of this second fragment showed a homology of 87-89% with respect to ptov strains, 85% with respect to btov strains and only 57% with bev. negative water controls were analyzed in parallel with each primer set to discard sample cross-contamination ( fig. 2a and b, lanes 3 and 4) . thus, a new ptov strain has been identified in this work and it has been named as ptov-bres throughout this report. to establish the phylogenetic relationship of ptov-bres with other ptov strains, we amplified the sequences corresponding to the genes coding for m, he and n proteins from ptov-bres cdna. the amplicons were analyzed by agarose gel electrophoresis, purified from the gel, and cloned into pgemt ® -easy vector and at least 5 independent clones were sequenced in both directions for each gene. the n gene of ptov-bres was amplified as 505 bp fragment (fig. 2c, lane 1) that showed 91.7-93.1% homology with respect to other ptov strains, 91.3-92.1% with respect to btov and 70% with bev (fig. 3a) . the 700 bp dna fragment corresponding to m gene (fig. 2c, lane 2) showed 94.4-95.6% homology with other ptov strains, 80.6-81.1% with respect to btov strains, and 81.2% with respect to the bev isolate (fig. 3c) . the he gene from ptov-bres was amplified using a two-step strategy. first, we amplified a 1700 bp (fig. 2c, lane 3) . this fragment was sequenced and it showed to contain the 3 end from m gene, the intergenic region containing the canonical translation regulatory sequence (trs) and the complete he gene. a new primer, ptov-bres-he5 , was designed to amplify only the complete he gene without extra sequences (1.2 kb fragment) using the cdna as template (fig. 2c, lane 4) . consensus sequence for ptov-bres he gene shows 92% homology with the previously described p4 strain of ptov, and 75-78% homology with the other ptov strains. with respect to btov strains a 67-70% identity was obtained. only 52% homology was observed with respect to the htov-he gene sequence and 63% with respect to the he gene fragment conserved in bev (fig. 3b) . these results strongly suggest that ptov-bres is a new strain of ptov not detected previously. we have generated a recombinant baculovirus that expressed the nucleocapsid protein from ptov-bres fused at its n-terminus to a histidine tag (rbac-ptov-n). highfive insect cells infected with rbac-ptov-n showed a prominent 20 kda protein band when analyzed by sds-page and coomassie brilliant blue staining at 48 and 72 hpi (data not shown), and this protein was specifically recognized by ␣his (fig. 4a) and ␣bres (fig. 4b) antibodies in western blot. the estimated size of this main product was in agreement with the predicted size for the recombinant fusion protein. other protein bands of lower molecular weight, likely corresponding to degradation products, and a slower migrating protein were also observed by western blot. the recombinant n protein was solubilized by treatment of the infected cells with 6 m guanidine, and was purified by affinity chromatography using a cobalt-coated resin. the protein was recovered by three rounds of elution with imidazole in presence of 8 m urea. purity of the protein was analyzed by sds-page and coomassie brilliant blue staining. fig. 4c shows the analysis by sds-page of the different steps of the purification procedure. after elution, only the bands corresponding to the recombinant n protein were observed (fig. 4c, lanes 5-7) , indicating that the protein was highly purified. protein was stored in 8 m urea to keep it soluble. to generate an anti-ptov n protein polyclonal serum (r␣ptov-n) the purified protein was dialysed against distilled water, freeze-dried and resuspended in pbs before being inoculated in a rabbit. specificity of antibodies was analyzed by western blot using extracts from rbac-ptov-n infected and non-infected high-five insect cells (data not shown). purified recombinant n protein was used to develop an elisa to detect antibodies against ptov in porcine serum samples. optimal concentrations of reagents was determined by checkerboard titration using the above described polyclonal serum, r␣ptov-n, two porcine serum samples, ␣bres and cps, as positive controls, and five serum samples from spf animals that were used as negative controls. the optimal protein concentration that provided higher discrimination between negative and positive sera was 400 ng/well, and the optimal porcine serum dilution was 1:100 (data not shown). since there are no reference sera available for porcine torovirus, elisa cut-off value was established using 5 spf serum samples and 69 serum samples from cd/cd animals that had been collected between 0 and 36 days after birth. all these serum samples were analyzed for igg antibodies in five individual assays performed on different days. these control serum samples showed a mean elisa reactivity of 0.175 ± 0.03. therefore, elisa cut-off values were established as the mean value obtained from control animals plus three times the standard deviation (o.d. 492 nm = 0.270). none of these control serum samples showed elisa o.d. values above the cut off. lack of cross-reactivity between ptov and antibodies to other related viruses infecting pigs was confirmed by elisa, western blot and virus neutralization tests using ␣prrsv, ␣prcv, and ␣bres serum samples, purified ptov n protein or bev particles as toroviral antigens, and purified prrsv and tgev virions as related viruses (data not shown). in order to evaluate the potential use of the ptov-n protein as antigen for diagnostic elisa to detect antibodies against porcine torovirus, 45 serum samples were collected from three spanish farms located in galicia, navarra and aragon and were analyzed by elisa, virus neutralization assay and/or western blot. all 45 serum samples analyzed by elisa were positive against torovirus and these results were confirmed by western blot and/or serum neutralization test in 44 cases (93%). most serum samples from the farm in navarra showed moderate elisa values (between 0.5 and 1.5) and low neutralizing antibodies titers (log nd 50 < 1.5) (fig. 5a) . one sample showed positive elisa value (o.d. 492nm = 1.30) and a neutralizing titer close to the cut-off (log nd 50 = 0.63) (fig. 5a , arrow), however, it was positive by western blot (fig. 5a, strip 1) . from the farm located in galicia, more dispersed results were obtained by elisa (o.d. 492nm between 0.3 and 2.3), neutralization test (log nd 50 between 0.5 and 3.2) and western blot (fig. 5b ). all 15 serum samples were elisa positive, and that result was confirmed by western blot in 9 samples and by neutralization test in 14 samples. the only sample that was positive by elisa (o.d 492nm = 0.325) but negative by the other two tests (fig. 5b , strip 13 and arrow) had a neutralizing titer that was close to the cut-off value (log nd 50 = 0.54). serum samples from aragon showed high titers of antibodies against ptov-n (o.d. 492nm between 1.0 and 2.3), and accordingly, they were highly neutralizing (log nd 50 between 1.5 and >3.2), and for some of them we could not determine the neutralizing titer since the highest serum dilution used, 1:4096, still reduces the number of viral plaques to more than 50%, and they were all positive by western blot (fig. 5c ). the results obtained by the elisa test using ptov n protein as antigen are in good agreement with those obtained by neutralization assay, indicating that the elisa could be used instead for detection of antibodies to torovirus, providing a simpler and more feasible diagnostic system. toroviruses have been described as potential gastroenteritis causing agents that appear to infect many animal species, including humans. most torovirus epidemiological information comes from studies on btov performed in different countries, and they indicate that torovirus are broadly dispersed around the world, with high prevalences in animal populations. however, few studies have been reported about ptov, despite they indicate an important prevalence of ptov at least in european countries. the lack of a growing system for ptov has hindered its study and the development of diagnostic tools. this work reports the identification and partial characterization of a new ptov strain and the development of an elisa assay to detect antibodies against ptov in porcine serum samples. first, we describe a new primer pair, tovm5 and tovm3 , whose sequences are highly conserved between torovirus species. using this primer set we were able to detect a new strain, ptov-bres. in addition, primer set tovm5 and tovm3 was able to amplify bev and btov genomes from cell culture medium and a stool sample, respectively. moreover, although htov detection using tovm5 and tovm3 primer pairs has not been tested yet, these primers might also amplify htov since they were designed in conserved regions from all torovirus m gene available sequences. hence, we propose that this new primer set could be used for torovirus epidemiological trials. at present, little information about ptov genome sequence is available since only five ptov strains have been described (kroneman et al., 1998; matiz et al., 2002; smits et al., 2003) . genomic information about other ptov variants is needed to understand torovirus evolution and to develop new diagnostic tools. in this work we describe the amplification and sequencing of m, he and n genes from ptov-bres. interestingly, phylogenetic analysis of he gene showed a high sequence homology (92%) with the he gene from strain p4, but only 73-78% with other ptov strains. two ptov lineages had previously been described based on he gene sequences, which are represented by p4 and markelo strains. it has been suggested that these two ptov lineages were generated by recombination events into the he gene between a parental ptov and an unknown donor (smits et al., 2003) . ptov-p4 was detected in italy in 1990, during an enteritis outbreak (lavazza et al., 1996) , and since then no other strains have shown a phylogenetic relationship with it. therefore, p4 was considered as the likely ptov parental strain, and markelo-like strains (markelo, p78, p9 and p10) could then be considered as recombinant progeny originated from it. our finding of a new ptov strain phylogenetically related to p4 in a sample collected in 2002 suggests that at least these two ptov lineages might be currently circulating in porcine populations. antigenic differences between he proteins from both ptov lineages have not been determined but this possibility should be considered. in this regard, antigenic differences between the known btov lineages have been previously described (smits et al., 2005) . in this work, we also describe the expression of the n protein of ptov-bres and its use for the development of an elisa method to detect antibodies to torovirus in swine serum samples. to our knowledge, no specific serodiagnostic assay for ptov has been developed to date. antibodies against ptov were previously analyzed by bev serum neutralization test (weiss et al., 1984; liebermann, 1990; kroneman et al., 1998) , however, this method is time consuming and its sensitivity could be compromised by the fact that it is based on cross-reactivity between torovirus species. besides, two elisa assays to detect antibodies against btov and bev have been previously reported. both methods were developed using sucrose gradient purified virus, obtained either from faeces from gnobiotic calves infected with btov (brown et al., 1987; van kruiningen et al., 1992) or from culture medium from bev infected cells (weiss et al., 1984) . however, these approaches cannot be applied to porcine torovirus, since ptov cannot be propagated in cell cultures yet, and no experimental infection protocol has been reported. therefore, alternative strategies to obtain specific ptov antigens are needed. expression of viral proteins in insect cells to develop diagnostic assays have been reported for other viruses (pelosi et al., 1999; sestak et al., 1999; guo et al., 2001; deng et al., 2003; timani et al., 2004; farkas et al., 2005; shin et al., 2007) . the torovirus nucleocapsid protein is the most abundant protein in the virions, and is highly antigenic as shown by the strong antibody response developed against it in gnobiotic calves experimentally infected with btov. the n and he proteins from btov strain brv-1 were expressed previously in e. coli and in insect cells (duckmanton et al., 1999) , respectively, and both proteins showed immunoreactivity to specific antisera and field serum samples by western blot. however, these proteins were not used to develop any diagnostic assay. therefore, we sought to use n protein from ptov as antigen to develop diagnostic methods. thus, recombinant nucleocapsid protein from ptov-bres isolate was expressed in insect cells and purified by affinity chromatography. using this approach, large amounts of highly purified protein were achieved. recombinant ptov-bres-n protein was used to develop an elisa assay to detect antibodies against torovirus in swine serum samples. moreover, given the high degree of n protein conservation, the developed assay may also be useful to study the seroprevalence to bovine and even human toroviruses. elisa optimization was carried out using a serum sample from a pig naturally infected by ptov (␣bres), a commercial serum mixture from healthy pigs (cps) and serum samples from spf pigs inoculated or not with prrsv or prcv. the assay prove to be very reproducible since very little variation was observed in the o.d. values of positive and negative control sera among assays performed in different days. cross-reactivity of ptov with other related viruses infecting pigs has been discarded in the three assays tested (elisa, western blot and virus neutralization) and these results confirm that these viruses are serologically unrelated, and are in agreement with previous results described by other authors (woode et al., 1982 (woode et al., , 1985 lamouliatte et al., 1987) . however, cps antibodies recognized both ptov-n and bev-n proteins (not shown), indicating that the animals used to obtain the serum had been infected by, or, exposed to ptov. similar observations had previously been described with different batches of commercial newborn calf serum that were shown to react with bev (vanopdenbosch et al., 1992b) . due to the lack of reference sera for ptov, elisa cut-off value was empirically determined using serum samples from spf and cd/cd animals. overall, elisa results from adult animals from different spanish farms located in aragon (n = 20) and navarra (n = 10) suggest a high prevalence of antibodies in spanish pig population, since all 30 adult pigs analyzed from these farms were positive by elisa, and these results were confirmed by neutralization test and western blot. on the other hand, the high prevalence of antibodies in serum samples from piglets from the farm from galicia suggests that ptov infection occurs early in piglets. this prevalence rate is higher than others previously reported for ptov in the netherlands (81%) using neutralization assay (kroneman et al., 1998) and for btov (brown et al., 1987; koopmans et al., 1989) . however, these ptov prevalence rate data can only be considered as preliminary, since both, our, and the study performed in the netherlands, were carried out with a short number of animals. further epidemiological studies are needed to obtain definitive information regarding ptov prevalences in spain and in other countries such as italy, where ptov strains seem to normally circulate. pathogenic potential of ptov as diarrhea causing agent remains unclear. in this regard, pigs from the farm in aragon, where an acute gastroenteritis outbreak was reported without an identified causing agent, show high levels of antibodies against torovirus. more studies are needed to understand the potential pathogenesis of ptov. in conclusion, we have developed an elisa for torovirus diagnosis that could be very useful for future epidemiological studies, to determine seroprevalence to porcine torovirus, but also to bovine and even human toroviruses. moreover, the nucleocapsid protein expressed by the baculovirus expression system could also be evaluated in immunization protocols to study its potential application for vaccination purposes against toroviruses. in this regard, protective responses elicited against the nucleocapsid protein have been reported for different coronaviruses (wasmoen et al., 1995; seo et al., 1997; cavanagh, 2003) . in addition, we have reported a new ptov strain and three genes coding for structural proteins have been fully sequenced providing information 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antibodies to nucleocapsid and spike proteins of the sars-associated coronavirus in sars patients protection of cats from infectious peritonitis by vaccination with a recombinant raccoon poxvirus expressing the nucleocapsid gene of feline infectious peritonitis virus purification and partial characterization of a new enveloped rna virus (berne virus) antibodies to berne virus in horses and other animals morphogenesis of berne virus (proposed family toroviridae) longitudinal profile of immunoglobulin g (igg), igm, and iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus breda and breda-like viruses: diagnosis, pathology and epidemiology studies with an unclassified virus isolated from diarrheic calves comparative studies on three isolates of breda virus of calves we thank raquel blanco and juan ramón rodríguez for critical reading of the manuscript. we also want to thank r.j. de groot (utrecht university, utrecht, the netherlands) for providing the e. derm cells and bev virus, s. van gucht (gent university, belgium) for providing the serum samples from spf piglets, j. segales for providing the serum samples from cd/cd animals and l. enjuanes for supplying tgev and prrsv viruses.this work was supported by grants cicyt-bio2006-10988 and consolider-porcivir csd2006-00007 to d.r. j.p. was recipient of fpi fellowship from the spanish ministry of education and science. key: cord-278939-z6kiee09 authors: mani, janice s.; johnson, joel b.; steel, jason c.; broszczak, daniel a.; neilsen, paul m.; walsh, kerry b.; naiker, mani title: natural product-derived phytochemicals as potential agents against coronaviruses: a review date: 2020-04-30 journal: virus res doi: 10.1016/j.virusres.2020.197989 sha: doc_id: 278939 cord_uid: z6kiee09 coronaviruses are responsible for a growing economic, social and mortality burden, as the causative agent of diseases such as severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), avian infectious bronchitis virus (ibv) and covid-19. however, there is a lack of effective antiviral agents for many coronavirus strains. naturally existing compounds provide a wealth of chemical diversity, including antiviral activity, and thus may have utility as therapeutic agents against coronaviral infections. the pubmed database was searched for papers including the keywords coronavirus, sars or mers, as well as traditional medicine, herbal, remedy or plants, with 55 primary research articles identified. the overwhelming majority of publications focussed on polar compounds. compounds that show promise for the inhibition of coronavirus in humans include scutellarein, silvestrol, tryptanthrin, saikosaponin b(2), quercetin, myricetin, caffeic acid, psoralidin, isobavachalcone, and lectins such as griffithsin. other compounds such as lycorine may be suitable if a therapeutic level of antiviral activity can be achieved without exceeding toxic plasma concentrations. it was noted that the most promising small molecules identified as coronavirus inhibitors contained a conjugated fused ring structure with the majority being classified as being polyphenols. the dramatic change of events with the recent unprecedented coronavirus pandemic declared by the world health organisation (who) has prompted an exponential increase of scientific interest in coronaviruses globally. as of april 22 nd 2020, the pandemic has resulted in 2,553,112infections, with 177,286 deaths worldwide, which continues to drastically increase as we write (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). coronaviruses (covs) belong to the family coronaviridae, subfamily coronavirinae and are large (genome size 26-32 kb; wu et al., 2020a) , enveloped, positive-sense single-stranded ribonucleic acid (rna) viruses that can infect both animals and humans ( figure 1 ). based on their genotypic and serological characteristics, the viruses are subdivided into four genera: alpha-, beta-, gamma-, and deltacoronavirus (chu et al., 2020; lu et al., 2015) . at present, all identified covs that are capable of infecting humans belong to the first two genera. these include the alphacoronaviruses (αcovs) hcov-nl63 (human cov-nl63) and hcov-229e and the betacoronaviruses (βcovs) hcov-oc43 (human cov-oc43), hku1 (human cov), sars-cov (severe acute respiratory syndrome cov), and mers-cov (middle eastern respiratory syndrome cov) (lu et al., 2015) . in the past two decades there have been three epidemics caused by the betacovs, namely sars in 2002-03, mers in 2012 and covid-19, first identified in 2019 . sars-cov emerged in 2002-03 in southern china, causing a global threat and infecting more than 8,000 people, with approximately 800 fatalities recorded, largely in china and the surrounding regions (lu et al., 2015; paraskevis et al., 2020) . mers-cov emerged in the middle east, spreading to several countries to infect close to 2,300 individuals, resulting in 845 deaths as of july 2019 (world health organization, 2019) . the present cov pandemic resulting from sars-cov-2, which causes covid-19 (coronavirus disease), was identified in wuhan city, in the hubei province of southern mainland china on the 31 st december 2019 (sohrabi et al., 2020) . the genome of sars-cov-2 is approximately 70% identical to that of sars-cov , hence leading to its current name. the major druggable targets of sars-cov-2 include 3-chymotrypsin-like protease (3clpro), papain like protease (plpro), rna-dependent rna polymerase, and spike (s) proteins (wu et al., 2020b) . the s proteins interact directly with human angiotensin-converting enzyme (ace) 2, allowing the virus to enter the cells. at present, no preventive vaccines or established antiviral therapies are available for coronaviruses (sohrabi et al., 2020) . however, several synthetic compounds have shown promise, including hydroxychloroquine and choloroquine phosphate (cortegiani et al., 2020; gao et al., 2020) , which act through several mechanisms, including alkalisation of the host cell phagolysosomes. newer antiviral medications such as lopinavir (yao et al., 2020) , remdesivir (holshue et al., 2020; wang et al., 2020) , and arbidol (khamitov et al., 2008) also show promise. other suggested treatment options include lopinavir/ritonavir, nucleoside analogues, neuraminidase inhibitors, and peptide ek1 (lu, 2020) . a detailed list of current and planned clinical trials investigating various drugs for the treatment of sars-cov-2 was provided by pang et al. (2020) , with updated results available from clinicaltrials.gov (2020). in addition, traditional herbal medicines and purified natural products may guide the development of novel antiviral drugs. in other words, more efficient drugs can often be designed based on the structure of natural compounds that exhibit the desired activity. classic examples of this drug discovery pathway include emetine, an isoquinoline alkaloid isolated from cephaelis ipecacuanha and used as an amoebicidal drug; quinine, derived from the bark of cinchona trees; and numerous other drugs modified from natural compounds, such aspirin, morphine and paclitaxel, an antineoplastic drug used for the treatment of cancer (ganjhu et al., 2015) . indeed, half of all drugs approved between 1981 and 2014 were derived from or mimicked a natural compound (newman and cragg, 2016) . furthermore, in the current outbreak of covid-19, many patients appear to be turning to complementary or traditional medicinal therapies, albeit using them almost exclusively in conjunction with western medicine. for example, one study suggested that almost 92 % of 135 hospitalised patients in northeast chonqing (china) received traditional chinese medicine in addition to western medicine . however, based on the many studies conducted on this topic, it is hard to separate the potential effects of, and interaction between, traditional chinese herbal medicine and western medicine. recent reviews have suggested that traditional chinese medicine could be used for the prevention or treatment (yang et al., 2020a ) of covid-19; while still acknowledging that many studies involving clinical trials are poorly designed or controlled, and the choice of treatments is largely empirically based. as previous work has highlighted the potential of traditional chinese medicines as a source of potential novel drugs (ling, 2020) , we have not included details on such studies investigating the antiviral activity of remedies comprising portions of numerous plant species in this review. rather, our aim is to collate data on the broad spectrum of natural phytochemicals from individual plant species that may have therapeutic potential. naturally occurring antiviral agents acting against general coronaviruses were briefly reviewed by lin et al. (2014) six years ago, while more recent reviews by pang et al. (2020) and lu (2020) on therapies for covid-19 made only brief mention of natural therapeutics and did not explore the active compounds or their mechanism of action. in light of the current covid-19 pandemic, this review aims to gather and consolidate information on extracts and compound(s) derived from natural products which show potential antiviral bioactivity for the inhibition of coronaviruses. it is hoped that the information presented may guide the naturally-derived drug discovery process in finding a treatment for sars-cov-2. the pubmed database (www.ncbi.nlm.nih.gov/pubmed/) was used to locate articles including the following combination of terms: (coronavirus, sars or mers) and (traditional medicine, herbal, remedy or plants). papers primarily focussed on the antiviral activity of prepared chinese traditional medicines, which typically comprise multiple plant species, were considered out of scope of this review. all articles up to and including 25 march 2020 were considered, yielding a total of 659 results. two of the authors independently screened the results and identified relevant articles, yielding a total of 35 primary articles on human coronaviruses and 22 on animal coronaviruses were found to be pertinent and thus included in this review (total = 58 papers). of these, two papers (6%) were on sars. the majority of studies on human coronaviruses (69%; n=24) included sars-cov, with only 3 (9%) including mers-cov and 8 (23%) other human coronaviruses. it should be noted that one study included both sars-cov and mers-cov (o'keefe et al., 2010) while another included both mers-cov and hcov-229e (müller et al., 2018) , hence these percentages do not add to 100%. table 1 summarises the studies reporting the inhibition of various human coronavirus strains using compounds derived from plant sources. the table is arranged by viral strain in order to better compare the bioactivity of compounds from different studies upon the same viral genotypes. where identified, the key compounds responsible for the antiviral activity and their identified mechanisms of action are presented. it should be noted the term ec50 (effective concentration) applies to cell-based assays, while ic50 (inhibitory concentration) applies to enzyme-or biochemical-based assays. few studies report on sars-cov-2, as expected given the short time since its emergence. however, a number of studies report on use of computer modelling for screening purposes (liu and zhou, 2005; lung et al., 2020; toney et al., 2004; wang et al., 2007; zhang et al., 2020) . typically, these models determine the free binding of energy between a ligand and a receptor (forli et al., 2016) , with a lower free binding energy indicating a stronger bond between the ligand and receptor. although obtaining consistent results via different modelling approaches can be challenging (aldeghi et al., 2016) , computer modelling nevertheless allows for comparison of the relative binding affinity of bank of molecules toward the receptor in question. in addition to reducing the high costs and length of time associated with physically screening large banks of compounds or plant extracts for bioactivity (chen et al., 2017) , the speed and versatility of this method may be particularly valuable for rapidly finding a potent inhibitor of sars-cov-2. compounds that are highlighted through this method can then be forwarded on cell-based assays to assess their in vitro effectiveness and toxicity, before continuing to animal and clinical trials. lung et al. (2020) virtually screened 83 compounds found in chinese traditional medicines for activity against the rna-dependent rna polymerase of sars-cov-2, identifying theaflavin, an antioxidant polyphenol, as a potential inhibitor. similarly, zhang et al. (2020) virtually screened 115 compounds found in chinese traditional medicines, highlighting 13 for further studies. several of these were naturally occurring polyphenolic compounds such as quercetin and kaempferol, which have already received considerable interest for the treatment of other disease types (cassidy et al., 2019; khan et al., 2019; tome-carneiro and visioli, 2016) . given the relatively large amount of research that has been performed searching for inhibitors of sars-cov, antiviral agents that successfully inhibit this viral strain may provide a good starting point for identifying compounds that are active against sars-cov-2. several authors have utilised virtual computer docking models to screen for potential compounds that could bind to and inhibit key proteins present in sars-cov (liu and zhou, 2005; toney et al., 2004; wang et al., 2007) , highlighting the potential antiviral activity of compounds such as sabadinine and aurantiamide acetate. compounds may be screened against a number of binding sites in order to test for potential inhibition of coronaviruses; the main sites that are typically used are the chymotrypsinlike protease (3clpro), papain like protease (plpro), spike proteins and rna-dependent rna polymerase. several large in vitro screening studies searching for inhibitory activity of naturally occurring compounds against sars-cov have been performed, mainly on chinese medicinal herbs (li et al., 2005; wang et al., 2003) . while the results highlight the potential of selected plant extracts against sars-cov, they also demonstrate that such work can be akin to searching for a 'needle in a haystack'. for example, li et al. (2005) screened over 200 ethanol/chloroform extracts of chinese medicinal herbs and found only four (lycoris radiata, artemisia annua, pyrrosia lingua and lindera aggregata) with moderate to high antiviral activity using a cpe assay (ec50 values ranging from 2.4 ± 0.2 to 88.2 ± 7.7 µg/ml). of these, a single compound (lycorine) from one plant species (l. radiata) was earmarked as a potential drug candidate against sars-cov. the antiviral efficacy of lycorine was quite high (ec50 of 15.7 ± 1.2 nm), with a selectivity index greater than 900. although the authors did not make mention of this fact, lycorine can cause toxic effects at low dosage levels (around 1 mg/kg in dogs) (kretzing et al., 2011) , hence great caution would be required for further development of this compound as a drug candidate. yu et al. (2012) screened the activity of a library of 64 naturally occurring compounds against sars-cov helicase, which plays a key role in the viral genome replication, transcription, and translation. the polyphenolics myricetin and scutellarein ( figure 2 ) were identified as the most promising candidates (ic50 values of 2.71 ± 0.19 and 0.86 ± 0.48 µm, respectively). although the antiviral activity of the compounds was not assessed in cell-based assays, the authors did report that neither compound was toxic to normal (non-tumorigenic) breast epithelial cells. myricetin is found in reasonably high concentrations in fruits such as cranberry (häkkinen et al., 1999) as well as in several vegetables such as calamus scipionum and garlic (miean and mohamed, 2001) . scutellarein was isolated from scutellaria baicalensis (chinese skullcap), which has been traditionally used in the treatment of inflammation and respiratory infections, amongst other uses (zhao et al., 2016) . both compounds were found to inhibit sars-cov helicase (nsp13) through the inhibition of atpase activity, but did not directly inhibit helicase activity. this appeared to be the only publication identifying naturally occurring compounds as inhibitors of sars-cov helicase. the need to isolate and synthesise more active agents as part of the development pipeline was highlighted in a study by runfeng et al. (2020) . the authors reported that the chinese herbal medicine lianhuaqingwen (comprised of a mixture of plant species) showed antiviral activity against sars-cov-2; however, the ec50 was quite high (~411 µg/ml). for comparison, the commercial drug remdesivir showed an ec50 of 0.651 µm (approx. 0.39 µg/ml) using the same assay (runfeng et al., 2020) . this also underscores the potential lack of potency in some traditional medicines promoted for the treatment of coronavirus symptoms. one family of compounds that demonstrate antiviral activity across a number of studies is the polyphenols. for example, quercetin showed an ic50 of 8.6 ± 3.2 µm against sars-cov plpro (park et al., 2017) . no cell-based assay of antiviral activity was performed. quercetin ( figure 2 ) is a flavonoid found in many foods, but in particularly high levels in certain berries and herbs (justesen and knuthsen, 2001; kaack and austed, 1998) . as previously mentioned (section 3.2.2), the structurally similar polyphenolics myricetin and scutellarein ( figure 2 ) display reasonable levels of inhibitory activity against sars-cov helicase (yu et al., 2012) . bioassay-guided fractionation of the ethanolic extract obtained from psoralea corylifolia seeds has also identified polyphenolics as the bioactive compounds responsible for the activity of this plant species against sars-cov plpro (kim et al., 2014) . furthermore, six phenolic phytochemicals were isolated from the ethanolic extracts -identified as bavachinin, neobavaisoflavone, isobavachalcone, 4'-o-methylbavachalcone, psoralidin and corylifol a -with their antiviral activity varying widely (ic50 values between 4.2 -38.4 µm). again, no cell-based antiviral assays were performed. isobavachalcone and psoralidin (figure 2 ) showed the greatest antiviral activity, with both found to be mixed, reversible inhibitors of plpro through a type i mechanism (i.e. preferentially bind to the free enzyme, rather than the enzyme substrate complex) (kim et al., 2014) . plant lectins, which are proteins that can bind specifically and reversibly to carbohydrate groups (mitchell et al., 2017) , are another group of naturally occurring compounds that may inhibit sars-cov. lectins have shown promise as antiviral agents against viruses such as influenza and herpes simplex virus (hwang et al., 2020) , as well as ebola (covés-datson et al., 2019; michelow et al., 2011) . remarkably, elevating the plasma levels of recombinant human mannose-binding lectin in mice allowed them to survive otherwise fatal ebola infections (michelow et al., 2011) . keyaerts et al. (2007) screened the activity of a broad range of plant lectins (33 in total) against sars-cov using a cytopathicity effect (cpe) assay, finding ec50 values as low as 0.45 ± 0.08 µg/ml for lycoris radiata agglutinin. although the exact mechanism of action was not determined, activity at the stage of viral attachment or the end of the infectious viral cycle were deemed to be the most likely targets. in clinical trials, other lectins have demonstrated reasonable to good tolerability (petersen et al., 2006) , hence with further testing, they may prove to be one of the more promising classes of naturally derived compound(s) for the treatment of sars-cov-2 and other coronavirus infections. whilst many natural derived compounds show considerable promise for the inhibition of sars-cov and other human coronaviruses, few approach the level of efficacy and/or selectivity required for commercial drugs. for example, the calpain inhibitor mdl28170 has been shown to have an ic50 value of just 2.5 nm (~1 ng/ml) in its activity against the sars-cov enzyme cathepsin-l (simmons et al., 2005) . cathepsin-l is a cysteine protease which is an important lysosomal endopeptidase enzyme involved in the initiation of protein degradation (sudhan and siemann, 2015) and mediates entry and infection of sars-cov in its host cells (huang et al., 2005) . chemical modification of naturally-derived compounds may be required to increase the potency of their antiviral activity to levels suitable for therapeutic application. for the drug discovery process, beginning with the understanding of the structural conformity (i.e. structure including potential isomers) of the naturally derived compound(s) can reduce timelines and greatly increase the chance of finding effective viral inhibitors. working on this principle, hoever et al. (2005) found that modification of glycyrrhizin, naturally found in liquorice and previously used for the treatment of sars-cov (haiying et al., 2003) , could increase its viral inhibition activity. for example, adding 2-acetamido-β-d-glucopyranosylamine to the glycoside chain of glycyrrhizin increased its antiviral activity in a cpe assay by 10-fold (decrease in ec50 from 365 ± 12 µm to 40 ± 13 µm), through increased attraction to the s proteins. amides and conjugates with amino acid residues and free cooh increased activity of glycyrrhizin by up to 70-fold (ec50 for cpe assay ranging from 5 ± 3 µm to 139 ± 20 µm), albeit with significantly reduced selectivity. cho et al. (2013) demonstrated that tomentins a-e (all naturally occurring compounds) showed viral inhibition activity against sars-cov greater as compared to their non-geranylated precursor compounds. similarly, quercetin-7-rhamnoside ( figure 2 ) demonstrates over 100 times higher antiviral activity against an animal coronavirus strain compared to its parent compound, quercetin (choi et al., 2009) . while the examples of modifications presented here vary, it should not be considered that substitutions are added in a random fashion, but rather performed in the context of targeting a specific receptor or biochemical pathway. for example, the addition of glycosides and specific amino acid residues to glycyrrhizin was performed with the intent of increasing its affinity for the highly glycosylated spike proteins of sars-cov. with this in mind, future studies that identify an effective natural inhibitor of coronavirus should also consider the design and testing of potential modifications or synthetic derivatives that could increase its desired activity. a difference in the efficacy of natural therapeutics, mirroring that observed for commercially/synthetically available drugs, has been observed between coronavirus species/strains. for example, park et al. (2017) found that the compound 3'-(3-methylbut-2-enyl)-3',4,7trihydroxyflavane, isolated from broussonetia papyrifera, showed good inhibition of plpro from sars-cov (ic50 of 3.7 ± 1.6 µm), but not against plpro from mers-cov (ic50 of 112.5 ± 7.3 µm). in contrast, the polyphenolics kazinol f and broussochalcone a isolated from the same species showed better efficacy against mers-cov plpro. similarly, o'keefe et al. (2010) found that the efficacy of the compound griffithsin against sars-cov was highest for the urbani and tor-ii strains (ec50 of 0.61 µg/ml using a cpe inhibition assay) and lowest for the frank strain (ec50 of 1.19 µg/ml). griffithsin, isolated from the red alga griffithsia sp., is a lectin possessing three large identical carbohydratebinding domains orientated as an equatorial triangle, which enable multivalent binding (o'keefe et al., 2010) . although the source of these observed differences in the inhibition activity of griffithsin against different sars-cov strains was not explored by o' keefe et al., (2010) , it may be attributable to genomic differences in the amino acid sequences of the spike proteins between sars-cov strains, leading to varying multivalent interactions with the carbohydrates-binding domains and thus differing affinity for binding to the spike proteins. nevertheless, these studies highlight the importance of conducting in vitro tests of potential naturally-derived therapeutics on sars-cov-2 prior to animal or clinical trials. only a handful of studies have investigated the potential of natural products as therapeutic agents against mers-cov. silvestrol (figure 2) , a phytochemical from aglaia sp., was found to be a more potent inhibitor of mers-cov replication (ec50 of 1.3 nm) (müller et al., 2018) . however, no cell-based antiviral assays were performed. silvestrol is a specific inhibitor of rna helicase eif4a, thus inhibiting viral replication and leading to the inhibition of expression of cov proteins and preventing the formation of replication/transcription complexes (müller et al., 2018) . griffithsin (figure 2) , a 12.7 kda lectin found in the griffithsia genus (red algae), is one of the most promising inhibitors of mers-cov. it comprises three carbohydrate-binding domains which allow it to bind specifically to glycans on cov protein spikes and inhibit viral attachment to host cells, with high potency found in in vitro trials against mers-cov (ec50 of ~0.125 µm) (o'keefe et al., 2010) and several hcov strains (ec50 of 0.0032-0.33 µm) (millet et al., 2016) . griffithsin also appears to have a low systematic toxicity, with its specificity index against hcov cells (compared to human colorectal adenocarcinoma or fibroblast cell lines) has been estimated at between 30 -3100 (o' keefe et al., 2010) , hence showing the potential to be considered as one of the prime candidates for animal and clinical trials against sars-cov-2. a recent study highlighted the potential anti-hcov-nl63 activity of the methanolic extract of strobilanthes cusia leaf and its major phytochemicals (tsai et al., 2020) . the s. cusia extract effectively reduced virus yield (ec50 = 0.64 µg/ml) in cells infected with hcov-nl63 in a dose-dependent manner. of the six key compounds isolated, purified and identified via nmr spectroscopy, two exhibited the most potent antiviral activity against hcov, namely tryptanthrin ( figure 2) , a natural alkaloid containing the basic indoloquinazoline moiety, and indigodole b (5ar-ethyltryptanthrin), an indole alkaloid derivative. the ec50 values against virus yield from infected cells were 1.52 µm and 2.60 µm for tryptanthrin and indigodole b, respectively. the increased antiviral activity of tryptanthrin in comparison to indigodole b may result from the double bond at c5a in the former, as compared to the additional ethyl moiety in the latter. this highlights that for compounds based on a tryptanthrin structural conformity, addition of a double bond in the quinazoline ring could significantly increase their antiviral activity. tryptanthrin has previously been found to have a broad spectrum of biological activities including anticancer, anti-inflammatory, antiprotozoal, antiallergic, antioxidant and antimicrobial action (kaur et al., 2017) . through manipulation of varying modes of time-ofaddition/removal assay, it was found that tryptanthrin prevented the early and late stages of hcov-nl63 replication, particularly by blocking the viral rna genome synthesis and papain-like protease 2 activity, and inhibiting the post-entry stage of hcov replication (tsai et al., 2020) . intriguingly, both tryptanthrin (ec50 = 0.06 µm) and indigodole b (ec50 = 2.09 µm) exhibited strong viricidal activity directly against hcov-nl63. as the hcov-nl63 spike protein (s protein) targets the ace2 receptor, showing a highly conserved sequence and structural similarity to sars-cov and sars-cov-2 (letko et al., 2020) , tryptanthrin has the potential to be explored as a possible bioactive agent against sars-cov-2 and other human coronaviruses. another recent study on hcov-oc43 focussed on the antiviral activities of the bis-benzylisoquinoline alkaloids tetrandrine, fangchinoline, and cepharanthine (cpe assay ec50 values of 0.33 ± 0.03, 1.01 ± 0.07 and 0.83 ± 0.07 µm, respectively) (kim et al., 2019) . these compounds were the primary bioactive phytochemicals identified in stephania tetrandra and related species. all compounds were found to inhibit virus-induced cell death, through suppressing viral replication, expression of viral s and n proteins (nucleocapsid protein), and the virus-induced host response. however, the effective inhibitory concentrations were not reported for the inhibition of viral protein expression or host response. in addition, tetrandrine activated the p38mapk pathway in mrc-5 cells, improving the aforementioned host response. weng et al. (2019) tested the ethanolic extracts of sambucus formosana (elderberry) stems against the human coronavirus strain hcov-nl63, finding quite high efficacy (ec50 of 1.17 ± 0.75 µg/ml) for viral yield reduction. with further investigation of the phenolic composition of the extracts and antiviral assays performed on the main individual phenolic acids present, caffeic acid ( figure 2 ) was identified as the most potent compound present (ec50 of 3.54 ± 0.77 µm; or ~0.64 ± 0.14 µg/ml). although no specific binding sites were identified, caffeic acid was found to inhibit the attachment of hcov to host cells, indicating potential binding to s proteins. caffeic acid has also been found to inhibit other viruses such as hepatitis b (wang et al., 2009) . notably, extracts of sambucus nigra (black elderberry), another species in the same genus as s. formosana, have been commercialised for the treatment of cold and flu symptoms. therefore, it is likely that extracts from s. formosana would prove similarly nontoxic and suitable for human use, although clinical trials would be required to confirm this. nevertheless, the bioavailability/delivery mechanism of such extracts would need to be considered in order to reach therapeutic plasma concentrations for viral inhibition (wittemer et al., 2005) . cheng et al. (2006) examined the anticoronaviral activity of saikosaponins (a, b2, c and d) and their mode of action against hcov-229e in vitro. saikosaponins represent a group of pentacyclic triterpenoid derivatives, usually present as glycosides, that have been isolated from medicinal plants such as bupleurum spp., heteromorpha spp. and scrophularia scorodonia, previously found to possess efficacy against several viruses, including human immunodeficiency virus (hiv) (chiang et al., 2003; ushio and abe, 1992) . all saikosaponins tested showed good to moderate anti-coronavirus activity, with saikosaponin b2 (figure 2) showing the greatest potency (ec50 of 1.7 ± 0.1 µm). subsequent timeof-addition studies indicated that saikosaponin b2 inhibited viral attachment and penetration. interestingly, the same compound has been found to inhibit hepatitis c entry (lin et al., 2015) as well as multidrug resistance-associated drug transporters present on the cell surface (zhao et al., 2019) , indicating that it has potential to display a broad spectrum of bioactivity. this may be advantageous in developing nations where hepatitis c prevalence is high (pawlotsky, 2014) . finally, two compounds that have shown efficacy against mers-cov (section 3.3) also show promise against hcov. müller et al. (2018) found that silvestrol (figure 2 ) inhibited the translation of hcov-229e proteins, with an ec50 of 3 nm. a follow-up study confirmed that silvestrol can inhibit hcov-229e in an ex vivo bronchial epithelial cell system, with its mechanism of action being the specific inhibition of rna helicase eif4a (müller et al., 2020) . another compound active against mers-cov, griffithsin, also has shown great efficacy against several hcov strains (ec50 of 0.0032-0.33 µm) (millet et al., 2016) . this again underscores the prospect of exploring griffithsin for antiviral activity against sar-cov-2. animal coronavirus strains are responsible for severe morbidity events across a wide range of domestic animals and livestock, incurring major economic demise worldwide (jackwood et al., 2010; lelesius et al., 2019; mccutcheon et al., 1995) . the genomic diversity, coupled with the ability of coronaviruses to rapidly adapt and mutate, presents unique challenges in the development of novel antiviral agents, hence exploring alternative methods of controlling these viruses could potentially be effective across many or all serotypes. as natural phytochemicals have been shown to have activity across a wide range of viral pathogens (chen et al., 2014) , they form the basis of the studies reviewed here. the majority of plant-derived compounds considered in this review are direct-targeting antivirals, acting through direct inhibition of some part of the virus, such as proteases or spike proteins. for example, silvestrol prevents viral replication occurring through the specific inhibition of rna helicase eif4a (müller et al., 2018) , while griffithsin binds directly to the s protein, preventing viral entry to the host cell (o'keefe et al., 2010) . however, host-targeting antivirals form another important group of antiviral compounds. for example, extracts of cinnamomi sp. inhibit the clathrindependent endocytosis pathway, thus preventing viral entry to the host cells (zhuang et al., 2009) . other host-targeting antiviral compounds may stimulate the immune response (lau et al., 2008) . table 2 presents a summary of studies reporting anti-viral activity of plant-derived isolates against a range of animal coronavirus strains. where available, the key compounds responsible for the antiviral activity and their mechanism of action are provided. the antiviral activity of extracts from plant species against the avian ibv viral strains have been extensively studied (chen et al., 2014; jackwood et al., 2010; lelesius et al., 2019; li et al., 2011; nguyen et al., 2015; yang et al., 2010; yang et al., 2011; yin et al., 2011) . considering all studies on avian ibv that have established the mechanism of viral inhibition, the main mechanisms of action appear to be through viral envelope disruption or interference with the spike protein (table 2) . a notable exception are the terpenoid compounds 1,8-cineole, (-)-α-pinene and (-)-β-pinene, which bind to the ibv nucleocapsid protein (n-protein) (yang et al., 2010; yang et al., 2011) , inhibiting its interaction with the viral genomic rna and breaking the ibv replication cycle. these observations were reinforced by conformational models of the terpenoids binding to the active site at the n terminus of the n protein, which indicated that these compounds have the potential to bind strongly to five amino acid residues at this location (tyra92, proa134, phea137, aspa138 and tyra140) (yang et al., 2010; yang et al., 2011) . as these residues are highly conserved between ibv strains (yang et al., 2010) , these terpenoids would be expected to show high efficacy against most or all ibv strains, making them a sensible target for further research. nevertheless, the envelope protein (e protein) of avian ibv remains the key target for most researchers. the coronavirus e protein is integral to many stages of the viral life cycle, including assembly, budding, envelope formation, and pathogenesis (schoeman and fielding, 2019) . interestingly, chen et al. (2014) reported the inactivation of two distinct enveloped viruses (avian ibv and influenza) following treatment with sambucus nigra extract, suggesting that s. nigra extract may have the potential to display a broad spectrum of antiviral effects against many other enveloped viruses. although the authors raised the possibility of synergistic action of inhibitory compounds within the extract, no fractionation, identification or characterisation of the components was performed. however, flavonoids or lectins were suggested as the likely aetiological agents of the antiviral activity. feline coronavirus is another coronavirus in the alphacoronavirus group, causing a fatal disease in cats with no effective antiviral treatments available. in one study, galanthus nivalis agglutinin, another lectin, was identified as a potent inhibitor of feline coronavirus (fcov) (hsieh et al., 2010) . with an ec50 of just 0.0088 nm and a high selectivity index (>218), this carbohydrate-binding protein outperformed all synthetic antiviral agents tested for comparison purposes. for example, the protease inhibitor nelfinavir displayed an ec50 of 8.19 µm and a selectivity index of just 1.4. as with other agglutinins, galanthus nivalis agglutinin binds to the spike and membrane proteins of fcov, preventing their attachment to the host cells. mcdonagh et al. (2014) conducted a screening study against 19 compounds, focusing on those demonstrated to have previous antiviral effects against coronaviruses or other rna viruses. several naturally occurring compounds were included in this study, such as quercetin, curcumin, rutin, glycyrrhizic acid, hesperidin, hesperitin, baicalin and artemisinin. however, none of these compounds reached ec50 at the concentrations tested. glycyrrhizic acid was the most promising, causing a 26.7% reduction in cpe at a concentration of 25 µm. as only a single concentration was tested for each compound (ranging from 10-50 µm, depending on the compound), further investigation of these compounds against fcov is required. theerawatanasirikul et al. (2020) adopted a computer-aided approach to their screening study against feline infectious peritonitis virus (fipv), a mutated form of the parental enteric form of fcov. firstly, compounds from a chemical library were virtually screened for potential binding to the protease 3cl. the 15 most promising compounds were then evaluated in vitro using a protease inhibitor assay against fipv 3cl pro . of the naturally occurring compounds tested, the lowest ic50 values were shown by 7-methylluteolin (28.5 ± 4.2 µm) and stictic acid (29.4 ± 4.6 µm). quercetin 7-rhamnoside ( figure 2 ) also showed moderate inhibition (ic50 of 77.2 ± 13.8 µm); however 7-benzyl luteolin and steviol showed no inhibition (ic50 > 500 µm). subsequent testing of the active compounds using a cytopathic effect (cpe) assay indicated that only stictic acid protected cells from viral-induced death (ec50 for previral entry treatment of 16.24 ± 1.33 µm; selectivity index of 23). porcine endemic diarrhoea virus is another serious coronavirus from the alphacoronavirus group. several studies have investigated the activity of phytochemicals against murine coronaviruses (strain mhv-a59), the most extensive of which were performed by kim et al. (2008) and kim et al. (2010) . nevertheless, none of the studies of this viral strain have conclusively managed to determine the specific compounds responsible for viral inhibition, but rather suggested possible compounds or classes of compounds based on their abundance in the extracts tested. the low inhibitory concentrations (<1 µg/ml) of extracts obtained from sophorae sp., acanthopanacis sp. and torilis sp. appear to show significant potential. in particular, the high viral specificity (si = 696) of sophorae sp. root extracts suggests that it could be considered as a prime candidate for future studies on the screening and isolation of compounds from the aforementioned species. the authors suggested that the antiviral activity of these three species are likely to be occurring through inhibition of rnadependent rna polymerase or other protease activity. six studies considered natural agents active against porcine endemic diarrhea virus (pedv), with the majority of these identifying the compounds with for their antiviral activity. in particular, the anti-pedv activity of griffithsin and quercetin ( figure 2 ) and its derivatives deserve particular discussion, given that these compounds have also been found to have antiviral activity against human covs (millet et al., 2016; o'keefe et al., 2010; wen et al., 2011) . notably, quercetin was also one of the compounds shortlisted by zhang et al. (2020) in their virtual screening of inhibitors of sars-cov-2 proteases. against pedv, quercetin-7-rhamnoside, a disaccharide glucoside, provided 50 % inhibition of viral activity at a concentration of just 0.03 µm, approximately 187 times lower than quercetin alone (choi et al., 2009) . significantly, the specificity of quercetin-7-rhamnoside was exceptionally high (si = 7143), indicating its potential for application in future animal or clinical trials. this study again highlights the importance of considering all possible structural conformities of a compound, including glycosides, in order to identify the most bioactive chemical species. although the mechanism of action was not determined for quercetin or quercetin-7-rhamnoside against pedv, previous computer modelling work has indicated that quercetin binds to and inhibits the sars-cov proteases plpro and 3clpro . as previously determined, griffithsin binds to the spike protein of cov, preventing attachment to host cells (millet et al., 2016; o'keefe et al., 2010) . in general, most authors recommend testing multiple coronavirus strains when searching for antiviral activity in naturally occurring compounds. similarly important is determining their specific mechanism of action. as highlighted by several studies, a selection of compounds that are active against animal coronaviruses are also active against human coronavirus strains (e.g. griffithsin, quercetin). this underscores the potential of utilising compounds with identified activity against animal coronaviruses to guide the discovery of drugs against human coronaviruses, in addition to identifying drugs for the treatment of economically significant animal coronaviruses such as pedv and avian ibv. across both human and animal cov strains, a clear trend toward the use of chemically polar compounds is evident (figure 2) . of the 30 studies that specified the solvent extraction protocols used, ethanol or an ethanol/water combination was the most commonly used (50 % of all studies), followed by methanol or a methanol/water combination (27 %). a further 17 % of relevant literature used a water-based extraction protocol, with only three studies using relatively non-polar solvents (diaminopropane, chloroform and acetone). this is consistent with a wide body of research indicating that relatively polar extracted fractions generally contain a greater level of bioactive and antimicrobial compounds compared to their nonpolar counterparts (han et al., 2007; tian et al., 2009; wigmore et al., 2016) . this may also be indicative of increased bioactivity of highly polar glycosylated compounds, similar to the vast difference in antiviral activity observed between quercetin and quercetin-7rhamnoside (choi et al., 2009 ). in the case of compounds being administered orally, more polar compounds are subject to compartmentalisation within the body, reducing their rate of elimination. however, the potential chemical changes that could occur as a result of highly acidic stomach conditions and the level of absorption in the intestine would need to be considered individually for each compound. naturally occurring phytochemicals provide a valuable and powerful resource of chemical compounds displaying antiviral properties. further chemical modification of these structures, guided by computerbased docking simulations, may also increase their potency and/or selectivity. some of the key compounds that show promise for the treatment of coronavirus in humans include scutellarein, silvestrol, tryptanthrin, saikosaponin b2, lectins such as griffithsin, lycorine and polyphenolicsincluding quercetin, myricetin, caffeic acid, psoralidin and isobavachalcone. needless to mention, these compounds may be toxic at certain levels, and hence in vitro and in vivo testing is required to determine safe and therapeutic levels for each compound before clinical trials in humans can be performed. initial studies could focus on compounds which have previously been 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flowering cherry fruits (prunus serrulata l. var. spontanea) identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 in silico screening of chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus scutellaria baicalensis, the golden herb from the garden of chinese medicinal plants saikosaponin b2 enhances the hepatotargeting effect of anticancer drugs through inhibition of multidrug resistance-associated drug transporters procyanidins and butanol extract of cinnamomi cortex inhibit sars-cov infection (hoever et al., 2005) page 27 of 34 clpro = chymotrypsin-like protease; cpe assay = cytopathogenic effect assay; n/a = not applicable to this study; nd = no data; plpro = papain-like protease. page 31 of 34 (li et al., 2019) key: cord-301151-f6vya3qh authors: zhu, xiaojuan; ge, yiyue; wu, tao; zhao, kangchen; chen, yin; wu, bin; zhu, fengcai; zhu, baoli; cui, lunbiao title: co-infection with respiratory pathogens among covid-2019 cases date: 2020-05-11 journal: virus res doi: 10.1016/j.virusres.2020.198005 sha: doc_id: 301151 cord_uid: f6vya3qh accumulating evidence shows that microbial co-infection increases the risk of disease severity in humans. there have been few studies about sars-cov-2 co-infection with other pathogens. in this retrospective study, 257 laboratory-confirmed covid-19 patients in jiangsu province were enrolled from january 22 to february 2, 2020. they were re-confirmed by real-time rt-pcr and tested for 39 respiratory pathogens. in total, 24 respiratory pathogens were found among the patients, and 242 (94.2 %) patients were co-infected with one or more pathogens. bacterial co-infections were dominant in all covid-19 patients, streptococcus pneumoniae was the most common, followed by klebsiella pneumoniae and haemophilus influenzae. the highest and lowest rates of co-infections were found in patients aged 15–44 and below 15, respectively. most co-infections occurred within 1–4 days of onset of covid-19 disease. in addition, the proportion of viral co-infections, fungal co-infections and bacterial-fungal co-infections were the highest severe covid-19 cases. these results will provide a helpful reference for diagnosis and clinical treatment of covid-19 patients. respiratory illness caused by a novel coronavirus was first noted in december of 2019 in wuhan, hubei province, china . the novel coronavirus is now referred to as severe and critical acute respiratory syndrome coronavirus-2 (sars-cov-2) by the international committee on taxonomy of viruses (ictv). the sars-cov-2 was transmitted through respiratory tract and could induce pneumonia (chen, et al. 2020; chu, et al. 2020) . by 6 april, who has reported of 1, 210, 956 laboratory-confirmed cases of sars-cov-2 infection and 67,594 deaths worldwide (who, 2020) . the current outbreaks of coronavirus infection remind us that covs are still a severe and critical threats to global public health. there are no proven antiviral therapies or vaccines till now. thus, the best way to deal with severe and critical infections of sars-cov-2 is to control the source of infection, early diagnosis, quarantine and supportive treatments. it is difficult for physicians to distinguish causative agents without a laboratory diagnosis due to the similar clinical presentations of different pathogens. therefore, the fast and accurate diagnosis of sars-cov-2 are particularly important for patients. however, most studies only focused on sars-cov-2, while the co-infection with sars-cov-2 has been somewhat neglected. co-infection with certain pathogens may also hinder accurate disease diagnosis. wang et al. presented the latest status of the sars-cov-2 co-infection in china and added details on combined bacterial and fungal infections . however, the types of co-infected pathogens and the proportion of co-infection in sars-cov-2-positive patients are unclear. in this study, the clinical features of covid-19 patients were analyzed, then 39 respiratory pathogens in their throat swab were detected by specific real-time rt-pcr. this study will provide a reference for epidemic prevention and clinical treatment in wuhan and other areas combating this epidemic. from jan 22 to feb 2, 2020, 257 throat swab samples with initial positive real-time rt-pcr results were collected from local hospital in jiangsu province. the samples were taken immediately from each https://doi.org/10. 1016/j.virusres.2020.198005 received 7 april 2020; received in revised form 29 april 2020; accepted 30 april 2020 covid-19 patient on admission. the clinical, laboratory, and outcome data were obtained from medical records. the following parameters were recorded on admission: age, sex, clinical presentation classification. all procedures conducted in this study involving human materials were approved by the jiangsu provincial center for disease control ethics committee. firstly, sars-cov-2 was re-confirmed by real-time rt-pcr using the same protocol described previously (chu, et al. 2020; huang, et al. 2020) . other 39 respiratory pathogens including influenza a (flua), influenza b (flub), human respiratory syncytial virus (rsv), parainfluenza types 1, 2, 3 and 4 (piv1, 2, 3, 4), human metapneumovirus ( were also detected using pathogen specific real-time rt-pcr as previously described ). kruskal-wallis h-test was used to compare continuous variables among multiple groups, and dunnett test was used for pair-wise comparison; categorical variables were expressed as number (%) and compared by chi-square test or fisher's exact test among multiple groups. a value of p < 0.05 was considered to be significant. spss 19.0 software and graphpad 7.0 were used for statistical analysis. baseline characteristics of all cases and different groups are described in table 1 . 257 patients were diagnosed with the sars-cov-2 infection and their clinical severity was classified according to national health commission of the people's republic of china revised criteria for diagnosis and treatment of novel coronavirus infection pneumonia (trial version fifth, revised version). these patients were categorized into four clinical types, of which 22 (8.5 %) were asymptomatic cases (a total of 19 patients showed symptoms after sampling), 78 (30.4 %) were mild cases, 140 (54.5 %) were moderate cases, and 17 (6.6 %) were severe/critical cases (including 14 severe cases and 3 critical cases). there was no death of all the cases in our study, 3 cases of critical patients were admitted to intensive care unit (icu). the age of patients in moderate category was significantly lower than in mild category (p < 0.01), whereas asymptomatic and severe/critical category did not significantly differ in age from the moderate category. totally, more men (53.7 %) than women were observed, but most of the patients were female (68.2 %) in asymptomatic category. below 15 years of age, a total of 11 (4.3 %) were diagnosed with the sars-cov-2 infection and there were no case in severe/critical category. a total of 243 (94.2 %) patients had viral, bacterial and fungal coinfections. as shown in fig. 1, 9 viruses, 11 bacteria and 4 fungi were detected. 81 patients (31.5 %) had viral co-infection, 236 (91.8 %) had bacterial co-infection, and 60 (23.3 %) had fungal co-infection ( table 2 ). the co-infection pathogens were as follows: s.pneumoniae table 2 ). the rates of viral co-infection (6, 35.3 %), fungal co-infection (5, 29.5 %) and bacterial-fungal co-infection (5, 29.5 %) were the highest in severe/critical category (p > 0.05; table 2 ). of the 138 male patients, 128 (92.8 %) were co-infected, while 113 (95.0 %) females had co-infection. below 15 years of age, there were 11 pathogens were found and hmpv only appeared in this group ( fig. 2a) . the rates of kp, aspergillus, e. coli, and hrv co-infection were the lowest. between 15 and 44 years of age, 22 pathogens were discovered except for hmpv and cryptococcus. among these, influenza a and hbov were only found in this group ( fig. 2a) . the rates of s.pneumoniae, kp, hi, aspergillus, ebv, e. coli, s. aureus, p.aeruginosa, hadv, a. baumannii, mucor, influenza b and mp co-infection were the highest. between 45 and 64 years of age, 19 pathogens were discovered and cryptococcus only appeared in this group (fig. 2a) . the rates of hrv, cp and mc co-infection were highest. above 65 years of age, there were 14 pathogens ( fig. 2a) . the rates of ebv, s. aureus, p.aeruginosa and mc co-infection were the lowest. however, the co-infection rates had no statistical significant difference among different age groups x. zhu, et al. virus research 285 (2020) 198005 (p > 0.05). the pathogen distribution was also analyzed along with disease course. the results showed that there were 19 pathogens in -4-0 days after onset, and cmv and hmpv co-infection only appeared in this group (fig. 2b) . the rates of e. coli and hrv co-infection were the highest. 21 pathogens were discovered in 1-4 days after onset. hbov and candida only appeared in this group. the rates of s.pneumoniae, kp, hi, aspergillus, ebv, s. aureus, p.aeruginosa, mc, hadv, hsv, a. baumannii and cp co-infection were the highest. there were 18 pathogens in 5-8 days after onset, and cryptococcus only appeared in this group (fig. 2b) . the rates of influenza b co-infection were highest. only 9 pathogens were found in 9-15 days after onset (fig. 2b) . no significant differences were found by days after onset (p > 0.05). there were 10, 22, 20 and 13 pathogens were found in symptomatic, mild, moderate, and severe/critical cases, respectively (fig. 3) . s.pneumoniae, kp, hi, aspergillus, ebv, e. coli, and s. aureus were simultaneously found in four clinical groups. s.pneumoniae, kp, hi, aspergillus, ebv were most frequently found. mc and a. baumannii were found in asymptomatic category, mild category and moderate category. although no statistical significance was found, the nucleic acid-positive rate of mc (3, 13.6 %) was the highest in mild category. hadv, hrv, hsv and p.aeruginosa were found in mild category, moderate case, severe/critical category. the positive rates of hrv (8, 10.3 %) were highest in mild category. cmv was discovered in asymptomatic category and moderate category. influenza a, influenza b, cp, mp and mucor were detected in mild case and moderate category. the positive rates of influenza a (1, 1.3 %), influenza b (2, 2.6 %), and mp (3, 3.9 %) were higher in mild category, whereas cp (4, 2.9 %) and mucor (4, 2.9 %) were higher in moderate category. bordetella pertussis and candida were determined in moderate category, severe/critical category. co-infection may significantly inhibit the immune system of host, increase antibacterial therapy intolerance, and be harmful to the prognosis of the disease (li and zhou, 2013) . in our study, 94.2 % of covid-19 patients could be co-infected with one or more other pathogens, including 9 viruses, 11 bacteria and 4 fungi. there was no death of all the cases. only 3 patients were admitted to the icu. the proportions of the co-infection had no statistical significant difference between icu and non-icu patients. the results suggested that there was no specific relationship between co-infection and icu admission, as well as the occurrence of death. the prognosis of patients with co-infection needs to be further studied. bacterial co-infection in the setting of viral pneumonia is known as major cause of mortality (guo, et al. 2019) . our results showed that bacterial co-infection were dominant in all covid-19 patients. s.pneumoniae, kp, and hi were the most common bacterial co-infection in our study. although some of them are co-colonization, they are conditional pathogen. they might also pathogenic when the immune function of covid-19 patients were decreased. it suggested that pneumococcal conjugate and polysaccharide vaccines would offer an .3) 1 (4.5) 11 (14.1) 11 (7.9) 1 (5.9) bacteriavirusfungi 23 (8.9) 1 (4.5) 11 (14.1) 10 (7.1) 1 (5.9) the viruses in the table refer to viruses other than sars-cov-2. a included "virus only" and "virus-fungi or bacteria-virus or bacteria-virusfungi". b included "bacteria only" and "bacteria-fungi or bacterial-virus or bacteriavirus-fungi". c included "fungi only" and "fungi-virus or fungi-bacterial or bacteria-virusfungi". x. zhu, et al. virus research 285 (2020) 198005 effective approach to preventing the most common co-infection during the covid-19 pandemic. in addition to bacteria, fungi was also a major factor of co-infection secondary to viral infections such as seen in influenza infection in which a. baumannii, aspergillus, candida and kp were discovered as common secondary infections (gao, et al. 2013; guo, et al. 2019) . in this study, the highest rates of co-infection with bacteria and fungi were found among severe and critical category. some of pathogens are known antibiotic resistance which may make the treatment of covid-19 patients more difficult. wang et al. studied 104 patients with sars-cov-2 infection and found that 3 (2.88 %) patients were co-infected with other coronavirus, 2 (1.94 %) were co-infected with influenza a . in this study, we also found a few of influenza a, influenza b co-infections, but no coronavirus was detected. these results indicated that the co-infections with influenza a, influenza b or coronavirus were not common in covid-19 patients, though the seasonal influenza is now prevalent. however, influenza a, influenza b co-infections may increase the risks of covid-19 patients. influenza vaccination is still recommended. it was notable that the positive rates of hadv and hrv were higher than that of influenza viruses. hadv and hrv are associated with lower mortality, but significant higher morbidity, causing a huge economic burden (fendrick, et al. 2003) . ebv and hsv were the common pathogens carried by adults. the effects of their roles in the process of covid-19 need to be explored. cmv, hbov, and hmpv were important pathogens of respiratory tract infection in children. however, only a few of them were found co-infected with sars-cov-2. the virus co-infection could cause both upper and lower respiratory tract infections, and demonstrate overlapped clinical presentations. thus, other viruses should also be considered while diagnosing and treating sars-cov-2. similar co-infection rate and pathogens in females was found when compared with males which mean both man and women are susceptible to other respiratory pathogens. the species of co-infected pathogens were higher between patients of 15-64 years old than that of below 15 years and above 65 years of age. we also found that the rates of influenza, hrv, and hadv co-infection were higher among cases of 15-64 years old which was different from the previous studies in which the pathogens were found mainly affecting the young and aged people. it may be due to the decreased immunity after sars-cov-2 infection. the highest co-infection rate and the most pathogen species were detected in cases of 1-4 days after onset. along with the course of disease, both the rates and pathogen species of co-infection among covid-19 patients were decreased significantly, which may due to the treatment x. zhu, et al. virus research 285 (2020) 198005 the patients received. we noticed that a high co-infection rate and pathogen species in patients of -4-0 days after onset, which indicated asymptomatic category also have high co-infection opportunity although the pathogen species was the lowest, and early treatment should be taken for them. pathogen species was also not high in severe/critical category, which may attribute to all of them have been taken medical intervention. in this study, we tested for the presence of 24 types of respiratory pathogens in 257 covid-19 patients. at present, more and more patients of infectious diseases were characterized by an increased multipathogen co-infection, which has increased the difficulty in clinical diagnosis and treatment. therefore, while testing sars-cov-2, we needed simultaneously screen other respiratory pathogens which was very important to for the appropriate treatment and diagnosis. x. zhu, et al. virus research 285 (2020) 198005 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia the economic burden of non-influenza-related viral respiratory tract infection in the united states clinical findings in 111 cases of influenza a (h7n9) virus infection clinical features predicting mortality risk in patients with viral pneumonia: the mulbsta score clinical features of patients infected with 2019 novel coronavirus in wuhan co-infection of tuberculosis and parasitic diseases in humans: a systematic review clinical diagnosis of 8274 samples with 2019-novel coronavirus in wuhan covid-19) situation report-77 clinical features and molecular characteristics of influenza b viral pneumonia in elderly patients in nanjing city a novel coronavirus from patients with pneumonia in china the authors declare no conflict of interest. key: cord-260422-z22t57ju authors: godet, julien; boudier, christian; humbert, nicolas; ivanyi-nagy, roland; darlix, jean-luc; mély, yves title: comparative nucleic acid chaperone properties of the nucleocapsid protein ncp7 and tat protein of hiv-1 date: 2012-06-26 journal: virus res doi: 10.1016/j.virusres.2012.06.021 sha: doc_id: 260422 cord_uid: z22t57ju rna chaperones are proteins able to rearrange nucleic acid structures towards their most stable conformations. in retroviruses, the reverse transcription of the viral rna requires multiple and complex nucleic acid rearrangements that need to be chaperoned. hiv-1 has evolved different viral-encoded proteins with chaperone activity, notably tat and the well described nucleocapsid protein ncp7. we propose here an overview of the recent reports that examine and compare the nucleic acid chaperone properties of tat and ncp7 during reverse transcription to illustrate the variety of mechanisms of action of the nucleic acid chaperone proteins. rna chaperones are proteins that interact with rna molecules to solve the rna folding problem (herschlag, 1995; cristofari and darlix, 2002; schroeder et al., 2004) . rna molecules are synthesized as linear polymers that require appropriate folding to reach their active (native) conformations. along the folding trajectory, rna navigates through a rugged funnel-like landscape biased toward few native structures (fontana et al., 1993; chen and dill, 2000; russell et al., 2002) . intermediate conformations with stabilities close to the native functional states usually coexist such that a fraction of the molecules rapidly folds into their native structures while many others are kinetically trapped in misfolded intermediate conformations ( fig. 1) (thirumalai et al., 1997) . rna molecules are thus prone to adopt stable and persistent alternative secondary structures which have to overpass thermodynamic barriers to correctly fold (treiber and williamson, 1999) . this is why assistance appears to be necessary to reach the active rna conformations. rna chaperones were thus postulated to direct the correct folding of rna molecules and to resolve rna misfoldings (herschlag, 1995) . today's view is that rna chaperones are nucleic acid binding proteins present in all living organisms, including viruses, where they abbreviations: hiv-1, human immunodeficiency virus-type i; ncp7, nucleocapsid protein of hiv-1; zf, zinc finger; na, nucleic acid; tar, transactivation response element; pbs, primer binding site; aa, amino acids. * corresponding author. tel.: +33 03 90 24 42 63; fax: +33 03 90 24 43 12. e-mail address: yves.mely@unistra.fr (y. mély). perform multiple functions (cristofari and darlix, 2002; schroeder et al., 2004) . questioning the structure-function paradigm (wright and dyson, 1999; dyson and wright, 2005) , it was found that the prevalence of functional regions in rna chaperones without a well defined 3d-structure was very high and such intrinsically disordered domains (idd) were proposed to support the chaperone activity (tompa and csermely, 2004; tompa, 2005) . since rna chaperones do not share common sequences, motifs or structures, their identification is hardly predictable, and thus requires adequate assays to establish their chaperoning activity. several in vitro assays are routinely used (cristofari and darlix, 2002; rajkowitsch et al., 2005) and include annealing, helix destabilization, strand displacement (cristofari and darlix, 2002; rajkowitsch et al., 2005 rajkowitsch et al., , 2007 schroeder, 2007a, 2007b; godet and mély, 2010) , cis or trans-splicing (coetzee et al., 1994; zhang et al., 1995; mayer et al., 2002; semrad et al., 2004; belisova et al., 2005; grohman et al., 2007) and hammerhead-ribozyme rna cleavage (bertrand and rossi, 1994; herschlag, 1995) . fewer in vivo assays also exist, such as intron folding trap or transcription antitermination (clodi et al., 1999; lorsch, 2002; rajkowitsch et al., 2005) . the latter tests investigate simultaneously several if not all the components that account for the chaperoning activity. in vitro studies show that rna chaperones function by repetitive transient interactions with rna (cruceanu et al., 2006a; wu et al., 2010; doetsch et al., 2011b) . this property results in transient destabilization of the metastable na conformations and in accelerating the annealing of complementary sequences. the fast binding kinetics is thus likely to be a critical component of the chaperoning activity. monitoring the fast association and dissociation rates of scheme illustrating the rna energy landscape. rna energy landscape is rugged with many local minima that correspond to suboptimal kinetic trap foldings (b and c). rna-chaperones smoothen the landscape by destabilizing metastable conformations and lowering the activation energy between the different conformations, thus directing the folding of rna towards the native conformation. chaperones from na substrates is a challenging issue but none of the currently used assays can yet provide direct measurements of the on-and off-binding rates. indirect evidence of this property has been nevertheless gained from single molecule stretching experiments investigating the chaperone properties of hiv-1 gag and nc (williams et al., 2001; cruceanu et al., 2006a) , and the nucleocapsid protein of the yeast ty3 retrotransposon (chaurasiya et al., 2012) . at the same time, rna chaperones promote molecular aggregation, also known as molecular crowding. rna-chaperones usually present basic domains, ensuring that a significant part of the binding is provided by nonspecific electrostatic interactions. rna chaperones can bind to highly diverse nucleic acid (na) sequences and by so doing can coat na molecules, which is required for function. this has been termed the window of rna chaperone activity (reviewed in darlix et al., 2011) . this binding mode causes the formation of high molecular weight nucleoprotein complexes, where fuzziness appears to be a major trait in addition to molecular crowding (fuxreiter and tompa, 2012; ivanyi-nagy and darlix, 2012) . fast binding kinetics, molecular crowding and fuzziness cause a high local concentration of the partners and a smoother energy landscape resulting in a large increase in the rna folding rate (woodson, 2010) (fig. 1) . multiple functions have been assigned to cellular rnachaperones, such as transcription regulation, rnp assembly, pre-mrna processing, rna nuclear export and translation, and in mirna metabolism. rna-chaperones are also present in the viral world, where they have major roles in virus structure and replication (zúñiga et al., 2009 ). the first reported viral rna-chaperones are the nucleocapsid (nc) proteins of avian and murine retroviruses (prats et al., 1988) . later, a similar chaperone activity was shown for the hiv-1 nc protein (darlix et al., 1990; de rocquigny et al., 1992; barat et al., 1993; dib-hajj et al., 1993; lapadat-tapolsky et al., 1993) . the intense research efforts on hiv/aids benefited to the characterization of chaperones since a large part of our current understanding was gained during the past 20 years from studies on hiv-1 nc protein. other virus-encoded rna chaperones have since been characterized, notably the nucleoprotein n of coronavirus (zúñiga et al., 2007) , the 3ab protein of poliovirus (destefano and titilope, 2006) , the core protein of flaviviridae (cristofari et al., 2004; ivanyi-nagy et al., 2006 sharma et al., 2011) , the nucleoprotein n of hantavirus panganiban, 2005, 2006) or the small delta antigen sdag of hepatitis d virus (huang and wu, 1998; huang et al., 2003 huang et al., , 2004 wang et al., 2003) (for a review see zúñiga et al., 2009) . moreover, viruses are capable of highjacking cellular factors with rna chaperoning activity in the course of viral rna synthesis and translation (zúñiga et al., 2009) . all these examples highlight the variability of chaperone proteins selected through evolution to solve the folding problem and to assist the large nucleic acid rearrangements occurring throughout viral life cycles. here, we review the mode of action of hiv-1 ncp7 in parallel with that of the trans-activator of transcription tat, another potent nucleic acid chaperone encoded by hiv (kuciak et al., 2008; boudier et al., 2010) to illustrate the variety of the mechanisms of action of the nucleic acid chaperone proteins. hiv-1 ncp7 is a small (55 amino acids) basic protein resulting from the protease-mediated cleavage of the pr55gag polyprotein precursor. ncp7 is characterized by two conserved 'cx 2 cx 4 hx 4 c' zinc fingers (zfs) ( fig. 2a) , flanked by small domains rich in basic residues. interestingly the basic zf linker and the n-and c-terminal domains appear to be disordered indicating that ncp7 belongs to the idp family . at the same time, a hydrophobic plateau can form at the top of the zfs in the native protein (morellet et al., 1992 mély et al., 1994) , which is composed of residues val13, phe16, thr24 and ala25 of the proximal zf and residues trp37, gln45 and met46 of the distal zf. this zf plateau plays a key role in the selection of the viral sequences during genomic rna packaging and contributes to ncp7 chaperoning properties (godet and mély, 2010; darlix et al., 2011) . ncp7 can cause nucleic acid destabilization (bernacchi et al., 2002; azoulay et al., 2003; beltz et al., 2003; cosa et al., 2004 cosa et al., , 2006 and can transiently interact with nucleic acids with fast binding and dissociation kinetics (cruceanu et al., 2006a) . ncp7 also activates the annealing of complementary sequences you and mchenry, 1994) and the cleavage of a cognate substrate by a hammerhead ribozyme (bertrand and rossi, 1994; kuciak et al., 2008) , and efficiently rescues the splicing of a group i intron mutant in the t4 phage td gene in vivo (clodi et al., 1999) . the destabilizing activity of ncp7 mainly relies on the zfs (beltz et al., 2005) while the nucleic acid annealing and condensation properties are largely dependent on the basic residues of the disordered regions ( fig. 2a ) stoylov et al., 1997; williams et al., 2001) . the importance of the various ncp7 amino acids can be easily seen on the sequence logo plot resulting from the alignment of 3120 ncp7 hiv sequences of the lanl hiv database (http://www.hiv.lanl.gov/). fig. 2b shows that the information content associated to the amino acids composing the two zfs and the linker are close to their theoretical maximum (log 2 (20) ≈ 4.32 bits). in contrast, the n-terminal domain is less conserved although the positions of the positively charged amino acids are essentially conserved, suggesting that a precise distribution of basic amino acids in the n-terminal and basic linker domains is required for nc function doetsch et al., 2011a) . the gag precursor also exhibits nucleic acid chaperone activity (rein, 2010; wu et al., 2010) . the comparative chaperone activities of recombinant gag (or more precisely gag p6 ) and of the differently processed cleavage products have been investigated. gag can bind nucleic acids with higher affinity than ncp7, while in contrast, the nucleic acid chaperone activity improves greatly as the gag precursor is progressively processed to give ncp15, ncp9 and ncp7 (cruceanu et al., 2006b; rein, 2010; wu et al., 2010) . modification of the chaperone activity of nc during the viral cycle likely accounts for the modifications in the virus structure and function consecutive to the gag cleavage events during maturation (mirambeau et al., 2010) . in particular, critical architectural changes of the viral core result from the nucleoproteic complex condensation as gag is progressively processed (mirambeau et al., 2007) . the progressive decrease of binding affinity as gag is processed is likely ascribed to the loss of the specific high affinity binding mode required for selective vrna encapsidation to favour a less specific and transient binding mode mandatory to facilitate the nucleic acid rearrangements during reverse transcription. the mature ncp7 is characterized by highly dynamic binding properties (cruceanu et al., 2006a) . effective strand annealing activity is notably correlated with nc's ability to rapidly bind and dissociate from nucleic acids. indeed, nc variants with slow on/off rates are poorly efficient in rearranging nucleic acids, even though they are still capable of binding with high affinity to nucleic acids and to aggregate nucleic acids (cruceanu et al., 2006b; stewart-maynard et al., 2008) . in the inner core of the mature viral particle, approximately 1500-2000 copies of ncp7 are coating the genomic rna (briggs et al., 2004; chertova et al., 2006; chen et al., 2009) , thus corresponding to an average occupancy of 5-7 nt per ncp7 molecule. such an occupancy value was found to be required for optimal nc chaperoning activities in vitro (reviewed in darlix et al., 2011) , notably in the annealing reactions taking place during viral dna synthesis by rt. during reverse transcription which occurs in this viral nucleoprotein assemblage called the reverse transcription complex (rtc), ncp7 is thought to direct most of the critical nucleic acid rearrangements, namely primer trnalys,3 annealing to the viral pbs (barat et al., 1989 (barat et al., , 1993 de rocquigny et al., 1992; hargittai et al., 2004) , the first and second dna strand transfers (you and mchenry, 1994; lapadat-tapolsky et al., 1995; auxilien et al., 1999; ramalanjaona et al., 2007; godet et al., 2011) and to assist the formation of the central dna flap (charneau et al., 1994; hameau et al., 2001) . in addition, nc appears to assist the viral dna polymerase activity of rt throughout viral dna synthesis, notably by relieving rt pausing at the initiation step corresponding to ss-cdna synthesis (liang et al., 1998; rong et al., 2001; bampi et al., 2004; liu et al., 2010) , by increasing the polymerization rate and stimulating the rt-rnase h activity (bampi et al., 2006; grohmann et al., 2008) , and by promoting the fidelity of plus-strand priming (jacob and destefano, 2008; post et al., 2009 ) as well as that of (−) and (+) strand dna synthesis (kim et al., 2012) . thus, the overall efficiency of reverse transcription is increased by ncp7 which by forming a condensed but highly mobile ribonucleoproteic complex increases the molecular crowding of the nucleic acids generated during reverse transcription and thus, facilitate their annealing tanchou et al., 1995) . however, direct demonstration of these nucleic acid rearrangements in the viral context is still missing. tat is a small basic protein that has multiple key roles in virus replication and pathogenesis (karn, 1999) . tat is encoded for by two exons and is composed of 101 (clinical isolates) or 86 amino acids (laboratory isolates). the full-length tat is composed of six different regions which composition and functions are briefly described below (fig. 3) . region i (aa 1-20) is acidic and proline rich and is involved in tat-mediated immune suppression (wrenger et al., 1997) . region ii (aa 21-40) contains seven conserved cys residues, where all of them but cys31 are required for transcription transactivation (kuppuswamy et al., 1989; jeang et al., 1999) . these cys residues interact with two zn 2+ ions (frankel et al., 1988a (frankel et al., , 1988b , conferring to tat the property to trigger apoptosis (egelé et al., 2008) . region iii (aa 41-48) represents the highly conserved core that is critical for tat binding to tubulin (chen et al., 2002) , which triggers the mitochondrial pathway of apoptosis and neuronal cytoskeletal changes leading to aids-associated dementia (chen et al., 2002; giacca, 2005) . region iv (aa 49-64) is arg-rich and mediates the binding of tat to the 5 tar sequence (kuppuswamy et al., 1989; betti et al., 2001) . this region also contains the tat nuclear localization signal (vivès et al., 1997) . the c-terminal region contains a glutamine-rich domain (aa 57-72) that contributes to the transactivating activity of tat (kuppuswamy et al., 1989) . this region is also involved in induction of apoptosis in t-lymphocytes upon binding to tubulin (campbell et al., 2004) . finally, region vi (aa 73 to 86 or 101) can interact with the integrin-mediated sites of cellular adhesion and with integrin and fibronectin receptors. it is also involved in the cell penetration properties of tat (barillari et al., 1993) . tat binding to tar rna (trans acting responsive element) activates viral dna transcription initiation and elongation from the 5 ltr promoter (laspia et al., 1989; feinberg et al., 1991) . the tat/tar nucleoprotein complex promotes the recruitment of a series of transcription factors leading to the formation of very active elongating transcription machinery (chun and jeang, 1996; okamoto et al., 1996) . beyond its role in viral dna transcription, tat/rna interactions were reported to influence viral mrna capping and splicing, and translation (chiu et al., 2002; berro et al., 2006; charnay et al., 2009) . moreover, tat could possibly act as a rna silencing suppressor via interactions with dicer, trbp, sirna and mrna (bennasser et al., 2005; bennasser and jeang, 2006) . tat belongs to the idp's family (shojania and o'neil, 2006) . conformational disorder and flexibility may confer to tat its ability to interact with numerous viral and cellular partners (dunker et al., 2005; dosztányi et al., 2006) . together with its nucleic acid binding properties, the disordered nature of tat suggests that this protein is also a nucleic acid chaperone (kuciak et al., 2008) . tat and several tat-derived peptides were found to efficiently activate dna annealing, ribozyme-mediated rna cleavage and rna transsplicing in vitro (kuciak et al., 2008) . tat was also found to induce the displacement of an imperfect dna strand by a perfectly complementary sequence in a dna exchange assay (kuciak et al., 2008) , while no strand-displacement activity was found in assays using complementary rna sequences (doetsch et al., 2011a) , suggesting a nucleation-limited strand exchange activity. the inability of tat to exchange rna strands is likely explained by the limited ability of tat to destabilize rna structures. therefore, tat likely promotes nonspecific nucleic acid annealing reactions when destabilization is not or poorly required. amino acids responsible for the chaperone activity spanned from residues 44 to 61. tat(44-61) was found to be the shortest known sequence with nucleic acid chaperoning activity. interestingly, a panel of alanine-scanning mutations from amino acids 45 to 54 evidenced a striking correlation between the conservation of these amino acids (fig. 3 ) and the positions of the mutations that prevent virus to initiate the natural endogenous reverse transcription (nert) in a cell-free virus supernatant assay (apolloni et al., 2003) . the selection pressure on the central stretch of basic amino acids may thus be related to its implication in reverse transcription. the substantial nucleic acid chaperone properties exhibited by tat may account for its ability to promote the annealing of the primer trna to the viral rna (kameoka et al., 2002) and intervene in the first strand transfer (boudier et al., 2010) and by this way, to stimulate rtion as does ncp7 (harrich et al., 1997; ulich et al., 1999; apolloni et al., 2007) . during reverse transcription, major and complex nucleic acid rearrangements are required to allow the full-length genomic dsdna, also called vdna, to form. all along these steps, the nucleic acid chaperone activity of ncp7 was evidenced to assist and to facilitate the synthesis of the vdna. tat was also found to promote most of these steps in vitro. in the following sections, we will briefly outline the chaperone properties of tat and ncp7 regarding their mechanism to chaperone in vitro the rearrangements of the nucleic acid sequences involved in critical steps of reverse transcription. a. during initiation, the trna lys,3 is placed on the vrna and serves as a primer to be further elongated by rt. b. during the first strand transfer, the minus-strand strong-stop dna is translocated to the 3 end of the viral rna genome, in a reaction mediated by base-pairing of the repeat r sequences at the 3 ends of the rna (containing tar) and cdna (containing ctar) reactants, to allow reverse transcription to resume. c. the second strand transfer involves (1) the synthesis of plus strand strong-stop dna that terminates after copying the 3 end of the trna sequence; (2) trna lys,3 primer removal and (3) the annealing of (−)pbs to the (+)pbs sequence. the annealing of the two complementary pbs dna stem-loops enables rt to complete viral dna synthesis. at the early events of vdna synthesis, the reverse transcriptase (rt) elongates a primer trna annealed to the viral primer-binding site (pbs), a 18-nt conserved sequence in the 5 leader region of the genomic rna, to later synthesize the minus-strand strong-stop cdna (minus ss-cdna) (fig. 4a) . these steps are facilitated by different viral chaperones. 3.1.1. positioning the replication primer trna onto genomic rna during assembly, cellular trna lys isoacceptors are selectively incorporated into virions (jiang et al., 1993; mak and kleiman, 1997; pavon-eternod et al., 2010) . the chaperone properties of the nc domain in pr55gag are thought to facilitate the specific placement of the trna lys,3 primer on the primer binding site (pbs) of the viral rna (cen et al., 1999; feng et al., 1999; cruceanu et al., 2006b; guo et al., 2009; wu et al., 2010) . in vitro, ncp7 directs the annealing of the trna lys,3 primer to the pbs (li et al., 1996) , by facilitating the strand exchange at the level of the trna acceptor stem and by unlocking in the presence of the complementary genomic rna sequence, the highly stable interactions at the level of the t c loop (chan and musier-forsyth, 1997; tisné et al., 2003 tisné et al., , 2004 hargittai et al., 2004; tisné, 2005; barraud et al., 2007) . the kinetics of the trna lys,3 annealing on the pbs sequence follow a nucleation-limited bimolecular reaction. the reaction is enhanced by five orders of magnitude by ncp7, largely due to its ability to strongly promote duplex nucleation (hargittai et al., 2004) . the nc zfs specifically interact with the t c loop. although nc zfs do not promote unwinding of trna lys3 , the truncated form of ncp7 was found to destabilize two base pairs that could serve as nucleation points for the annealing of trnalys3 to the viral rna (tisné et al., 2001) . contrary to nc zfs, a sshs mutant of nc, which lacks the folded zfs, poorly destabilizes the trna tertiary core (hargittai et al., 2001) . it was nevertheless able to anneal more efficiently than ncp7 the trna lys,3 primer onto the pbs (hargittai et al., 2004) . mutants with complete zf deletions are also able to efficiently anneal trna lys,3 primer to the pbs, provided that the basic n-or cterminal domains are present . these zf mutants suggest that the destabilization of the trna core does not appear to be critical in vitro and that multivalent cationic peptides might be sufficient for efficient trna primer annealing to the pbs. this conclusion is also supported by the minimal alterations of the annealing kinetics induced by mutations which alter the secondary or tertiary structure or the stability of the trna (hargittai et al., 2004) and by the greater trna-annealing activity of an n-terminal extended form of ncp7 protein in vitro (roldan et al., 2005) . though the zfs appear dispensable in vitro, it cannot be excluded that as for the second strand transfer (see below), the zfs can induce a specific reaction pathway that is critical for a timely and controlled trna lys,3 /pbs annealing reaction. in full line with its ability to facilitate rna annealing, tat was also reported to increase the efficiency of primer trna placement onto genomic rna. in vitro, tat could even replace ncp7 at this step (kameoka et al., 2002) in a largely electrostatically driven trna annealing promotion. this was confirmed by mutational analysis showing that deletion of the basic domains of tat resulted in the loss of the annealing property (kameoka et al., 2002) . nonetheless, initiation of reverse transcription from tat-annealed trna lys,3 occurred less efficiently than from heat-annealed trna lys,3 (kameoka et al., 2002) . specific formation of the initiation complex is mediated by extended interactions between the hiv-1 rna and trna lys,3 (goldschmidt et al., 2002) . although variable among hiv strains (goldschmidt et al., 2004) , these extended interactions (isel et al., 1996 (isel et al., , 1998 (isel et al., , 1999 (isel et al., , 2010 li et al., 1996; beerens and berkhout, 2002; huthoff et al., 2003) are decisive for the efficiency of the initiation of reverse transcription. formation of the initiation complex requires rearrangements in the 5 utr vrna that may be promoted by the fully processed nc (iwatani et al., 2003; guo et al., 2009) . indeed, trna lys,3 annealed by gag exhibits a strongly reduced ability to initiate reverse transcription and binds less tightly to viral rna than the ncp7-annealed trna lys,3 . this necessary chaperoning of reverse transcription priming thus appears as a key regulatory step which can only be catalysed when a significant amount of ncp7 has been processed. once the primer is placed, the subsequent synthesis of vdna can start. the primer is initially extended by 6 nt in a slow and distributive initiation phase where the dna polymerization is characterized by a rapid dissociation of rt and early kinetic pausing events at positions +3 or +5 nt (isel et al., 1996; lanchy et al., 1996 lanchy et al., , 1998 . ncp7 reduces rt pauses at these positions, although with different efficiencies. interestingly, an additional pause at +1 nt was observed in vitro when the trna was heat-annealed on pbs but not when the annealing reaction was performed using ncp7. in contrast, the pause at +1 position was not affected with zf mutants of nc, suggesting again that all the chaperone properties of the native ncp7 protein are important for promoting an active trnalys,3/vrna initiation complex . on the contrary to the +1 nt pause, nc was not able to help rt to escape the +3 pausing event (liang et al., 1998; rong et al., 2001; liu et al., 2010) . the strong +3 nt pause has been proposed to originate from the template structure and notably from the folding of the a-rich stem-loop located upstream the pbs (liang et al., 1998; liu et al., 2010) . ncp7 induced a partial and transient disruption of the stem secondary structure, as evidenced by a decrease of the high-throughput shape footprinting reactivity and a broadening of the stem end-to-end fret distribution (wilkinson et al., 2008; liu et al., 2010) , but this destabilization appeared nevertheless not sufficient to prevent the +3 nt pause event. in sharp contrast to ncp7, multimerization of gag or gag-related proteins dramatically compromises reverse transcription since the cooperative binding and the slow dissociation rate of the multimerized gag proteins impaired rt processivity (wu et al., 2010) . similarly, full-length two-exon tat (86-or 101-amino acid) but not the truncated oneexon tat (72 amino acid) was found to suppress the elongation of the trna lys,3 /vrna initiation complex (kameoka et al., 2001 (kameoka et al., , 2002 . interestingly, peptides resulting from the cleavage of tat by the hiv-1 protease were able to enhance the synthesis of the (−)ssdna in a natural endogenous reverse transcription assay (apolloni et al., 2003) . thus, as envisioned for gag, the protease cleavage of tat is also needed for promoting primer elongation in vitro. tat was therefore hypothesized as a reverse transcription accessory factor involved in the spatio-temporal control of the reverse transcription. whether or not these findings are biologically relevant, they clearly underline the need for small basic proteins which can bind transiently and remain mobile on the nucleic acid lattice to allow primer extension. taken together, these observations suggest that if trna positioning can be promoted by different partners, primer extension requires a structurally well-defined initiation complex and a dynamic reverse transcription complex, which are likely promoted by the highly dynamic ncp7 (cruceanu et al., 2006a) . initiation of reverse transcription could thus constitute a key regulatory step of which timing is finely regulated by the processing of the gag precursor. this hypothesis is ascertained by observations showing that most of the annealed trna lys,3 in immature extracellular particles are only extended by a few nucleotides (oude essink et al., 1996; huang et al., 1997) . the first strand transfer constitutes a critical step in reverse transcription. this transfer occurs from region r at the 5 -end of the genome to a redundant r region at the 3 -end (fig. 4b) . in hiv-1, the r region consists of two strongly structured hairpins, namely the tar and poly(a) hairpins (baudin et al., 1993; watts et al., 2009) . tar is especially critical for efficient strand transfer (berkhout et al., 2001) . indeed, the antisense ctar dna (further referred as ctar) of the (−)ssdna has to anneal to the complementary tar rna sequence located at the 3 end of the vrna to allow dna synthesis to resume on the acceptor strand (negroni and buc, 2000; basu et al., 2008) . ctar and tar are imperfect stem-loops defined by double-stranded segments separated by numerous conserved bulges, mismatches and an internal loop (baudin et al., 1993) . reacting tar and ctar in vitro does spontaneously lead to the ctar/tar duplex, but at an extremely slow rate (you and mchenry, 1994; godet et al., 2006; vo et al., 2006 vo et al., , 2009 . through its chaperone activity, ncp7 plays a major role in promoting the annealing reaction (tsuchihashi and brown, 1994; you and mchenry, 1994; rein et al., 1998; guo et al., 2000) by enhancing the annealing rate by about 3000-fold at physiological temperature and salt conditions you and mchenry, 1994; driscoll and hughes, 2000; urbaneja et al., 2002) . the stability of tar and ctar local structures appeared of key importance to modulate the strand transfer (wu et al., 2007) . in full line with its ability to bind more transiently and dynamically to nucleic acids than gag, the fully processed ncp7 promotes the first strand transfer more efficiently than gag or gag-derived proteins at low protein concentrations (wu et al., 2010) . whereas the annealing reaction was facilitated as the ncp7 concentration increased, the strand transfer was drastically inhibited in the presence of increasing quantities of gag or gag-derived proteins. as gag and partially processed gag proteins containing the nc domain showed destabilizing and annealing activities almost as effective as ncp7, the inhibition of the strand transfer likely results from a strong restriction of the elongation step, suggesting that gag reduces the ability of rt to traverse the template ('roadblock' mechanism). once again, the ability of ncp7 to remain highly flexible and mobile within the nucleoproteic reverse transcription complex appears critical for reverse transcription to proceed (levin et al., 2005) . detailed mechanistic investigations of the ctar/tar annealing reaction showed that ctar and tar anneal in the absence of ncp7 through formation of an unstable loop-loop interaction that further converts into an extended duplex (vo et al., 2006 (vo et al., , 2009 kanevsky et al., 2011) . ncp7 switches the reaction pathway by directing the hybridization of these sequences through the end of their doublestranded stems, as a consequence of ncp7 ability to destabilize the structure of the ctar stem (godet et al., 2006; vo et al., 2009) . the destabilizing activity of ncp7 induces complex secondary structure fluctuations of the ctar ends cosa et al., 2004 cosa et al., , 2006 , leading to the formation of the open reactive species required for the annealing process. the ncp7-induced mechanistic switch shows that the reaction pathway is selected on the basis of the available reactive intermediates and is governed by the intermediates which require the least bp melting prior annealing (vo et al., 2009) . these observations constitute a nice example of how nucleic acid chaperones may remodel annealing reactions to facilitate the formation of the most stable nucleic acid conformations. the mechanism by which tat and tat-derived peptides activate the annealing of the complementary tar sequences (in the form of dna) was also thoroughly investigated (kuciak et al., 2008; boudier et al., 2010) . like ncp7 or gag, tat(1-86) promotes the hybridization of ctar to tar dna. tat peptides corresponding to the n-terminal acidic domain and to the cys-rich domain are poorly active in annealing. on the contrary, peptides tat(44-61) and tat(48-86) promote tar dna/ctar annealing, tat(44-61) being the most active of the two. this suggests that the basic rna binding domain of tat is critical to promote the annealing reaction. unlike ncp7, neither tat nor tat(44-61) are able to destabilize ctar. tat(44-61) is however able to modify the annealing mechanism of ctar with tar (in a dna/dna annealing context at least) since the analysis of ctar and tar dna mutants clearly evidenced that the reaction is initiated at the bottom of the two reacting species (boudier et al., 2010) . in addition, tat(1-86) and the mature nc were found to show comparable efficiency in promoting the annealing of ctar with the dna equivalent of tar. taken together, data on tat indicates that in the ctar/tar dna reaction, the stimulatory effect of tat on the first strand transfer resembles that of ncp7, though the hybridization reaction is differently nucleated. furthermore, tat and ncp7 were shown to cooperatively activate the annealing of ctar to tar dna, supporting a potent accessory role of tat in the stimulation of the reverse transcription. a second strand transfer reaction is required for reverse transcription to resume. the plus strand transfer involves a sequence of synchronized events (fig. 4c ) consisting in (i) the synthesis of plus strand strong-stop dna that terminates after copying the 3 end of the trna sequence, (ii) the necessary removal of the trna primer and (iii) the subsequent annealing of the minus (−) and plus (+) dna copies at the level of the 18 nt primer binding site (pbs) sequence (basu et al., 2008) . ncp7 chaperoning assistance is involved in many of the steps above described but we will focus here only on the (+)/(−)pbs annealing reaction. the dna pbs sequence folds into a bulged stable 4-bp stem-loop hairpin with a partially ordered pentanucleotide loop and a 4-nt single-strand overhang . using fluorescently labelled (+)pbs with various (−)pbs mutants, the properties of ncp7 and tat on the (+)/(−)pbs hybridization were comparatively investigated. in vitro, (+)pbs can spontaneously anneal to (−)pbs (ramalanjaona et al., 2007) . the pbs(+)/(−) annealing reaction proceeds mainly through the single-strand overhangs of the pbs sequences while nucleation through loop-loop interaction appears negligible in the absence of peptides (fig. 5a) . this was ascribed from a pbs mutational analysis showing that hybridization rates of loop mutants were poorly affected while mutations that decreased the sequence complementarity in the ss overhangs almost completely impaired the reaction (fig. 5b) . in sharp contrast to the nucleic acid sequences involved in the first strand transfer, ncp7 does not melt the stable (−)pbs stem (egelé et al., 2004 (egelé et al., , 2005 . nevertheless, ncp7 strongly promotes the annealing of (+)/(−)pbs stem-loops by increasing the annealing rate by about 60-fold, mainly by accelerating the loop pathway. as a consequence, ncp7 modifies the mechanism of the (+)/(−)pbs annealing reaction by activating the loop-loop kissing pathway that is negligible without ncp7 (ramalanjaona et al., 2007) (fig. 5a and b) . the ability of ncp7 to switch the annealing reaction from the single strand overhang pathway to the loop-loop kissing pathway strongly correlates with the ability of ncp7 to rearrange the pbs loop and to restrict the dynamics of the pbs loop bases (bourbigot et al., 2008; godet et al., 2011) . the latter were investigated using 2-aminopurine, a structural fluorescent probe that minimally affects the folding of the pbs loop and its binding parameters with ncp7. in full line with nmr data (bourbigot et al., 2008) , comparison of the 2ap fluorescence quantum yields in the absence and in the presence of ncp7 clearly evidenced that ncp7 significantly rearranged the pbs loop, notably by decreasing base stacking. time-resolved fluorescence anisotropy also revealed that ncp7 restricts the picosecond to nanosecond dynamics of the pbs loops by limiting the overall flexibility of the loops and freezing the local mobility of the bases where ncp7 is bound. local structural rearrangement and freezing of the local base dynamics of the loop are strictly dependent on the integrity of the zinc finger hydrophobic platform and constitute general features of the destabilizing activity of ncp7 (avilov et al., 2008 (avilov et al., , 2009 godet et al., 2011) . thus, the destabilizing activity of ncp7 is directly responsible for the switch to the loop-loop annealing pathway. as for the first strand transfer, tat(44-61) is able to strongly promote the annealing reaction of (+)/(−)pbs. in the presence of only 2 tat(44-61) molecules per pbs, the annealing rate constant was found six times faster than the one observed for nc(11-55) under saturating concentrations (fig. 5a) to reach a rate close to that observed in the presence of the full-length ncp7. but in sharp contrast to ncp7, tat(44-61) was not able to stimulate the annealing pathway through the loop pathway ( fig. 5a and b) , likely due to its very limited ability to rearrange the pbs loops (unpublished data). tat(44-61) shows striking similarities with sshs 2 nc or sshs 2 nc(11-55), two nc mutants where the cysteines have been substituted by serines to prevent zinc binding and which consequently do not exhibit destabilizing activity. like tat, these two zinc finger mutants do not modify the odn dynamics and structure . thus, tat(44-61) stimulates the (−)/(+)pbs annealing through already available existing pathways in the absence of peptides (i.e. through the ss overhangs). taken together, these data show that tat is able to promote the annealing reaction of the complementary pbs sequences, albeit through a different mechanism from that observed in the presence of ncp7. the activities of tat-derived peptides and ncp7 in the different in vitro models reviewed here are macroscopically very similar. both tat peptides and ncp7 promote the annealing of different complementary sequences, as well as the placement of primer trna on the viral rna. nonetheless, comparison of the mechanism of action of tat and ncp7 evidences striking differences in their ability to chaperone nucleic acid rearrangements. the most important differences result from the inability of tat, unlike nc, to destabilize transiently nucleic acids and modify the local dynamics of the nucleobases. so, even if both proteins are able to aggregate nucleic acids and promote their annealing, the destabilizing activity of ncp7 mediated through its folded zinc fingers is responsible for specific nucleic acid rearrangements and annealing pathways. taken together, these data evidence that chaperoning mechanisms are multiple and if the rna-chaperone concept is relatively straightforward, its expression appears quite diverse. a direct consequence is that no simple or consensual assay exists for evidencing and characterizing the rna chaperone activity. moreover, characterization of the hiv-1 chaperone activities in vivo appears highly challenging due to the fact that the hiv-1 genome encodes for at least three proteins (ncp7, tat and vif) exhibiting redundant na chaperone activities, highlighting the critical necessity of chaperones for viral replication. due to the differential abundance of these proteins along the viral life cycle, it is likely that these proteins exhibit their na chaperoning activities at different steps of viral replication. therefore, spatio-temporal regulation (henriet et al., 2007) and possible cooperativity (boudier et al., 2010) within these different "chaperone solutions" selected to solve the "folding problem" represent very exiting fields to explore. the authors declare no conflict of interest human immunodeficiency virus type 1 protease regulation of tat activity is essential for efficient reverse transcription and replication the hiv-1 tat protein stimulates reverse transcription in vitro role of post-transcriptional modifications of primer trnalys,3 in the fidelity and efficacy of plus strand dna transfer during hiv-1 reverse transcription site-specific characterization of hiv-1 nucleocapsid protein binding to oligonucleotides with two binding sites probing dynamics of hiv-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine destabilization of the hiv-1 complementary sequence of tar by the nucleocapsid protein through activation of conformational fluctuations nucleotide excision repair and template-independent addition by hiv-1 reverse transcriptase in the presence of nucleocapsid protein the chaperoning and assistance 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components of human ro rnps impact of the terminal bulges of hiv-1 ctar dna on its stability and the destabilizing activity of the nucleocapsid protein ncp7 structural determinants of hiv-1 nucleocapsid protein for ctar dna binding and destabilization, and correlation with inhibition of self-primed dna synthesis hiv-1 tat interaction with dicer: requirement for rna evidence that hiv-1 encodes an sirna and a suppressor of rna silencing structural features in the hiv-1 repeat region facilitate strand transfer during reverse transcription hiv-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of tar and its complementary sequence acetylated tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32 facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of hiv-1 and the heterogeneous nuclear ribonucleoprotein a1 characterization of hiv-1 tat proteins mutated in the 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retrotransposon ty3 hiv-1 tat targets microtubules to induce apoptosis, a process promoted by the pro-apoptotic bcl-2 relative bim rna folding energy landscapes fluorescence fluctuation spectroscopy on viral-like particles reveals variable gag stoichiometry proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages tat stimulates cotranscriptional capping of hiv mrna requirements for rna polymerase ii carboxyl-terminal domain for activated transcription of human retroviruses human t-cell lymphotropic virus i and hiv-1 assaying rna chaperone activity in vivo using a novel rna folding trap escherichia coli proteins, including ribosomal protein s12, facilitate in vitro splicing of phage t4 introns by acting as rna chaperones secondary structure and secondary structure dynamics of dna hairpins complexed with hiv-1 nc protein evidence for non-two-state kinetics in the nucleocapsid protein chaperoned opening of dna hairpins the ubiquitous nature of rna chaperone proteins the hepatitis c virus core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand rna in vitro rapid kinetics of protein-nucleic acid interaction is a major component of hiv-1 nucleocapsid protein's nucleic acid chaperone function nucleic acid binding and chaperone properties of hiv-1 gag and nucleocapsid proteins cis elements and trans-acting factors involved in the rna dimerization of the human immunodeficiency virus hiv-1 flexible nature and specific functions of the hiv-1 nucleocapsid protein first glimpses at structure-function relationships of the nucleocapsid protein of retroviruses trans-activation of the 5 to 3 viral dna strand transfer by nucleocapsid protein during reverse transcription of hiv1 rna viral rna annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers poliovirus protein 3ab displays nucleic 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nucleocapsid protein in modulating the specificity of plus strand priming multifaceted activities of the hiv-1 transactivator of transcription identification of trnas incorporated into wild-type and mutant human immunodeficiency virus type 1 a mechanism for plus-strand transfer enhancement by the hiv-1 nucleocapsid protein during reverse transcription the tat protein of human immunodeficiency virus type 1 (hiv-1) can promote placement of trna primer onto viral rna and suppress later dna polymerization in hiv-1 reverse transcription role for human immunodeficiency virus type 1 tat protein in suppression of viral reverse transcriptase activity during late stages of viral replication structural determinants of tar rna-dna annealing in the absence and presence of hiv-1 nucleocapsid protein tackling tat nucleocapsid protein annealing of a primer-template enhances (+)-strand dna synthesis and fidelity by hiv-1 reverse transcriptase the hiv-1 transcriptional activator tat has potent nucleic acid chaperoning activities in vitro multiple functional domains of tat, the trans-activator of hiv-1, defined by mutational analysis binding and kinetic properties of hiv-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription contacts between reverse transcriptase and the primer strand govern the transition from initiation to elongation of hiv-1 reverse transcription interactions between hiv-1 nucleocapsid protein and viral dna may have important functions in the viral life cycle analysis of the nucleic acid annealing activities of nucleocapsid protein from hiv-1 hiv-1 tat protein increases transcriptional initiation and stabilizes elongation nucleic acid chaperone activity of hiv-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism human immunodeficiency virus type 1 nucleocapsid protein (ncp7) directs specific initiation of minusstrand dna synthesis primed by human trna(lys3) in vitro: studies of viral rna molecules mutated in regions that flank the primer binding site mechanistic studies of early pausing events during initiation of hiv-1 reverse transcription initiation complex dynamics direct the transitions between distinct phases of early hiv reverse transcription rna chaperones exist and dead box proteins get a life primer trnas for reverse transcription folding of the td pre-rna with the help of the rna chaperone stpa spatial proximity of the hiv-1 nucleocapsid protein zinc fingers investigated by time-resolved fluorescence and fluorescence resonance energy transfer the hantavirus nucleocapsid protein recognizes specific features of the viral rna panhandle and is altered in conformation upon rna binding characterization of the rna chaperone activity of hantavirus nucleocapsid protein hiv-1 protease and reverse transcriptase control the architecture of their nucleocapsid partner features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function conformational behaviour of the active and inactive forms of the nucleocapsid ncp7 of hiv-1 studied by 1 h nmr determination of the structure of the nucleocapsid protein ncp7 from the human immunodeficiency virus type 1 by 1 h nmr copy-choice recombination by reverse transcriptases: reshuffling of genetic markers mediated by rna chaperones transactivation by human immunodeficiency virus tat protein requires the c-terminal domain of rna polymerase ii hiv-1 reverse transcriptase discriminates against non-self trna primers profiling non-lysyl trnas in hiv-1 fidelity of plus-strand priming requires the nucleic acid chaperone activity of hiv-1 nucleocapsid protein small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer trna onto genomic rna rna chaperones, rna annealers and rna helicases coupling rna annealing and strand displacement: a fret-based microplate reader assay for rna chaperone activity dissecting rna chaperone activity assays for the rna chaperone activity of proteins investigating the mechanism of the nucleocapsid protein chaperoning of the second strand transfer during hiv-1 dna synthesis nucleic acid chaperone activity of retroviral gag proteins nucleic-acid-chaperone activity of retroviral nucleocapsid proteins: significance for viral replication a hiv-1 minimal gag protein is superior to nucleocapsid at in vitro annealing and exhibits multimerization-induced inhibition of reverse transcription hiv-1 nucleocapsid protein and the secondary structure of the binary complex formed between trna(lys.3) and viral rna template play different roles during initiation of (−) strand dna reverse transcription exploring the folding landscape of a structured rna strategies for rna folding and assembly rna chaperone activity of large ribosomal subunit proteins from escherichia coli analysis of the rna chaperoning activity of the hepatitis c virus core protein on the conserved 3 x region of the viral genome hiv-1 tat is a natively unfolded protein: the solution conformation and dynamics of reduced hiv-1 tat-(1-72) by nmr spectroscopy retroviral nucleocapsid proteins display nonequivalent levels of nucleic acid chaperone activity ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein formation of stable and functional hiv-1 nucleoprotein complexes in vitro kinetic partitioning mechanism as a unifying theme in the folding of biomolecules structural bases of the annealing of primer trna(3lys) to the hiv-1 viral rna heteronuclear nmr studies of the interaction of trna(lys)3 with hiv-1 nucleocapsid protein specific recognition of primer trna lys 3 by hiv-1 nucleocapsid protein: involvement of the zinc fingers and the n-terminal basic extension the annealing mechanism of hiv-1 reverse transcription primer onto the viral genome the interplay between structure and function in intrinsically unstructured proteins the role of structural disorder in the function of rna and protein chaperones exposing the kinetic traps in rna folding dna strand exchange and selective dna annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein functional domains of tat required for efficient human immunodeficiency virus type 1 reverse transcription hiv-1 nucleocapsid protein as a nucleic acid chaperone: spectroscopic study of its helix-destabilizing properties, structural binding specificity, and annealing activity a truncated hiv-1 tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus mechanistic studies of mini-tar rna/dna annealing in the absence and presence of hiv-1 nucleocapsid protein hiv-1 nucleocapsid protein switches the pathway of transactivation response element rna/dna annealing from loop-loop kissing to zipper nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen architecture and secondary structure of an entire hiv-1 rna genome high-throughput shape analysis reveals structures in hiv-1 genomic rna strongly conserved across distinct biological states mechanism for nucleic acid chaperone activity of hiv-1 nucleocapsid protein revealed by single molecule stretching taming free energy landscapes with rna chaperones the n-terminal structure of hiv-1 tat is required for suppression of cd26-dependent t cell growth intrinsically unstructured proteins: re-assessing the protein structure-function paradigm fundamental differences between the nucleic acid chaperone activities of hiv-1 nucleocapsid protein and gag or gag-derived proteins: biological implications effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of hiv-1 nucleocapsid protein human immunodeficiency virus nucleocapsid protein accelerates strand transfer of the terminally redundant sequences involved in reverse transcription escherichia coli protein stpa stimulates self-splicing by promoting rna assembly in vitro role of rna chaperones in virus replication coronavirus nucleocapsid protein is an rna chaperone this work was funded by agence nationale de la recherche (contract anr-2010-blan-1529-01) and agence nationale de recherche sur le sida (anrs). key: cord-268337-o6lo55o8 authors: lloyd, richard e. title: translational control by viral proteinases date: 2005-11-21 journal: virus res doi: 10.1016/j.virusres.2005.10.016 sha: doc_id: 268337 cord_uid: o6lo55o8 most rna viruses have evolved strategies to regulate cellular translation in order to promote preferential expression of the viral genome. positive strand rna viruses express large portions, or all of their proteome via translation of large polyproteins that are processed by embedded viral proteinases or host proteinases. several of these viral proteinases are known to interact with host proteins, particularly with the host translation machinery, and thus, encompass the dual functions of processing of viral polyproteins and exerting translation control. picornaviruses are perhaps the best characterized in regards to interaction of their proteinases with the host translation machinery and will be emphasized here. however, new findings have shown that similar paradigms exist in other viral systems which will be discussed. translation can be divided into the three phases of initiation, elongation and termination. most translation regulation mechanisms regulate the initiation phase, including viral regulation schemes, thus, initiation will be emphasized here. most cellular mrnas are translated by mechanisms that are dependent on the 5 cap structure. de novo initiation of typical mrna requires recognition of the 5 m7gpppn cap structure by the trimeric translation factor complex eif4f, and subsequent recruitment of a 43s ribosomal subunit (containing a 40s ribosomal subunit, eukaryotic initiation factors (eifs) eif1, eif1a, eif2, eif5, eif3 and met-trnai met ) and other initiation factors to form a 48s ribosomal preinitiation complex. the 48s complex is functional for scanning the mrna sequence in a 5 -3 direction for initiation codons in a favorable consensus sequence. there is no clear evidence that the cap structure is released by eif4e during the scanning process, although it is often depicted this way (see fig. 3 ). the initiation phase of translation is completed when the 60s ribosomal subunit has joined, and then the 80s ribosome completes the translation of the mrna. for recent reviews on this complex topic, see (gebauer and hentze, 2004; merrick, 2004; preiss and hentze, 2003; rogers et al., 2002; schneider and mohr, 2003) . note that for clarity, only * tel.: +1 713 798 8993; fax: +1 713 798 5075. e-mail address: rlloyd@bcm.tmc.edu. initiation factors that play a role in viral proteinase-mediated translation regulation mechanisms are discussed further below. eif4f is a heterotrimeric complex consisting of eif4g, eif4e and eif4a and can be isolated as a salt-stable complex from mammalian cells. eif4g is a multivalent scaffolding protein that contains binding domains for cap-binding protein eif4e and the prototype dead-box helicase eif4a. eif4g also contains binding sites for poly(a)-binding protein (pabp) and mnk-1 kinase (fig. 1 ). eif4f is also associated with eif4b, which interacts with eif4a in rna unwinding assays but may not be required for cap-binding functions (grifo et al., 1984; ray et al., 1985; rozen et al., 1990) . there are two major forms of eif4g, termed eif4gi and eif4gii that share only 46% homology but are highly conserved in key regions that bind other translation factors (gradi et al., 1998a) . eif4gi is the dominant form in hela cells, comprising approximately 90% of total eif4gi (marissen and lloyd, 1998) . further, a complex translation initiation scheme involving alternate initiation codon selection at five augs and alternate splicing produces a set of five isoforms of eif4gi that vary at the n-terminus. the smallest isoform lacks the pabp-binding site (byrd et al., 2002 (byrd et al., , 2005 . the primary function of eif4f is to facilitate binding of 40s ribosomal subunits to the 5 cap structure of mrna and then aid ribosomal scanning. because eif4g can simultaneously bind all these initiation factors, it performs two critical linking or bridging functions. first, eif4g-mediated linkage of eif3 (which is bound to 40s ribosomal subunits), and eif4e, completes a molecular bridge which binds the mrna to the fig. 1 . schematic illustrating scaffolding protein eif4gi (and eif4gii) (shown in red) as an extended structure bearing a series of binding domains for other translation factors pabp, eif4e, eif4a (two sites), eif3 and mnk-1. the nh2 and cooh-termini of eif4g are shown. the locations of known protease cleavage sites are depicted with arrows. a central region of eif4g linking eif4e-and eif3-binding domains has been expanded in the box to illustrate locations of individual proteinase cleavage sites. ribosome (fig. 3a) . second, eif4g-mediated linkage of pabp and eif4e simultaneously provides a second molecular bridge linking 5 and 3 ends of the mrna in a pseudo-circularized structure (wells et al., 1998) . thus, eif4g is in many ways the centerpiece of the translation initiation complex. therefore, it is not surprising that many viruses have evolved mechanisms to modify eif4g functions in their bid to control cellular translation. several non-proteolytic translation regulation mechanisms involving eif4gi are detailed in other chapters herein. this chapter reviews only mechanisms involving cleavage of eif4g and other factors. picornavirus rna does not contain a cap structure to aid ribosome binding. to compensate for this, the 5 untranslated region (5 utr) contains a large rna structure called an internal ribosome entry sequence (ires) that recruits ribosomes to bind to internal sites in the rna. ires structures have been found in a wide range of virus and cellular mrnas and are quite variable in sequence and structure. the mechanism of ribosome binding by hcv and picornavirus iress are best understood and they involve variable subsets of the canonical initiation factors, depending on the ires. in addition, certain rna-binding proteins such as la, ptb, unr and pcbp2 have been described that stimulate the functional activity of certain ires elements in biochemical assays (bedard et al., 2004; blyn et al., 1996 blyn et al., , 1997 boussadia et al., 2003; costa-mattioli et al., 2004; hellen et al., 1993; hunt et al., 1999; meerovitch et al., 1989 meerovitch et al., , 1993 . such ires-transactivating factors (itafs) are thought to play a role in the selective pathogenesis and variable replication of several picornaviruses in different cell types and tissues (pilipenko et al., 2000 (pilipenko et al., , 2001 . poly(a)-binding protein (pabp) binds the poly(a) tail on mrna via four conserved rna-recognition motifs (rrms) and contains a highly conserved c-terminal domain (ctd) linked by a proline-rich domain. like eif4gi, pabp also binds a large number of proteins, including eif4g, eif4b, translation termination factor erf3, and three regulatory proteins, unr, and pabp-interacting proteins 1 and 2 (paip1 and paip2) ( fig. 2) (bushell et al., 2001; chang et al., 2004; imataka et al., 1997 imataka et al., , 1998 khaleghpour et al., 2001; kozlov et al., 2004; roy et al., 2002) . the interaction between eif4e/eif4g and pabp is sufficient to circularize the mrna, and provides the fig. 2 . schematic illustrating pabp (shown in green) with its multiple binding partners. the structure of pabp is illustrated with four numbered rrm domains that interact with rna, a flexible proline-rich linking domain and a structured cooh-terminal domain that contains a binding cleft for erf3, eif4b and paip2. rrms 2 and 3 interact with eif4g and unr. a region near rrm 4 interacts with 60s ribosomal subunits in yeast and may be involved in pabp oligomerization. the location of viral proteinase cleavage sites is indicated. other binding sites for paip1 and paip2 are not shown for clarity. primary basis for 5 -3 interactions in mrnas (wells et al., 1998) . many, if not most translating mrnas in the cell are thought to be arranged in such a quasi-circular configuration. this "closed loop model" was suggested over 30 years ago as the most efficient means to recycle ribosomes during translation (baglioni et al., 1969; philipps, 1965) . it has been proposed that circularization of mrna stimulates translation by increasing the binding affinity of eif4e for the cap structure or by providing a mechanism to recycle terminating ribosomes to the 5 end of the mrna sachs and varani, 2000) . further, enhanced translation on circularized mrnas promotes translation of only intact mrnas, and pabp-eif4g interaction may stabilize mrna by inhibiting cap-and poly(a)-oriented mrna decay mechanisms (coller and parker, 2004; jacobson, 2004; meyer et al., 2004) . the presence of poly(a) tails (with pabp bound), stimulate joining of both the 60s ribosomal subunit and 40s ribosomal subunit to mrna during initiation in yeast and reticulocyte lysates (munroe and jacobson, 1990; sachs and davis, 1989) . also, pabp stimulates translation of mrna that is missing a cap structure, during so-called poly(a)-dependent initiation, even if the 5 end of mrna is blocked. this 3 -mediated translation still requires eif4g . thus, functional interaction between 5 and 3 ends of the mrna is considered to be crucial, particularly in conditions in vivo where mrnas must compete for ribosomes. the confirmed interaction between pabp and erf3 is very interesting as it may place pabp in space and time in the same location as a terminating ribosome and the stop codon (hoshino et al., 2000; kozlov et al., 2001; uchida et al., 2002) . this suggests that often lengthy 3 utr may be looped out to allow for this interaction (fig. 3a) . this configuration may also enhance translation by facilitating recycling of ribosomes. it may be possible for ribosome subunits to hop or shunt from stop codons, or 3 utr regions to the 5 utr, thus providing a way for ribosomes to continually translate the same mrna without undergoing more rounds of de novo initiation. direct evidence for ribosome recycling has not been demonstrated, however, has been inferred from some experiments that will be discussed below. it is not known if the biochemical requirements for ribosome recycling would differ from the requirements for de novo ribosome binding and scanning. therefore, the concept and mechanism of re-initiation of ribosomes is under investigation. these new models of translation control impact our understanding of viral regulation of translation. virus infection of most cell types by human enteroviruses such as poliovirus (pv), rhinovirus (hrv) and coxsackie b viruses (cvb3) induces a rapid and nearly complete inhibition of host cell protein synthesis. the mechanism that blocks host translation does not affect translation of virus rna during the first 4-5 h of infection, however, virus translation does suffer a rapid decline about 2 h after host translation is inhibited. the early translational switch is temporally accompanied by dis-aggregation of polysomes containing cellular mrna followed by reformation of larger polysomes containing exclusively viral mrna. the mechanism of the translational switch is largely based on lack of a cap structure on viral rna and the presence of an ires that mediates cap-independent translation initiation. the actual site for host translation inhibition was long ago localized to the initiation step of protein synthesis, prompting a search for inactivated initiation factors in infected cells. in 1982 etchison and hershey made the seminal discovery that eif4g (then called p220) was cleaved during pv infection, thus inactivating eif4f complexes in cells (etchison et al., 1982) . other reports demonstrated that eif4e, eif4a, eif4b, eif3 were not cleaved in pv-infected cells (duncan et al., 1983; etchison et al., 1984; lee et al., 1985) , however, pabp was independently reported by two groups to be cleaved by pv and cvb3 in 1999 (joachims et al., 1999; kerekatte et al., 1999) . no other canonical translation factors are known to be cleaved during pv infection. eif4gi is cleaved by 2a proteases of pv, hrv and cvb3 at position (amino acid 681/682) sommergruber et al., 1994) . cleavage of eif4g effectively splits the eif4f complex in half, separating the eif4e-binding domain of eif4g from the eif3-binding domain (lamphear et al., 1995) ( fig. 1) . thus, the bridging function of eif4g that brings capped mrnas to the 40s ribosome is neatly abrogated by the virus. however, the second bridging function of eif4g that connects 5 -3 ends of mrna is retained. pv infection also activates at least two cellular proteinase activities that cleave eif4gi very near the 2a pro cleavage site and at another site 43 amino acids upstream . combined, these multiple proteinase activities rapidly cleave eif4gi during the first 2-3 h of infection. efforts to purify eif4g cleavage activities from pv-infected cells revealed that fractions enriched with 2a pro had weak eif4g cleavage activity and most cleavage activity associated with cellular proteins (bovee et al., 1998a,b) . the preferred molecular target of hrv 2a pro was recognized to be eif4f (eif4e bound to eif4g) instead of purified eif4gi, likely due to formation of a protease-sensitive conformation (haghighat et al., 1996) . this model is supported by structural analysis of the region of eif4g that binds eif4e. this region is unstructured before binding but becomes folded into an alpha helix with two turns after binding eif4e (gross et al., 2003; marcotrigiano et al., 1999) . the issue of why pv 2a pro isolated from infected cells contains so little eif4gi cleavage activity has not been resolved, nor have the cellular eif4g-proteases been identified. hrv 2a pro cleaves eif4gi relatively rapidly in translation reactions in rabbit reticulocytes, requiring only 15 min and 1:12 molar ratios of enzyme to substrate (glaser and skern, 2000) . but recombinant pv and cvb 2a pro are less efficient in in vitro cleavage assays (bovee et al., 1998a) . eif4g is also cleaved by caspase 3 at two other sites during apoptosis ( fig. 1 ) (marissen and lloyd, 1998) . viral infection activates some apoptosis pathways, however, the caspase inhibitor zvad-fmk did not diminish eif4g cleavage during pv infection (roberts et al., 2000) . in contrast, another investigation found that zvadfmk did inhibit eif4g cleavage if viral rna replication was also blocked with 2 mm guanidine in order to limit 2a pro expression . the eif4g-protease is clearly not caspase 40s subunit is depicted on the aug codon after completing scanning. it is unclear if the cap structure is released by eif4f during scanning (dashed line) or the 5 utr is looped out (solid line). ribosomes that have reached the stop codon bind erf3 which may interact with pabp-ctd to facilitate recycling of 60s subunits to waiting 40s subunits on initiation codons, or alternatively, both subunits may recycle. the pabp-ctd may dynamically switch back and forth between binding erf3 and eif4b (white arrows). this recycling step can function after eif4g is cleaved. pabp oligomerization involving the ctd (yellow arrows) may prevent other pabp-ctd from interactions with eif4b and erf3. (b) model for translation shutoff in pv-infection. after cleavage of eif4g by 2a pro (orange arrows), de novo binding of 40s subunits to mrna via the cap structure is blocked and recycling of ribosomes is blocked by cleavage of pabp by 3c pro (green arrows). cleaved eif4g may still retain mrna in closed loop configuration. 3, which cleaves at different sites and is not activated early in infection belov et al., 2003; carthy et al., 1998; romanova et al., 2005) , however, this protease may be part of an early apoptotic response to viral infection. the eif4gi homolog, eif4gii, is also cleaved by 2a pro , however, cleavage kinetics are slower during hrv or pv infection than cleavage of eif4gi (gradi et al., 1998b) . the cleavage site on eif4gii has been mapped to amino acid 699, which is in the same region as the 2a pro cleavage site in eif4gi (gradi et al., 2003) . based on kinetics studies in infected cells, cleavage of both eif4gi and eif4gii was proposed to be required for complete translation shutoff during pv or hrv infection (gradi et al., 1998b; svitkin et al., 1999) . however, no mechanistic studies of eif4gii's contribution to cellular translation in comparison to eif4gi or pabp have been performed. the animal picornavirus foot-and-mouth disease virus (fmdv) also causes rapid cleavage of eif4g during virus infections. the 2a gene of fmdv is not a functional proteinase, rather cleavage of eif4g is accomplished by the leader proteinase (l-pro), an alternate papain-like protease encoded at the n-terminus of the viral polyprotein. this proteinase cleaves eif4gi at a distinct site, seven amino acids upstream of the 2a pro cleavage site, thereby accomplishing the same functional scis-sion of eif4e-and eif3-binding domains (kirchweger et al., 1994) . this cleavage reaction has been very well characterized and occurs extremely rapidly, presumably while ribosomes are translating the fmdv polyprotein (glaser and skern, 2000) . lproteinase also cleaves eif4gii, however, this scission is very rapid and efficient, unlike pv or hrv 2a pro , which cleave more slowly. the l pro cleavage site is located one amino acid downstream of the 2a pro cleavage site on eif4gii (gradi et al., 2004) . interesting work has shown that both fmdv l pro and hrv 2a pro initially recognize and bind to eif4gi in regions away from the scissile bond, between amino acids 600-674 (fig. 1) , which are located immediately to the n-terminal side, but do not include the amino acids where cleavage occurs. similarly, mutagenesis and binding studies have located eif4g-binding domains on each protease that are located away from the substrate binding cleft, thus representing exosites (foeger et al., 2002 (foeger et al., , 2003 . comparison of the 2a pro and l pro exosites on each protease reveal no obvious similarity, despite the fact they interact with the same general region of eif4gi. phylogenetic analysis of key residues in the hrv2 2a pro exosite shows a lack of conservation among pv and other enteroviruses in this region, suggesting that other viral 2a pro molecules may not share this feature (foeger et al., 2003) . thus, fmdv has evolved a separate l-proteinase in place of 2a pro to cleave eif4g, affirming the importance of this translation control strategy for the virus. however, it is important to recognize that not all picornaviruses encode proteinases that cleave eif4g, such as the cardioviruses encephalomycarditis virus (emcv) and mengovirus. the emcv 2a gene, which is not a proteinase, is localized to nucleoli during infection and may instead interfere with or alter ribosome assembly and transport to indirectly regulate translation. (aminev et al., 2003a,b) . recently several reports have described cleavage of eif4g by proteases of other viruses. human immunodeficiency virus-1 (hiv-1) infection of cells resulted in partial cleavages of eif4gi that was mapped to three sites in two regions on either side of the eif3-binding domain ( fig. 1) (ohlmann et al., 2002; ventoso et al., 2001) . similarly, proteases of hiv-2, human tcell leukemia virus (htlv-1), simian immunodeficiency virus (siv), moloney murine leukemia virus and mouse mammary tumor virus, also caused partial cleavage of eif4gi (alvarez et al., 2003) . cleavage of eif4gi at the downstream site inhibits de novo initiation of both capped and ires-driven mrnas in reticulocyte lysate assays (ohlmann et al., 2002) . translation assays based on luciferase reporter constructs in cells indicated that expression of hiv protease (hiv pr) primarily inhibited translation of capped mrnas. interestingly, comparison of translation function of eif4gi c-terminal cleavage products produced by l pro and hiv pr revealed that the slightly shorter hiv pr-derived fragment was defective in supporting translation of the pv-ires but not the emcv ires. this 40-amino acid segment of eif4g binds rna and was suggested to be critical for scanning (prevot et al., 2003) . hiv mrnas are capped, so why would a virus that expresses capped mrnas encode a function that cleaves eif4g and represses cap-dependent translation and scanning? similar to picornaviruses, several retroviruses, including hiv and siv, contain ires elements in the leader or gag gene that may help them escape this translation restriction or maintain expression in quiescent cells or during mitosis (brasey et al., 2003; buck et al., 2001; ohlmann et al., 2000) . hiv proteinase stimulated translation of hiv mrna in vitro (ventoso et al., 2001) and cleavage of eif4g by l pro activated the hiv leader ires (ohlmann et al., 2000) . interestingly, eif4gii is not cleaved by hiv pr (alvarez et al., 2003; ohlmann et al., 2002) . thus, the combined weak and late cleavage of eif4gi and lack of eif4gii cleavage insures that significant levels of intact eif4f will always be present to support cap-dependent translation during hiv infection. accordingly, hiv may be seen to modulate cap-dependent translation, though probably not aggressively shutoff translation like picornaviruses. although fmdv l pro is the most active eif4g-protease described, an alternate secondary cleavage of eif4gi, along with partial cleavage of eif4a occurs in infected cells late in infection in bhk cells . these cleavage events were shown to correlate with the shutdown of viral translation more than host translation and were induced by 3c pro instead of l pro . similar secondary cleavage does not occur with human eif4gi due to an amino acid change at the active site. the fmdv 3c pro cleavage site on eif4g was mapped 39 amino acids downstream of the fmdv l pro cleavage site (fig. 1) . the functional consequences of further cleavage of eif4gi are unclear, however, cleavage of eif4a, though partial, may produce a dominant negative mutant that blocks functions required for fmdv ires activity pause et al., 1994) . 2a pro -mediated cleavage of eif4g separates the nh2terminal eif4e-binding domain (mrna binding) and coohterminal eif3-binding domain (ribosome binding) of eif4g (lamphear et al., 1995) . this finding formed the basis for the attractive "eif4g cleavage model" for the mechanism of pvinduced shutoff of cap-dependent translation. eif4f complexes that are cleaved by 2a pro are not able to recruit ribosomes to capped mrnas liebig et al., 1993) . this hypothesis was influenced by early reports that transient expression of 2a pro alone in cells was sufficient to cause eif4g cleavage and translation inhibition in cells (aldabe et al., 1995; davies et al., 1991) and a drastic 50-fold decrease in translation rate of a reporter gene (sun and baltimore, 1989) , a finding that has not been repeated by others. more typically, expression of 2a pro in cells has been associated with less drastic effects on translation (e.g. 2-3 fold) and proposed to lead to apoptosis which also leads to translation inhibition via a variety of alternate mechanisms (aldabe et al., 1995; barco et al., 2000; tee and proud, 2002; zhao et al., 2003) . recently, the 2a pro cleavage site was mutated on eif4gi and cells ectopically expressing cleavage-resistant eif4g were able to partly restore translation inhibition from expressed 2a pro (zhao et al., 2003) . these data provide strong evidence that eif4gi cleavage is an important aspect of the shutoff mechanism, but do not indicate that other factors and events are not also required. many findings suggest that a revision of this eif4gi cleavage model for shutoff is in order. aaron shatkin was first to point out that the kinetics of eif4g cleavage in infected hela cells did not correlate closely with the onset of host cell shutoff during infection (shatkin, 1985) . instead, cleavage of eif4gi precedes translation shutoff by 30 or more minutes. several groups have shown that poliovirus infections carried out in the presence of inhibitors of viral rna replication (e.g. 2 mm guanidine-hcl, quercitin, monensin) result in complete cleavage of eif4gi, yet cap-dependent translation is only partly inhibited, usually by about 50% (bonneau and sonenberg, 1987; bovee et al., 1998b; irurzun et al., 1995; pã©rez and carrasco, 1992) . thus, some fundamental event was missing from the shutoff models that did not occur in the presence of guanidine. further, the cleaved c-terminal fragment of eif4gi can still support cap-dependent translation initiation, although with a limited efficiency (ali et al., 2001) . these results demonstrate that cleavage of eif4gi alone is not sufficient to completely block host cap-dependent translation in vivo. what could be the missing part(s) of the translation shutoff model? it is important to note that many biochemical experiments that tested the function of cleaved eif4g used conditions where only de novo translation was measured and there was less opportunity to measure the fate of translating polysomes, e.g. ribosomes that may be able to recycle. initially, gradi et al. demonstrated that eif4gii is more resistant to 2a pro cleavage during poliovirus infection than eif4gi, particularly in infections containing guanidine. similar correlations were observed in rhinovirus-infected cells (svitkin et al., 1999) . thus, earlier results in which eif4gi was found to be cleaved but translation continued at 40-50% levels was proposed to result from incomplete cleavage of eif4gii, retaining enough functional eif4f in the cell to sustain cap-dependent translation. this seems reasonable at first, however, eif4gii comprises only a small portion, approximately 10%, of the total cellular eif4g. it is unclear if a portion of uncleaved eif4gii, can support such high translation rates (50% of normal) in guanidine-pv-infected cells. however, the cleavage of both eif4gi and eif4gii would likely be required for complete translation shutoff to occur. what else could have been missing from the shutoff model? alternatively, it has been shown that pabp is also cleaved in pv-and cvb3-infected cells (joachims et al., 1999; kerekatte et al., 1999) . importantly, since cleavage of pabp is blocked in pv infections carried out with 2 mm guanidine-hcl, pabp cleavage also may be an important "missing event" in the model of translation shutoff. in infected cells the cleavage of pabp is substantial, but is not complete at times when the host translation shutoff is maximal. for instance, host translation is effectively shutoff by 3 h post-infection in pv-infected cells yet only 35% of pabp is cleaved at this timepoint. however, pabp cleavage typically progresses to 60-70% by 6 h post-infection (joachims et al., 1999; kuyumcu-martinez et al., 2002) . purified pabp can be directly cleaved in vitro with purified 2a pro and a single cleavage site for both pv and cvb3 2a pro was mapped in the c-terminal proline-rich region of pabp, splitting the m487-g488 peptide bond (joachims et al., 1999; kerekatte et al., 1999) . this cleavage separates the four n-terminal rnarecognition motifs (rrms) from the ctd and homodimerization domains (fig. 2) . pabp cleavage correlated with translation inhibition in infected hela cells and in some instances pabp cleavage correlated better than eif4g cleavage (kerekatte et al., 1999) since it is less abrupt. both initial reports focused on 2a pro as the mediator of pabp cleavage partly because expression of 2a pro alone was thought to be sufficient for translation inhibition (aldabe et al., 1995; davies et al., 1991; sun and baltimore, 1989) . however, all picornaviruses contain another proteinase, 3c pro , that performs most of the processing of viral polyproteins. new experimental results have established a significant role for 3c proteinase in translation regulation. in fact, most pabp in pv-infected cells is processed by 3c pro , not 2a pro . further mapping studies and examination of infected hela cell extracts with better pabp antibodies revealed that pv 3c pro can cleave pabp at three sites that flank the 2a pro cleavage site. these sites were mapped to q/t413, q/g438 and q/g538 (fig. 2) (kuyumcu-martinez et al., 2002) . thus, cleavage of pabp by 3c pro at any site and 2a pro at its single site, all result in separation of the rrms from the c-terminal peptide interaction domain (fig. 2) . the fact that pabp cleavage correlated with significant inhibition of translation, yet only a subset of cellular pabp was processed in cells, suggested that compartmentalization or alternate conformation may regulate pabp cleavage. indeed, pabp fractionates into several subcellular compartments. about a third of hela cell pabp was found in a soluble compartment that was not associated with other initiation factors or polysomes. this pabp fraction was very refractory to cleavage with either 2a pro or 3c pro (kuyumcu-martinez et al., 2002) . in contrast, pabp in ribosome-enriched fractions was preferentially cleaved in vitro and in vivo compared to pabp in other fractions. an interesting proteinase-selectivity toward certain pabp pools was noted. 3c pro preferentially processes most of the pabp in polysome fractions, not 2a pro . in addition, binding of pabp to poly(a) rna stimulated 3c pro -mediated cleavage and inhibited 2a pro -mediated cleavage (kuyumcu-martinez et al., 2002) . these findings provide evidence that pabp conformational changes or association with other factors in translation complexes activates pabp cleavage. the molecular details of the complexes and specific factors that modulate proteinase cleavage have not been identified. caliciviruses are single-stranded rna viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. like picornaviruses, calicivirus rna contains an 5 -linked vpg protein and a 3 poly(a) tail, however, there is only a very short 4-5 nucleotide 5 leader region and currently no evidence for an ires. calicivirus vpg is much larger (â�¼15 kda) than picornavirus vpg (â�¼2-5 kda) and has been shown to bind directly to eif3 (daughenbaugh et al., 2003) . this novel interaction may play a positive selective role in translation initiation by recruiting the 40s ribosomal subunit preferentially to calicivirus rna. further, calicivirus vpg itself contains sequence homology to translation factor eif1a, indicating that it potentially may complete with cellular host factors for ribosome binding (sosnovtseva et al., 1999) . eif1a is a small, highly conserved factor that functions in binding met-trnai to 40s ribosomes and in mrna binding and scanning (hershey and merrick, 2000) . the overall genomic arrangement of structural and nonstructural proteins of caliciviruses is reversed from picornaviruses, yet small regions of homology exist in non-structural proteins and caliciviruses encode a 3c-like proteinase that processes its orf1 polyprotein. there is no homologous 2a pro in caliciviruses. experiments with human norovirus (nv) or feline calicivirus (fcv) 3c-like proteinases show they do not cleave human or mouse eif4g in in vitro assays (kuyumcu-martinez et al., 2004a) . however, some eif4g cleavage does occur in fcvinfected feline kidney cells. the eif4gi processing occurred late in infection and only partly cleaved eif4gi at the timepoints when host translation shutoff was evident (willcocks et al., 2004) . the eif4gi processing profile was quite different than that generated by pv 2a pro . the identity of the eif4g-proteinase is unknown, though it could be a cellular activity activated by the infection. in contrast to the lack of 2a pro and drastic eif4gi cleavage, pabp is readily targeted by fcv and nv 3c-like proteinases (kuyumcu-martinez et al., 2004a) . interestingly, the nv and fcv proteinases cleave pabp at different sites, however, these sites are identical to the three cleavage sites used by pv 3c pro . thus, caliciviruses also remove the c-terminal domain of pabp that binds eif4b and erf3, establishing a common translation regulation theme between two distinct classes of rna viruses. cleavage of pabp correlated well with shutoff of host translation in fcv-infected cells (kuyumcu-martinez et al., 2004a) . the functional consequences to translation resulting from pabp cleavage during virus infection are only beginning to emerge. the primary effect of cleavage of pabp would be to remove the ctd, and thus separate the binding domains for erf3, eif4b and paip from the mrna/rnp. how would this be expected to affect translation? it is known that pabp-binding to eif4g transduces conformation changes through eif4g that enhance the binding of eif4e to the cap structure (gross et al., 2003) . similarly, in plants pabp-eif4g binding stimulates translation and can transduce changes that increase the binding affinity of eif4e to the cap structure (borman et al., 2000; luo and goss, 2001; wei et al., 1998) . however, recombinant fragments of pabp and eif4g bind tightly and 3c pro cleavage of pabp has little effect on the pabp n-terminal fragment binding to eif4gi in pull-down assays, which occurs via rrm2 (imataka et al., 1998; kuyumcu-martinez et al., 2004a) . further, cleavage of both eif4g and pabp are not expected to interrupt 5 -3 circularization of the mrna (fig. 3b) . since pv 3c pro does not cleave eif4g, the relative impact of pabp cleavage alone on cap-dependent translation can be measured by expression of 3c pro in translation assays. the use of hela cell lysate translation system that is both cap-dependent and poly(a)-tail-dependent has allowed evaluation of the importance of these effects. interestingly, when translation of endogenous hela mrna was measured in this system, cleavage of eif4gi and eif4gii by addition of excess 2a pro resulted in only about a 60% decline in total translation (kuyumcu-martinez et al., 2004b) . a portion of pabp was also cleaved by 2a pro . when 3c pro was added to the same system, over 60% decline in translation was also observed. this suggested that partial cleavage of the 3c pro -sensitive pool of pabp or complete cleavage of both eif4gi and eif4gii were equally effective in blocking translation. however, cleavage of either eif4g or pabp alone was insufficient to shutdown capped translation more than two-three-fold (kuyumcu-martinez et al., 2004b) , significantly less than the drastic inhibition observed in infected cells. further experiments with capped reporter rnas showed that 3c pro specifically inhibited translation of rna containing poly(a) tails and that addition of pabp to hela extracts treated with 3c pro restored translation (kuyumcu-martinez et al., 2004b) . addition of pabp to reticulocyte lysates treated with 2a pro also partly restored translation (kerekatte et al., 1999) . in related work, nv 3c-like proteinases added to in vitro translation systems caused inhibition of capped mrnas in a poly(a)-dependent manner, similar to pv 3c pro (kuyumcu-martinez et al., 2004a) . further, transient expression of 3c pro in hela cells resulted in partial pabp cleavage and similar inhibition of translation (kuyumcu-martinez et al., 2004b) . these findings illustrate the importance of the ctd of pabp in poly(a)-dependent translation in mammalian cells and suggest that blockage of ctd function can impact translation on polysomes to a similar extent as cleavage of eif4g. so how does proteinase cleavage of pabp affect translation? the results of kinetics experiments suggest that inhibition of ctd function by 3c pro might inhibit late steps in translation (kuyumcu-martinez et al., 2004b; uchida et al., 2002) , and it is tantalizing to speculate that the removal of the ctd of pabp may block ribosome recycling. very little is known about ribosome recycling via 5 -3 interactions, however, evidence is accumulating that recycling may account for a substantial proportion of total translation initiation or may compensate for loss of de novo initiation in certain circumstances. it was actually proposed years ago that de novo initiation and recycling (called re-initiation then) were distinct processes and that recycling was cap-independent. this was demonstrated by showing that m7gdp cap analog could effectively block initiation of translation in nucleased lysates in which globin mrna was added back, however, cap analog did not effectively block endogenous globin translation in non-nucleased lysates (asselbergs et al., 1978) . pabp is now recognized as an initiation factor since it participates in several steps in the translation initiation pathway and stimulates formation of 80s ribosomes, possibly by stimulating 60s subunit joining (kahvejian et al., 2005) . the recognition that pabp can influence these steps in de novo translation initiation experiments suggests that pabp could have similar effect in stages of ribosome recycling. it is difficult to distinguish de novo initiation and ribosome recycling experimentally. the c-terminal domain of pabp cleaved away by 3c pro interacts with initiation factor eif4b, paip1, paip2, and interestingly, translation termination factor erf3. since the same binding cleft of pabp interacts with peptides from eif4b and erf3, it suggests that pabp can only bind one factor at a time (kozlov et al., 2001 (kozlov et al., , 2004 . one can speculate that pabp toggles back and forth between eif4b and erf3 in transient reactions that are associated with ribosome recycling. if this is true in hela cells, it predicts that cleavage of eif4g by 2a pro may not block recycling ribosomes, thus 50-60% continued translation in guanidine-pv-infected cells after eif4g cleavage may actually reflect the percentage of total cellular translation that is due to ribosome recycling. some support for this model was suggested by kinetics experiments in vitro that showed cleavage of eif4g after polysomes were fully formed had only a slight inhibitory effect on translation whereas cleavage of eif4gi before polysomes formed strongly blocked initiation (fig. 4) . in contrast, 3c pro was more effective in inhibiting translation after polysomes formed. drastic translation inhibition required both 2a pro and 3c pro . this result provides some evidence for the tantalizing idea that translating ribosomes can recycle on formed polysomes and that this recycling does not require intact eif4g. thus, the biochemical requirements for de novo initiation may be distinct from the requirements for ribosome recycling. finally, it is likely that enteroviruses use a dual strategy for host translation shutoff, requiring cleavage of eif4g by 2a pro to block de novo ribosome initiation and cleavage of pabp by 3c pro to interrupt recycling ribosomes. what effects do viral proteinases have on translation of viral mrna? during infection the virus must first insure that its mrna is efficiently or preferentially translated in cells, thus many schemes to regulate host translation have been described. for picornaviruses it has been well documented that cleavage of eif4g can stimulate ires-dependent translation (hambidge and sarnow, 1992) . only the c-terminal region of eif4g (spanning amino acids 676-1600), is required for ires activity and may bind with other factors to the ires ohlmann et al., 1996; pestova et al., 1996) . 5 -3 interactions have been reported to stimulate pv-ires-mediated translation (bergamini et al., 2000) . however, cleavage of eif4g may inter-rupt this type of translation stimulation , even though overall ires translation is upregulated by eif4g cleavage. in contrast to mechanisms to stimulate virus translation, plusstrand rna viruses such as pv must also interrupt translation of the viral rna genome at some point in the infection to allow the rna replicase to utilize the genomic mrna as a template. viral polysomes must be cleared of ribosomes before rna replication can occur. for pv it is likely that 3c pro achieves this function by cleaving pabp and some of the itafs that are important for supporting ires-dependent translation. for instance, ptb, la autoantigen and pcbp2 have been shown to play important roles in stimulating pv-ires-mediated translation and may function as rna chaperones, stabilizing the ires in translationally active tertiary configurations (costa-mattioli et al., 2004; hellen et al., 1993; pilipenko et al., 2001) . both la and ptb are cleaved by 3c pro in infected cells but result in opposing effects on pv-ires function (back et al., 2002; shiroki et al., 1999) . both itafs are only partly cleaved during infection, similar to pabp. ptb is expressed as three isoforms (isoforms 1, 2, 4) and predominately isoforms 2 and 4 are cleaved. ptb cleavage was associated with a two-fold loss of itaf function in translation assays with luciferase reporter rnas in vivo (back et al., 2002) . in contrast, la autoantigen was found to be processed near its c-terminus by 3c pro , removing a domain containing a nuclear localization signal. this truncated form of la correlated with stimulated pv-ires translation, and 3c pro cleavage was proposed to result from an increased concentration of la in the cytoplasm to support viral translation (shiroki et al., 1999) . in contrast, pcbp2 is cleaved in pv infection into a form that no longer supports pv-ires-mediated translation (personal communication, semler). it will be interesting to determine if there is similar truncation of unr. because itaf cleavage generally inhibits viral translation, it is not surprising these cleavages are observed late during infection cycles. the relative importance of these itafs in supporting virus translation, and hence the relative effect of their cleavage has yet to be determined. viruses utilize some other mechanisms to regulate host translation that are indirect. theoretically, any attack on transcription, mrna processing and export or increase in mrna turnover may indirectly inhibit translation. human enteroviruses downregulate transcription within 2 h of infection, thus reducing the flow of mrna to the cytoplasm and upsetting homeostasis. dasgupta and colleagues showed that poliovirus 3c pro was able to inactivate pol 1, pol ii and pol iii transcription in hela cells (clark and dasgupta, 1990; clark et al., 1991; das and dasgupta, 1993; kliewer et al., 1990; rubinstein et al., 1992; yalamanchili et al., 1997a yalamanchili et al., ,b, 1996 . in the case of rhinoviruses and pv, 3cd proteases are localized to the nucleus and concentrated in the nucleolus via a localization signal in the n-terminal domain of the 3dpolymerase domain of 3cd (aminev et al., 2003a,b; amineva et al., 2004; sharma et al., 2004) . 3cd is the precursor for 3c pro and 3dpolymerase but its protease domain is very active in this precursor form and has a slightly altered substrate specificity from fully processed 3c pro . while proteases of many picornaviruses and caliciviruses are known to cleave several translation factors, what of other rna viruses? essentially all plus-strand rna viruses encode proteinases for polyprotein processing and potentially any of these could interact with translation factors and regulate translation, yet cleavage of translation factors has not been reported for other virus families such as coronaviruses, flaviviruses and pestiviruses. interestingly, not all picornavirus infections result in eif4gi cleavage. hepatitis a virus does not encode a 2a protease and its ires requires intact eif4gi for translation function . similarly, echovirus 22 does not cleave eif4gi during infection (coller et al., 1991) . infection of hela cells with sindbis virus, an alphavirus, also do not result in eif4gi cleavage (lloyd, unpublished data). although much is known about how picornaviruses control translation via their proteinases, much remains to be elucidated. by discovering many of the cellular substrates of viral proteinases we have learned a great deal about the mechanism and regulation of the translation process. manipulation of viral proteinases in kinetics experiments has provided new clues that ribosomes may indeed recycle. in turn, the old paradigm that eif4gi cleavage is the essential event for translation shutoff in pv-infected cells must be modified to account for new data. a model is emerging that many rna viruses that encode ires elements may also manipulate cap-dependent translation via eif4g cleavage, though cleavage may occur to various extents. also, a new paradigm has emerged that pabp cleavage may be used by many viruses to manipulate poly(a)-dependent translation. since most plus-strand rna viruses have poly(a) tails on their genomes and mrnas, pabp cleavage would have the dual effect of inhibiting both cellular translation and viral translation. these viruses must eventually block translation on their genomes to allow rna replication. one important aspect of this model yet to be discovered is how the virus regulates cleavage of pabp or other factors so that viral translation is promoted just long enough to produce ample replicase proteins and then shutoff translation. the relatively weak cleavage of pabp and itafs by 3c pro (compared to eif4g cleavage) combined with local concentration effects from the accumulation of protease and replicase proteins at the microenvironment of viral polysomes may accomplish this regulation. further, viruses that cleave pabp would also be expected to have other mechanisms to promote viral translation over cellular translation. this is represented by eif4g cleavage (pv) or specific binding of vpg-rna to eif3 (calicivirus). finally, translation is a highly dynamic process that has evolved to regulate what, where and when proteins are synthesized. work with 2a proteinase did much to uncover mechanistic steps in de novo initiation process on the 5 end of mrna. now, the 3 end of mrna has also turned out to be surprisingly important in regulating translation. important future work with viral proteinases that inactivate specific translation functions will hopefully elucidate more secrets about how the recycling of ribosomes for multiple rounds of translation on the same mrna may occur and be regulated. competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle expression of poliovirus 2a pro in mammalian cells: effects on translation truncated initiation factor eif4g lacking an eif4e binding site can support capped mrna translation the eukaryotic translation initiation factor 4gi is cleaved by different retroviral proteases encephalomyocarditis viral protein 2a localizes to nucleoli and inhibits cap-dependent mrna translation encephalomyocarditis virus (emcv) proteins 2a and 3bcd localize to nuclei and inhibit cellular mrna transcription but not rrna transcription rhinovirus 3c protease precursors 3cd and 3cd' localize to the nuclei of infected cells diminished sensitivity of re-initiation of translation to inhibition by cap analogues in reticulocyte lysates translation of polioviral mrna is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3c(pro) the role of ribosomal subunits in mammalian cells a stable hela cell line that inducibly expresses poliovirus 2a pro : effects on cellular and viral gene expression multimerization of poly(rc) binding protein 2 is required for translation initiation mediated by a viral ires the major apoptotic pathway activated and suppressed by poliovirus foot-and-mouth disease virus 3c protease induces cleavage of translation initiation factors eif4a and eif4g within infected cells picornavirus ireses and the poly(a) tail jointly promote cap-independent translation in a mammalian cell-free system poly(rc) binding protein 2 binds to stem-loop iv of the poliovirus rna 5 noncoding region: identification by automated liquid chromatography-tandem mass spectrometry requirement of poly(rc) binding protein 2 for translation of poliovirus rna proteolysis of the p220 component of the cap-binding protein complex is not sufficient for complete inhibition of host cell protein synthesis after poliovirus infection intact eukaryotic initiation factor 4g is required for hepatitis a virus internal initiation of translation elf4g and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and ires-containing mrnas biochemical characterisation of cap-poly(a) synergy in rabbit reticulocyte lysates: the eif4g-pabp interaction increases the functional affinity of eif4e for the capped mrna 5 -end unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus direct cleavage of eif4g by poliovirus 2a protease is inefficient in vitro the predominant eif4g-specific cleavage activity in poliovirus-infected hela cells is distinct from 2a protease the leader of human immunodeficiency virus type 1 genomic rna harbors an internal ribosome entry segment that is active during the g2/m phase of the cell cycle the human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site disruption of the interaction of mammalian protein synthesis initiation factor 4b with the poly(a) binding protein by caspase-and viral protease-mediated cleavages generation of multiple isoforms of eukaryotic translation initiation factor 4gi by use of alternate translation initiation codons translation of eif4gi proceeds from multiple mrnas containing a novel cap-dependent ires that is active during poliovirus infection caspase activation and specific cleavage of substrates after coxsackievirus b3-induced cytopathic effect in hela cells unr, a new partner of poly(a)-binding protein, plays a key role in translationally coupled mrna turnover mediated by the c-fos major coding-region determinant a transcriptionally active form of tfiiic is modified in poliovirus-infected hela cells poliovirus proteinase 3c converts an active form of transcription factor iiic to an inactive form: a mechanism for inhibition of host cell polymerase iii transcription by poliovirus cap-binding complex protein-p220 is not cleaved during echovirus-22 replication in hela cells eukaryotic mrna decapping la autoantigen is necessary for optimal function of the poliovirus and hepatitis c virus internal ribosome entry site in vivo and in vitro identification of the cleavage site and determinants required for poliovirus 3cpro-catalyzed cleavage of human tatabinding transcription factor tbp the genome-linked protein vpg of the norwalk virus binds eif3, suggesting its role in translation initiation complex recruitment the effect of poliovirus proteinase 2a pro expression on cellular metabolism protein synthesis eukaryotic initiation factors 4a and 4b are not altered by poliovirus infection of hela cells demonstration in vitro that eucaryotic initiation factor 3 is active but a cap-binding protein complex is inactive in poliovirus-infected hela cells inhibition of hela cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eukaryotic initiation factor 3 and a cap binding protein complex recognition of eukaryotic initiation factor 4g isoforms by picornaviral proteinases human rhinovirus 2 2apro recognition of eukaryotic initiation factor 4gi involvement of an exosite molecular mechanisms of translational control eif4 initiation factors: effectors of mrna recruitment to ribosomes and regulators of translation extremely efficient cleavage of eif4g by picornaviral proteinases l and 2a in vitro cleavage of eukaryotic translation initiation factor 4gii within foot-and-mouth disease virus-infected cells: identification of the l-protease cleavage site in vitro a novel functional human eukaryotic translation initiation factor 4g proteolysis of human eukaryotic translation initiation factor eif4gii, but not eif4gi, coincides with the shutoff of host protein synthesis after poliovirus infection human rhinovirus 2a proteinase cleavage sites in eukaryotic initiation factors (eif) 4gi and eif4gii are different rnastimulated atpase activity of eukaryotic initiation factors ribosome loading onto the mrna cap is driven by conformational coupling between eif4g and eif4e the eif4g-eif4e complex is the target for direct cleavage by the rhinovirus 2a proteinase translational enhancement of the poliovirus 5 noncoding region mediated by virus-encoded polypeptide-2a a cytoplasmic 57-kda protein that is required for translation of picornavirus rna by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein pathway and mechanisms of initiation of protein synthesis novel function of the eukaryotic polypeptide-chain releasing factor 3 (erf3/gspt) in the mrna degradation pathway unr, a cellular cytoplasmic rna-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus rna a newly identified n-terminal amino acid sequence of human eif4g binds poly(a)-binding protein and functions in poly(a)-dependent translation a new translational regulator with homology to eukaryotic translation initiation factor 4g monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage regulation of mrna decay: decapping goes solo cleavage of poly(a)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro the mrna closed-loop model: the function of pabp and pabp-interacting proteins in mrna translation mammalian poly(a)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms cleavage of poly(a)-binding protein by coxsackievirus 2a protease in vitro and in vivo: another mechanism for host protein synthesis shutoff? translational repression by a novel partner of human poly(a) binding protein, paip2 foot-and-mouth disease virus leader proteinase: purification of the lb form and determination of its cleavage site on eif-4 gamma loss of a phosphorylated form of transcription factor creb/atf in poliovirus-infected cells structural basis of ligand recognition by pabc, a highly specific peptide-binding domain found in poly(a)-binding protein and a hect ubiquitin ligase structure and function of the c-terminal pabc domain of human poly(a)-binding protein calicivirus 3c-like proteinase inhibits cellular translation by cleavage of poly(a)-binding protein efficient cleavage of ribosome-associated poly(a)-binding protein by enterovirus 3c protease cleavage of poly(a)-binding protein by poliovirus 3c protease inhibits host cell translation: a novel mechanism for host translation shutoff mapping of functional domains in eukaryotic protein synthesis initiation factor 4g (eif4g) with picornaviral proteases -implications for cap-dependent and cap-independent translational initiation mapping the cleavage site in protein synthesis initiation factor-eif-4g of the 2a proteases from human coxsackievirus and rhinovirus isolation and structural characterization of cap-binding proteins from poliovirus-infected hela cells purification of two picornaviral 2a proteinases -interaction with eif-4g and influence on in vitro translation homeostasis in mrna initiation: wheat germ poly(a)-binding protein lowers the activation energy barrier to initiation complex formation capdependent translation initiation in eukaryotes is regulated by a molecular mimic of eif4g eukaryotic translation initiation factor 4g is targeted for proteolytic cleavage by caspase 3 during inhibition of translation in apoptotic cells a cellular protein that binds to the 5 -noncoding region of poliovirus rna: implications for internal translation initiation la autoantigen enhances and corrects aberrant translation of poliovirus rna in reticulocyte lysate cap-dependent and cap-independent translation in eukaryotic systems messenger rna turnover in eukaryotes: pathways and enzymes messenger rna poly(a) tail, a 3 enhancer of translational initiation an internal ribosome entry segment promotes translation of the simian immunodeficiency virus genomic rna in vitro cleavage of eif4gi but not eif4gii by hiv-1 protease and its effects on translation in the rabbit reticulocyte lysate system the c-terminal domain of eukaryotic protein synthesis initiation factor (eif) 4g is sufficient to support cap-independent translation in the absence of eif4e dominant negative mutants of mammalian translation initiation factor eif-4a define a critical role for eif-4f in cap-dependent and cap-independent initiation of translation functional dissection of eukaryotic initiation factor 4f: the 4a subunit and the central domain of the 4g subunit are sufficient to mediate internal entry of 43s preinitiation complexes haemoglobin synthesis and polysomes in intact reticulocytes a cell cycle-dependent protein serves as a template-specific translation initiation factor cell-specific proteins regulate viral rna translation and virus-induced disease dual function of the messenger rna cap structure in poly(a)-tail-promoted translation in yeast starting the protein synthesis machine: eukaryotic translation initiation poly(a)-tail-promoted translation in yeast: implications for translational control characterization of a novel rna-binding region of eif4gi critical for ribosomal scanning lack of direct correlation between p220 cleavage and the shut-off of host translation after poliovirus infection atp-dependent unwinding of messenger rna structure by eukaryotic initiation factors caspases are not involved in the cleavage of translation initiation factor eif4gi during picornavirus infection eif4a: the godfather of the dead box helicases variability in apoptotic response to poliovirus infection paip1 interacts with poly(a) binding protein through two independent binding motifs bidirectional rna helicase activity of eucaryotic translation initiation factors 4a and 4f infection of hela cells with poliovirus results in modification of a complex that binds to the rrna promoter the poly(a)-binding protein is required for poly(a) shortening and 60s ribosomal subunit dependent translation initiation eukaryotic translation initiation: there are (at least) two sides to every story translation initiation and viral tricks nuclear entry of poliovirus protease-polymerase precursor 3cd: implications for host cell transcription shut-off mrna cap binding proteins: essential factors for initiating translation intracellular redistribution of truncated la protein produced by poliovirus 3cpro-mediated cleavage 2a proteinases of coxsackie and rhinovirus cleave peptides derived from eif-4g via a common recognition motif mapping of the feline calicivirus proteinase responsible for autocatalytic processing of the nonstructural polyprotein and identification of a stable proteinase-polymerase precursor protein human immunodeficiency virus tat-activated expression of poliovirus protein 2a inhibits mrna translation eukaryotic initiation factor 4gii (eif4gii), but not eif4gi, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection poly(a)-binding protein interaction with eif4g stimulates picornavirus ires-dependent translation caspase cleavage of initiation factor 4e-binding protein 1 yields a dominant inhibitor of cap-dependent translation and reveals a novel regulatory motif a novel role of the mammalian gspt/erf3 associating with poly(a)-binding protein in cap/poly(a)-dependent translation hiv-1 protease cleaves eukaryotic initiation factor 4g and inhibits cap-dependent translation wheat germ poly(a) binding protein enhances the binding affinity of eukaryotic initiation factor 4f and (iso)4f for cap analogues circularization of mrna by eukaryotic translation initiation factors cleavage of eukaryotic initiation factor eif4g and inhibition of host-cell protein synthesis during feline calicivirus infection inhibition of host cell transcription by poliovirus: cleavage of transcription factor creb by poliovirus-encoded protease 3cpro inhibition of basal transcription by poliovirus: a virus-encoded protease (3cpro) inhibits formation of tbp-tata box complex in vitro cleavage of transcriptional activator oct-1 by poliovirus encoded protease 3cpro multiple eif4gi-specific protease activities present in uninfected and poliovirus-infected cells protection of cap-dependent protein synthesis in vivo and in vitro with an eif4g-1 variant highly resistant to cleavage by coxsackievirus 2a protease this work was supported by nih grant ai50237. the author would like to thank dr. kyle sherrill for critical review. key: cord-290481-i2ppvsh5 authors: dolja, valerian v.; kreuze, jan f.; valkonen, jari p.t. title: comparative and functional genomics of closteroviruses date: 2006-03-09 journal: virus res doi: 10.1016/j.virusres.2006.02.002 sha: doc_id: 290481 cord_uid: i2ppvsh5 the largest extant rna genomes are found in two diverse families of positive-strand rna viruses, the animal coronaviridae and the plant closteroviridae. comparative analysis of the viruses from the latter family reveals three levels of gene conservation. the most conserved gene module defines rna replication and is shared with plant and animal viruses in the alphavirus-like superfamily. a module of five genes that function in particle assembly and transport is a hallmark of the family closteroviridae and was likely present in the ancestor of all three closterovirus genera. this module includes a homologue of hsp70 molecular chaperones and three diverged copies of the capsid protein gene. the remaining genes show dramatic variation in their numbers, functions, and origins among closteroviruses within and between the genera. proteins encoded by these genes include suppressors of rna silencing, rnase iii, papain-like proteases, the alkb domain implicated in rna repair, zn-ribbon-containing protein, and a variety of proteins with no detectable homologues in the current databases. the evolutionary processes that have shaped the complex and fluid genomes of the large rna viruses might be similar to those that have been involved in evolution of genomic complexity in other divisions of life. with their 20-30 kb genomes, the largest known rna viruses are no match for the champions of the dna virus world whose genomes reach 1.2 mb (iyer et al., 2006) , let alone cellular life forms. nevertheless, rna viruses play a prominent role in our understanding of life's origin and evolution. because rna is widely believed to predate dna as the genetic material, rna viruses could be living fossils of the primordial rna world (joyce, 2002; koonin and martin, 2005) . therefore, at least some genes of rna viruses are likely to encode extremely ancient, perhaps, primeval proteins involved in replication and metabolism of nucleic acids. from a somewhat different, comparative-genomic perspective, large rna viruses provide an opportunity to investigate problems of genome complexity and its evolution on a relatively modest, tractable scale. closteroviruses share a conserved core of genes involved in replication with other animal and plant viruses within the * corresponding author. tel.: +1 541 737 5472; fax: +1 541 737 3573. e-mail address: doljav@science.oregonstate.edu (v.v. dolja). alphavirus-like superfamily of positive-strand rna viruses (dolja et al., 1994) . however, closteroviruses stand alone in regard to their genetic capacity and variability, as well as the unique morphology of their particles. it is instructive to compare the genera closterovirus and tobamovirus that both belong to the alphavirus-like superfamily and share the helical virion architecture (fig. 1 ). all tobamoviruses have ∼6.5 kb genomes that code for four proteins, one of which assembles to form the rod-shaped virion (fig. 1b and c) . in contrast, the size of closterovirus genomes varies from ∼15.5 to ∼19.5 kb with a coding capacity of 10-14 proteins (figs. 1a, b and 2). filamentous virions of closteroviruses incorporate at least five proteins that are assembled into a long body of uniform morphology and a short segmented tail (fig. 1c) . thus, the larger amount of genetic material in closteroviruses translates into the increased structural complexity and genetic variation among individual viruses. here we explore the gene repertoire of the family closteroviridae in an attempt to reconstruct the evolutionary history of these viruses. we also use the available information on the gene functions and regulation to propose a working model of the infection cycle for a 'generic' closterovirus. met, hel, and pol, methyltransferase, rna helicase, and rna-dependent rna polymerase domains of the replicase, respectively; p6, a 6-kda protein; hsp70h, a hsp70-homologue; p64, a 64-kda protein; cpm and cp, the minor and major capsid proteins, respectively; p20 and p21, the 20 and 21-kda proteins, respectively. the sequence similarity between cp, cpm, and the c-terminal domain of p64 is illustrated with the same color. the protein functions are indicated above and below the diagram, while the evolutionary conservation patterns are shown at the bottom. (b) genome map, functions, and evolutionary connections of tmv. p30, a 30-kda protein. other designations are the same as in (a). (c) cartoons of the tmv and byv virions. the tmv virions are rigid helical rods of ∼300 nm in length and 18 nm in diameter. the byv virions are flexuous, helical filaments of ∼1400 nm in length and 12 nm in diameter with the ∼100 nm-long tails that are ∼8 nm in diameter. the protein composition and the length of the encapsidated viral rna is shown. as is the case for all positive-strand rna viruses, putative components of the closteroviral rna replicase are expressed directly from the virion rna (karasev et al., 1989) . the product of the 5 -terminal open reading frame (orf) contains rna methyltransferase (met) and rna helicase (hel) domains. the rna-dependent rna polymerase (pol) is encoded by a downstream orf that is presumably expressed via a +1 translational frameshift (agranovsky et al., 1994; . thus, translation of the genomic rna results in two large polyproteins, one spanning met-hel, and the other one encompassing met-hel-pol; this second, larger polyprotein is produced in much smaller quantities due to the low frequency of frameshifting. the met-hel-pol module of closteroviruses is universally conserved in the entire alphavirus-like superfamily ( fig. 1a and b) (koonin and dolja, 1993) . a peculiar feature of the closteroviral replicases is the presence of a large variable region between the met and hel domains. this region is processed by an unknown mechanism to produce separate met-and hel-containing products (erokhina et al., 2000) . because ectopic expression of the met-hel region in plants is sufficient to generate these separate products, the processing could involve either a cryptic proteolytic activity within the met-hel polyprotein itself, or a cellular protease (peremyslov and dolja, unpublished data) . in addition to their enzymatic functions, rna replicases of alphavirus-like viruses direct assembly of the replication compartments. the principal component of these compartments is a protein shell formed by the met-hel protein subunits (schwartz et al., 2002) . this shell is enveloped by a membrane derived from the endoplasmic reticulum (er) or other endomembrane. in accord with this paradigm, we found that closterovirus infection results in a drastic remodeling of the er network that is transformed into a large vesicular factory of viral rna. we also found that transient expression of the met-hel polyprotein alone is sufficient to restructure er (prokhnevsky et al., unpublished data) . as proposed by schwartz et al. (2002) , one or a few pol-containing replication complexes are present within each virus-induced vesicle to amplify the viral genome and to produce subgenomic messenger rna (sgrnas). (agranovsky et al., 1994) ; ctv, citrus tristeza virus ; liyv, lettuce infectious yellows virus (klaassen et al., 1995) ; spcsv, sweet potato chlorotic stunt virus (kreuze et al., 2002) ; glrav-3, grapevine leafroll-associated virus-3 (ling et al., 2004) . the designations and color code for the conserved proteins are the same as in fig. 1a . l1 and l2, tandem of the leader proteinases in ctv. the unique proteins in each genome are shown in colors as dissimilar as possible with their approximate molecular weight following common 'p' designator, except for rnase iii and alkb domain (see the text for further information). the extreme 5 -terminal region of the closterovirus rna encodes the papain-like leader proteinase (l-pro) that is released from the polyprotein via autoprocessing (agranovsky et al., 1994) . even though papain-like proteinases are common among alphavirus-like viruses, their typical role in the viral life cycle is distinct from that of l-pro. the papain-like proteases of alphaviruses, rubiviruses, or tymoviruses are located between the replicase domains and are responsible for multiple processing events within the polyprotein (dougherty and semler, 1993) . in contrast, the closterovirus l-pro is located upstream of the replicase polyprotein and is not required for its processing (see above). in addition to autocatalytic processing, l-pro plays a prominent role in the amplification of the viral genome: elimination of l-pro reduces the amount of viral rna to ∼0.1% of the wildtype level (peng and dolja, 2000) . because l-pro co-localizes with the replication vesicles (zinovkin et al., 2003) , its function in rna amplification could involve either activation of the viral replicase or protection of the rna from degradation by a host defense system. some closteroviruses possess a tandem of the leader proteinases that probably evolved via gene duplication. as was found using the gene swapping approach, one of these tandem proteinases (l1), but not the other (l2), can functionally substitute for a single l-pro by enhancing virus rna amplification (peng et al., 2001) . when expressed together, l1 and l2 act synergistically to provide for even higher levels of viral rna. interestingly, proliferation of the papain-like leader proteinases is also observed in some viruses of the order nidovirales that includes the family coronaviridae (gorbalenya et al., 2006) . this evolutionary convergence might reflect common requirements posed by the increase in the size of the viral genomes and encoded polyproteins (peng et al., 2002) . another feature shared by closteroviruses and nidoviruses is expression of the multiple internal genes via a nested set of the 3coterminal subgenomic (sg) rnas. usually, these sgrnas are functionally monocistronic, each expressing only one protein from the 5 -proximal orf. while many diverse positive-strand viruses produce from one to three subgenomic sgrnas (miller and koev, 2000) , nidoviruses make up to 9, and closteroviruses up to 10 sgrnas. although the details of transcription mechanisms may vary among the viruses of the order nidovirales, they share certain important features (gorbalenya et al., 2006) . the short, au-rich, transcription-regulating sequences (trs) are present upstream from the 5 -terminal and all internal orfs. the trss are thought to mediate termination of the minus-strand synthesis to produce templates for the positive-strand sgrnas. in arteriviruses and coronaviruses, termination at the trs of each internal orf is followed by the template 'jumping' such that transcription complex lands to 5 -proximal trs and copies the part of the 5 -leader region. this discontinuous transcription mode resulting in a mosaic structure of the sgrnas is a unique and remarkable feature of the nidovirus genome expression mechanism (gorbalenya et al., 2006; pasternak et al., 2001; sawicki et al., 2001; zuniga et al., 2004) . the closteroviruses do not employ discontinuous transcription and have no trs-like, common element in the regions that direct sgrnas synthesis (karasev et al., 1997; kreuze et al., 2002; peremyslov and dolja, 2002) . it is not known if, similar to smaller alpha-like viruses, closteroviral sgrna synthesis involves internal initiation using genome-size, negative-strand template (miller and koev, 2000) . in fact, occurrence of the minus strands of sgrnas in closterovirus-infected cells (dolja et al., 1990; hilf et al., 1995) is better compatible with the nidovirus-like, minus-strand termination model. additional support for this model comes from extensive analysis of the ctvspecific rna species. it was found that the controller elements located upstream from each internal ctv orf direct formation of not only the positive and negative strands of sgrnas, but also a set of the 5 -coterminal positive-strand rnas of subgenomic size (gowda et al., 2001) . these latter rnas terminate at variable positions upstream of the initiation site of the corresponding sgrnas. the simplest reconciliation of these data is that the sgrna controller elements, which contain hairpin structures, act as terminators during transcription of both positive, and negative strands of the viral rna. although there were no reports of two distinct sets of subgenomic rnas in nidoviruses, this issue seems to deserve more experimental scrutiny. although the functional significance, if any, of the 5coterminal, subgenomic-size, ctv rnas is unknown, one may wonder if coexistence in the infected cells of the multitude of rna species may promote formation of the recombinant rna molecules. indeed, plants infected with closteroviruses often contain defective rnas (drnas), some of which appear to result from recombination between sgrnas and their counterparts from the 5 -terminal region (che et al., 2002; rubio et al., 2000) . moreover, ctv drnas reminiscent of crinivirus genomic rnas 1 and 2 were described (che et al., 2003) suggesting that the sgrnas may facilitate closterovirus evolution, be it genome segmentation observed in criniviruses (klaassen et al., 1995; liveratos et al., 2004) , gene duplication (boyko et al., 1992; napuli et al., 2003) , or recombinational accretion of the viral or cellular genes kreuze et al., 2002) . even though sgrna controller elements of closteroviruses exhibit little or no sequence conservation within the individual virus genomes or between the related closteroviruses (gowda et al., 2001; kreuze et al., 2002; peremyslov and dolja, 2002) , they likely possess common features of the higher order structures. these elements function efficiently in a heterologous genetic background, i.e., they are recognized by heterologous replicases . the structural variability of the sgrna controllers seems to contribute to the regulation of the level and timing of viral gene expression hilf et al., 1995) . on the more practical side, the promiscuity of the rna replicases toward sgrna controllers facilitates utilization of closteroviruses as versatile gene expression vectors. these vectors can accommodate multiple expression cassettes and efficiently produce reporters or other beneficial pro-teins in the infected plants (peremyslov and dolja, unpublished data) . it seems that the most intriguing questions pertinent to closterovirus rna synthesis are the transcription mechanisms, the enigmatic function of the large, variable central domain of the viral replicase, and the mechanism by which l-pro enhances rna amplification. the answers to these questions will certainly contribute to our understanding of what does it take to replicate and express large infectious rnas. the replication gene block of closteroviruses is rank and file of the alphavirus-like superfamily, some deviations from the prototype notwithstanding. in contrast, the five downstream genes comprise a truly unique genetic module not found outside the family closteroviridae (dolja et al., 1994) . this quintuple gene block (qgb) codes for a ∼6-kda hydrophobic protein (p6), an hsp70 homologue (hsp70h), a ∼60-kda protein (p60), the minor capsid protein (cpm), and the major capsid protein (cp) (fig. 1a ). p6 is a single-span transmembrane protein that resides in er and functions in virus movement from cell to cell (alzhanova et al., 2000; peremyslov et al., 2004b) . because p6 is not required for virus replication or assembly (alzhanova et al., 2001; peremyslov et al., 1998) , it can be considered a conventional movement protein (mp). cp forms a long, helical body of the flexuous, filamentous virions, and encapsidates ∼95% of the viral rna (fig. 1c ). cpm is paralogous to cp and functions as a principal component of the short virion tail (agranovsky et al., 1995; tian et al., 1999) . in addition to cpm, the tail assembly requires hsp70h and p60, each of which is also an integral, although a minor tail component (alzhanova et al., 2001; napuli et al., 2000 peremyslov et al., 2004a; satyanarayana et al., 2000 satyanarayana et al., , 2004 . similar to cellular molecular chaperones of hsp70 family, closteroviral hsp70h possess a highly conserved, n-terminal, atpase domain and a less conserved c-terminal domain bork et al., 1992) . extensive mutation analysis revealed that each of these domains is intimately involved in the virion assembly (alzhanova et al., 2001; prokhnevsky et al., unpublished data) . it was also found that p60 possess the cp-like, virion-embedded, c-terminal domain, and the n-terminal domain, which is exposed at the virion's surface . hsp70h and p60 act cooperatively to facilitate incorporation of cpm and to define the proper tail length (satyanarayana et al., 2004; alzhanova et al., unpublished data) . the tail encapsidates the 5 -terminal, ∼700 nt-long, rna region ( fig. 1c ) that contains a packaging signal recognized by cpm (peremyslov et al., 2004a; satyanarayana et al., 2004) . interestingly, virion bodies and tails can be assembled independently of each other. atomic force microscopy of beet yellows virus (byv, genus closterovirus) virions showed that the tails are narrower than the bodies and exhibit three-segment structure (peremyslov et al., 2004a) . a pointed tip segment of the byv tails contains a 20-kda protein (p20) (fig. 1c ) that is dispensable for incorporation of other virion components. inactivation of p20 results in shorter tails and apparently normal bodies. because the orthologs of byv p20 are not found in other closteroviruses, it is not clear if the tail segmentation pattern is a common feature of the family. a salient functional aspect of the byv virions is that each of the five structural proteins is also required for virus transport (dolja, 2003) . in the case of cp, this requirement could stem, simply, from the need to protect the long and degradationprone viral rna during its transport. indeed, the cell-to-cell movement cycle of a closterovirus takes a day compared to two hours for tmv. however, it seems unlikely that the transportrelated function of the tail is merely protective. first, cp can encapsidate the entire genome if the tail formation is prevented by mutations (alzhanova et al., 2001) . second, the tail harbors hsp70h, the only viral protein that was found in plasmodesmata (pd), intercellular channels through which viruses translocate (medina et al., 1999) . third, p20 is dispensable for virion assembly and cell-to-cell movement, but is required for long-distance transport of byv through the vascular system (prokhnevsky et al., 2002) . all this prompted interpretation of the tail as a device that evolved to facilitate cell-to-cell and systemic transport of the large closterovirus genomes (dolja, 2003) . it should be noted that this concept awaits experimental separation of assembly and transport functions for at least some tail proteins unless there is a causal relationship between the two. even though the qgb genes are universally conserved within the family, their order is not. in the genera ampelovirus and crinivirus, the order of cpm and cp is reversed, and the cpm is much larger than in the genus closterovirus (fig. 2) (klaassen et al., 1995; kreuze et al., 2002; ling et al., 2004; melzer et al., 2001) . other examples of qgb variation include duplication and divergence of the cpm gene in grapevine leafroll-associated virus-1 (fazeli and rezaian, 2000) , reshuffling of qgb in little cherry virus (theilmann et al., 2002) , and occurrence of enigmatic additional orfs within qgb of criniviruses and certain ampeloviruses. it has been long accepted that all non-defective plant viruses must code for at least three functions: genome replication, encapsidation, and transport within infected plants. arguably, suppression of rna silencing has emerged recently as a fourth universal function encoded in plant viruses (baulcombe, 2004) . some plant viruses possess dedicated suppressors, while others delegate this function to replicational, structural, or transport proteins (silhavy and burgyan, 2004; voinett, 2005) . interestingly, most of the dedicated viral suppressors represent small protein families without detectable homologous relationship to any other viral or host proteins. this tendency is often taken as evidence of relatively recent origins of the suppressors that evolved to counteract a powerful system of host defense against parasitic rnas. the major components of rna silencing machinery are conserved between diverse eukaryotes suggesting that this machinery emerged not much later than eukaryotes themselves (anantharaman et al., 2002; zamore and haley, 2005) . search for rna silencing suppressors in closteroviruses yielded sev-eral findings that further highlighted the trends in suppressor evolution. screening of the byv genome for silencing suppressor functions showed that the 21-kda protein (p21) is a suppressor of rna silencing and that the orthologous proteins from other viruses of the genus closterovirus also possess suppressor activity (chiba et al., 2006; reed et al., 2003) . it has been found that p21 specifically binds double-stranded forms of the small interfering rna (sirna) or microrna (mirna) (chapman et al., 2004) . thus, the molecular mechanism of p21 action is similar to that of another well-studied suppressor, p19 of tomato bushy stunt virus (silhavy and burgyan, 2004) : both suppressors sequester small rna effectors of silencing and prevent their loading into the rna-induced silencing complex (risc). however, these suppressors cannot discriminate between sir-nas that target viral genomes and endogenous sirnas and mirnas that are involved in regulation of plant development. as a result, accumulation of p21 or p19 in plants induces developmental abnormalities many of which are identical to symptoms of viral infection (chapman et al., 2004) . it was concluded that, at least in part, viral pathogenicity is due to interference of silencing suppressors with developmental function of plant small rnas. despite their mechanistic similarity, p21 and p19 appear to be structurally and evolutionarily unrelated and neither has detectable homologues outside the respective virus genera (vargason et al., 2003; ye and patel, 2005) . although citrus tristeza virus (ctv) encodes p20, a p21like suppressor of rna silencing, screening of the ctv genome revealed an additional suppressor, p23, that has no homologues in other closteroviruses (fig. 2) (lu et al., 2004) . subsequent computer analysis of the p23 sequence showed that it has a specific form of the zn-ribbon module that is present in a variety of cellular proteins and also in several proteins encoded by viruses of the genera carlavirus and vitivirus (chiba et al., 2006) . it seems likely that the latter viral proteins are distant homologues of p23. should it be demonstrated that these proteins are suppressors of rna silencing, this will be the first viral suppressor family represented in diverse viruses and, possibly, derived from a common host ancestor. it was also found that both ctv p20 and cp can interfere with the systemic spread of silencing, while p23 can only suppress the local silencing (lu et al., 2004) . thus, ctv evolved a complex system of rna silencing suppression with three components targeting distinct facets of rna silencing response. another remarkable discovery in the area of closterovirus counter-defense is a two-component rna silencing suppression system in sweet potato chlorotic stunt virus (spcsv) (kreuze et al., 2005) . one of the components of this system is a 22-kda protein (p22) that possesses suppressor activity on its own (fig. 2) . this protein is unique to spcsv and has no detectable sequence similarity to other proteins. a second component is an rnase iii homologue containing both the endonuclease and the dsrna-binding activities typically found in bona fide cellular rnases iii including dicer and dicer-like proteins, which are involved in the generation and functioning of sirnas and mirnas. in addition to spcsv, rnase iii is encoded by several nucleocytoplasmic large dna viruses (iyer et al., 2006) where its function remains to be determined. by itself, spcsv rnase iii exhibits no suppressor activity. however, co-expression of rnase iii with p22 results in a dramatic increase of rna silencing suppression (kreuze et al., 2005) . although the mechanism of this suppression is not known, recruitment of a protein whose homologues function in eukaryotic rna silencing for the purpose of overcoming host defense is a remarkable example of viral resourcefulness. it seems likely that the analysis of additional closteroviruses will yield more examples of multi-component and multilevel counter-defense systems. what could be the reason for multiplication of diverse suppressors in closteroviruses as opposed to single suppressors of smaller viruses? one possibility is that the large and slowly replicating closterovirus rnas are more prone to degradation by the rna silencing machinery than those of the smaller viruses. alternatively, the increased complexity of the closterovirus genomes could be of direct relevance. given that the antiviral sirnas in infected plants are mostly derived from hairpin elements of the positive rna strands (molnar et al., 2005) , controller element-rich closteroviral rnas are likely to harbor more hairpins than the simpler genomes of other viruses. the closterovirus genomes are peppered with genes whose products, typically, have no identifiable orthologs in other viruses (fig. 2) . except for byv p20, the functions of these enigmatic proteins remain to be elucidated. perhaps, the most illuminating example of this kind is the alkb domain found in several plant viruses (aravind and koonin, 2001) . recent analysis identified alkb domains in the polyproteins of two ampeloviruses, grapevine leafroll-associated virus-3 (fig. 2) and little cherry virus, as well as in ∼20 other alpha-like plant viruses, which share filamentous particle morphology and belong to genera allexivirus, carlavirus, foveavirus, potexvirus, trichovirus, and vitivirus (koonin, personal communication) . in all these cases, alkb domains are located within the multidomain viral replicases upstream of the rna helicase domains. interestingly, in each of these genera, only a minority of the viruses encode alkb domain, suggesting that it has been acquired relatively recently via horizontal gene transfer. the function of alkb in viral infection is unknown, but it seems likely that, similarly to certain cellular alkbs, it might be involved in rna repair via methylation reversal (aas et al., 2003) . the fact that the majority of the alkb-encoding viruses infect either woody or perennial plants is compatible with this hypothesis. indeed, the extent of methylation damage is likely to be exacerbated by the prolonged virus propagation within the same infected organism and the need to be transported across long distances in the hostile phloem environment. the available functional information provides for a relatively detailed reconstruction of the events in a closterovirus infection. in nature, most closteroviruses are inoculated into plants by insect vectors (karasev, 2000) . the initiation of the replication cycle upon virus entry into the cell requires virion disassembly followed by translation of the input viral genome. association of the virion tail with the capped, 5 -terminal, rna region suggests a role of the tail proteins in the rna exposure to translation initiation machinery of the cell. as originally proposed for tmv, subsequent disassembly of the viral particle and liberation of the rna could be mediated by the elongating ribosomes. the translated polyprotein is processed to yield l-pro and replicase components that recruit er and viral rna to the membranebound, replication complexes. the genome-size rna minus strand is made and used for synthesis of the genome plus strands and sgrnas, which are exported to the cytosol. the timing and amounts of the synthesis of sgrnas are regulated to optimize subsequent events of the virus infection cycle. for instance, sgr-nas that encode rna silencing suppressors are produced early in infection to counteract a potent host defense system. translation of the nascent genomes results in proliferation of replication complexes concurrent with virion assembly that is triggered by accumulation of the structural proteins. interestingly, the apparently normal virion tails can be formed in the absence of virion body assembly (satyanarayana et al., 2004) , while inactivation of the tail formation results in encapsidation of the entire rna by cp (alzhanova et al., 2001) . these findings suggest that the body assembly follows the faster process of tail formation. although cpm alone can encapsidate long stretches of rna, assembly of the functional virion tails of the proper length requires cooperative action of cpm, hsp70h, and p60 (satyanarayana et al., 2004) . dispensability of byv p20 for incorporation of other tail components suggests that this protein is added to preformed tails, possibly, via interaction with hsp70h (peremyslov et al., 2004a; prokhnevsky et al., 2002) . it seems that the major reason for accumulation of large virion masses in the infected cells is to promote closterovirus acquisition and transmission by insect vectors. however, a subpopulation of the virions must be engaged in the processes of cell-to-cell movement via pd and systemic transport via the phloem. because incorporation of each of the five virion proteins is required for one or both of these processes to occur, at least some of the transport events should involve physical translocation of the mature virions. our understanding of the mechanistic contributions of the transport proteins remains patchy at best. it is not known how the er-associated p6 aids the viral cell-tocell movement. it is also not clear if cpm and p60 have specific roles in virion transport in addition to their requirement for the tail assembly. in contrast, the mounting evidence suggests that hsp70h plays a pivotal role in virion translocation to and through pd. it has been shown that hsp70h accumulates in pd of the byv-infected cells and can be targeted to pd in the absence of other viral components (medina et al., 1999; prokhnevsky et al., 2002) . moreover, this autonomous targeting has been shown to rely on the actomyosin motility system (prokhnevsky et al., 2005) . it is tempting to propose that one of the roles of hsp70h present in the virion tails is to interact with myosin and chaperone the virions toward pd. this would provide an added benefit of directionality, with the virion tails put in close proximity or even inserted into pd channels. although the form in which closteroviral genome traverses pd is not known, the apparent lack of cp within pd might suggest that virions are (partially) disassembled upon passing pd channels (medina et al., 1999) . if this is the case, tail proteins may provide for directional rna transport, with the 5 -terminal region entering the adjacent cell and being recruited by ribosomes. the rest of the rna could be pulled into the cytoplasm co-translationally. this speculative model is not without analogy that comes from the nuclear export of giant, ∼30 kb mrnas of certain fly species (daneholt, 2001) . these mrnas are co-transcriptionally assembled into the ribonucleoprotein particles that dock to the nuclear pores with their ends corresponding to the mrna's 5 -termini. the directional transport through the pore occurs concomitantly with particle thinning and shedding some of the associated proteins. upon entering the cytoplasm, polysomes are formed and translation begins. the cycles of genome expression and replication, virion assembly and cell-to-cell movement, each of which takes ∼1 day, are repeated until infection reaches companion cells that have pd connection to phloem sieve elements. loading to sieve elements allows virus to be transported long distances with the liquid flow and to unload to sink tissues of stems, leaves, and roots. in all likelihood, multiple cycles of the virus replication on the phloem route are required to ensure successful colonization of the entire plant. two proteins that play specific roles in systemic transport of byv are l-pro and p20 (peng et al., 2003; prokhnevsky et al., 2002) . because l-pro is a replicational enhancer, it is plausible that it aids rna accumulation in the phloem cells. the location of p20 at the tail tip suggests a possible role in either stabilizing virions in the phloem, or guiding them through pd interconnecting sieve elements with companion cells, or both. the onset of systemic disease takes at least 2 weeks for closteroviruses of herbaceous plants and up to a few months for those infecting woody hosts. although the mechanisms of closteroviral pathogenicity are not entirely understood, the preferential replication in the phloem must play an important role. the massive recruitment of the er to viral replication complexes might interfere with the normal er function and cause phloem cytotoxicity manifested in such symptoms as vein clearing, or leaf yellowing, rolling, and wilting. the virus-coded suppressors of rna silencing also contribute to disease phenotype by interfering with microrna-mediated developmental processes. the data on the last phase of the closterovirus infection cycle, the semi-persistent insect transmission, are extremely scarce (karasev, 2000) . it appears that isolated virions of at least some closteroviruses can be transmitted to plants by insect membrane feeding, meaning that virion proteins are sufficient for the process to occur. it has been proposed that the large cpm of criniviruses mediates the whitefly transmission (tian et al., 1999) , while preliminary results implicated p20 in the aphid transmission of byv (prokhnevsky and dolja, unpublished data) . however, slow compared to other plant viruses, the life styles of closteroviruses proved evolutionarily successful given the long and rapidly growing list of closteroviruses that infect a broad range of hosts from cucumbers to strawber-ries to grapevine to citrus (aguilar et al., 2003; martelli, 2002; tzanetakis et al., 2005a,b) . closterovirus genome sequencing produced a genetic tapestry illustrated in fig. 2 . among the positive-strand rna viruses, complexity and diversity comparable to those of closteroviruses are seen only in the order nidovirales. because nidoviruses and closteroviruses belong to distinct evolutionary lineages, we assume that their genomes evolved independently via gene accretion by the simpler respective progenitors. an alternative to this scenario, namely, the origin of the smaller viral genomes from a very complex common ancestor, which possessed most of the genes now seen among positive-strand rna viruses, via gradual, lineage-specific gene loss, appears to be much less plausible, if realistic at all. to gain insight into closterovirus evolution, we conducted phylogenetic analysis of the several proteins conserved in all extant family members. the rna-dependent rna polymerase is the only protein universally present in all positive-strand rna viruses (koonin, 1991) . as expected, our analysis confirmed the relationship of closteroviral polymerases with those of the other viruses from the alphavirus-like superfamily, as well as the separation of the closteroviral polymerases into three clades corresponding to the currently recognized genera (fig. 3a) . the viruses of the genera closterovirus, crinivirus, and ampelovirus are predominantly transmitted by aphids, whiteflies, and mealybugs, respectively. this correlation between the polymerase phylogeny and the type of insect vectors likely reflects vector adaptation as a driving force behind divergent evolution of closteroviruses (karasev, 2000) . analysis of the closteroviral l-pro sequences also revealed clades corresponding to the three genera (fig. 3b) indicating that rna polymerases and proteases coevolved since the divergence of closteroviruses from the common family ancestor. the second leader proteases of ctv and glrav-2 (l2 in fig. 3b ) clustered with the other proteases of the same genus in accord with their proposed origin by lineage-specific duplication of the l-pro gene (peng et al., 2001) . the topology of the phylogenetic tree for closterovirus hsp70h sequences also mimicked that of the rna polymerase tree (fig. 3c) , supporting the presence of hsp70h in the common ancestor of closteroviruses. in a broader perspective, closteroviral homologues showed no specific affinity to cellular hsp70s from any particular group of organisms or compartments of the eukaryotic cell (fig. 3c) (boorstein et al., 1994) . this result is indicative of the rapid evolutionary divergence due to the functional specialization of hsp70h within the viral genetic context. the only viruses besides closteroviruses that encode hsp70s are the mimiviruses of the protozoan acanthamoeba (raoult et al., 2004) . interestingly, both of the mimiviral hsp70s cluster with their eukaryotic kin suggesting their relatively recent origin from the host and/or slower evolution (fig. 3a) . in all likelihood, closteroviruses and mimiviruses acquired hsp70 genes independently. (zhu et al., 1998) ; cysdv, cucurbit yellow stunting disorder virus (aguilar et al., 2003) ; glrav-1, grapevine leafroll-associated virus-1 (fazeli and rezaian, 2000) ; pmwav-2, pineapple mealybug wilt-associated virus-2 (melzer et al., 2001) ; other designations are as in fig. 2. (b) dendrograms for the papain-like proteinase domains of the closterovirus leader proteinases. ctv and glrav-2 each possess two leader proteinases, l1 and l2. eav-nsp1, the nonstructural protein-1, a papain-like leader proteinase of equine arteritis virus used as outgroup. (c) dendrograms for the hsp70 homologues from the closteroviruses, the mimivirus, prokaryotes, and eukaryotes. (d) dendrograms for the cp-like domains present in closterovirus cps, cpms, and p60s. glrav-1 encodes two divergent variants of cpm, 1 and 2. pva-cp, capsid protein of potato virus a, an outgroup. sequences alignment and phylogenetic analysis were performed using the clustal x 1.8 program (thompson et al., 1997) and mega 3.1 (kumar et al., 2004) . trees were generated using the neighbour-joining method (saitou and nei, 1987) with correction for multiple substitutions and 1000 bootstrap replicates. in all cases, similar trees and bootstrap values were obtained using the maximum parsimony method (fitch, 1977) (not shown). the ncbi accession numbers for closteroviruses were as follows. glrav-3, nc004667; pmwav-2, af283103; glrav-1, af195822; byv, nc001598; ctv, nc001661; glrav-2, nc007448; liyv, nc003617 and nc003618; spcsv, nc004123 and nc004124; cysdv, nc004810 and nc004809. the major cps of closteroviruses belong to the protein family that includes cps of all filamentous viruses of plants . however, unlike other filamentous viruses, closteroviruses encode at least two additional proteins possessing cp-related domains, cpm and p60 (boyko et al., 1992; napuli et al., 2003) . the phylogenetic analysis of these proteins demonstrates clear separation of the three p60 clades corresponding to the family genera (fig. 3d) . similarly to what was found for l-pro and hsp70h, this supports p60 presence in the common ancestor of extant closteroviruses. in contrast, cpms of the viruses of the genera closterovirus and crinivirus cluster with their respective cps (fig. 3d ). this tree topology suggests that cpms have evolved independently in these two genera via duplication of the cp gene. this could also be the case for the genus ampelovirus, but the affinity between ampeloviral cpms and cps is less significant statistically, probably indicating faster evolution of cpms in this genus. this notion is also compatible with the fact that in one of the ampeloviruses, glrav-1, the cpm gene underwent an additional duplication (fazeli and rezaian, 2000) . using the results of phylogenetic analysis, we developed the following scenario for the evolution of the family closteroviridae (fig. 4) . we propose that the monophyletic closterovirus lineage descends from a plant virus of the alphavirus-like superfamily that possessed a typical met-hel-pol replicase, a p6-like movement protein, and a single cp forming a filamentous capsid. this progenitor evolved into the last common ancestor of closteroviruses by acquiring several new genes. among these, acquisition of the l-pro gene and a central domain of replicase facilitated rna replication and conditioned expansion of the progenitor's genome. concurrently, the hsp70 gene was incorporated via recombination with a cellular mrna, whereas the p60 gene originated via duplication and divergence of the cp gene. the co-evolution of hsp70h and p60 resulted in emergence of a prototype virion tail that empowered cell-to-cell transport of the expanded genome of the ancestral closterovirus. because most of the extant closteroviruses are insecttransmissible, it seems reasonable to propose that the closterovirus ancestor was also transmitted by an insect. the three current lineages of closteroviruses evolved by adaptation to the distinct types of insect vectors (karasev, 2000) . the cpms appeared at that time via duplication and divergence of the cp gene. in addition to cpm phylogenenies (fig. 3d) , this scenario is supported by the different order of cp and cpm genes in the genus closterovirus versus the genera crinivirus and ampelovirus (fig. 2) . the latter two genera could arise independently from the common family ancestor (fig. 4) . alternatively, criniviruses could evolve by the split of a single ampeloviral genomic rna to two segments (dashed arrow in fig. 4) . the same order of the cp and cpm genes and the larger size of cpms in ampeloviruses and criniviruses compared to closteroviruses are compatible with the latter scenario. interestingly, evolution subsequent to emergence of cpms produced very few additional genes that would be specific to and conserved within each genus. one example is the p21-like (tzanetakis and martin, 2004) ; pyvv, potato yellow vein virus (liveratos et al., 2004) ; spav, strawberry pallidosis associated virus (tzanetakis et al., 2005b) . nucleotide accession numbers were as follows: bpyv, nc005209; pyvv, nc006063 and nc006061; spav, nc005896; cysdv, see fig. 3 . graphic representation of the alignments was done using vector nti program (invitrogen). suppressor of rna silencing in the viruses of the genus closterovirus. another example is provided by the p8-like and p28-like proteins that are conserved in genus crinivirus (fig. 5) . the genus ampelovirus exhibits most genetic variation in the family. even though the replication and assembly/movement modules are present in all known representatives of this genus, they are often perturbed by incorporation of additional unique genes or changes in the gene order (theilmann et al., 2002) . the numerous other novel genes acquired by closteroviruses, in addition to replication module and qgb, are virus-specific. for instance, ctv genes encoding p33, p18, p13, and p23 have no homologues in byv or other known genus members (fig. 2 ) (pappu et al., 1994) . conspiciously, similar tendency of prolifiration of the virus-or lineage-specific genes is observed in coronaviruses and other large nidoviruses (gorbalenya et al., 2006) . because the gene repertoirs of smaller rna viruses tend to be more conserved in any given taxon, it seems that the unusual genetic plasticity of nidoviruses and closteroviruses is conditioned by their genome expansion. the resulting benefit of faster macroevolution via accretion of new genes may provide large rna viruses with ability to rapidly adapt to new hosts, vectors, or changing environmental conditions. the task of establishing the time scale for the closterovirus evolution meets the major challenges common for all viruses. these challenges include the lack of paleonthological record, the relatively fast and variable pace of molecular evolution, and the complex relations between evolution of viruses and their hosts. however, comparison of the evolutionary histo-ries of viruses and host organisms may provide some useful insights. as has been discussed previously, each of the three superfamilies of the positive-strand rna viruses contains viruses that infect plants, insects, and vertebrates, suggesting that these superfamilies evolved prior to separation of the animal and plant kingdoms (koonin and dolja, 1993) . this notion is further reinforced by finding of a plethora of picornalike viruses in the marine environment including viruses that infect diatoms (culley et al., 2003) . regardless of the adapted eukaryotic phylogeny, the specifics of which remain debated, plants, animals, and diatoms diverged early in the evolution of eukaryotes, and horizontal transfer of picornaviruses from land plants or animals to marine biota seems highly unlikely. therefore, the superfamilies of positive-strand rna viruses including alphavirus-like superfamily to which closteroviruses belong might have diverged at the dawn of eukaryotic evolution. the lack of information on rna viruses of red and green algae or primitive land plants limits our ability to deduce the time of origin for the last common ancestor of closteroviridae or other extant plant virus families. however, it seems that these ancestors should have been in place, at the latest, by jurassic (150-200 million years ago), when the great diversity of the flowering plants evolved to dominate the land (benton and ayala, 2003; kenrick and crane, 1997; palmer et al., 2004) . because this process was accompanied closely by evolution of the potential closteroviral insect vectors, such as aphids and other homoptera (gaunt and miles, 2002; martinez-torres et al., 2001) , it seems reasonable to think that diversification of the three closteroviral genera also came about near that time. what is the functional significance of the increase in genome complexity seen in closteroviruses relative to the simpler plant viruses? a comparison of tmv and byv, which both evolved from the alphavirus-like ancestors, shows that the large part of the ∼9 kb genomic surplus of byv is dedicated to facilitating the synthesis of the virion rna and multiple sgrnas. the rest of the surplus was invested in the formation of the complex virion tail that empowers virus transport within and transmission between the host plants and in suppression of rna silencing (fig. 1) . if this is the case, it almost looks like the complexity of closteroviruses arose to maintain itself. similar comparison between the more and less complex nidoviruses, such as sars virus and eav, respectively, reveals an increase in the number of rna metabolism enzymes in the former (gorbalenya et al., 2006) . in addition to enabling replication and transcription of the large viral genomes also seen in closteroviruses, further leap in complexity is thought to confer coronaviruses with a higher fidelity of rna synthesis and a higher degree of rna metabolism autonomy. a greater independence of the dna metabolism from the host cell is also one of the major tendencies in the evolution of the complex dna viruses (iyer et al., 2006) . why then the largest dna viruses with the ∼1200 kb genomes are so much larger than the largest rna viruses with the ∼30 kb genomes? the more or less obvious answer seems to lie in the chemical properties of rna, which is generally thought to be the original enzymatic and information-storage molecule of the ancient rna world. it is further believed that the limitations in the rna stability and replication fidelity resulted in the evolution of dna as the dedicated genomic material. it seems that ∼30 kb could be close to the maximum size of rna molecules that can function as autonomous genetic elements due to minimal stability requirements. no larger rnas are known to be used by any viruses, transposable elements, or prokaryotic cells; although eukaryotes do have some giant mrnas, these are likely to be short-lived and translated via special mechanisms such as coupling of translation with nucleo-cytoplasmic export (gregorio et al., 1999) . the plant hosts impose even stricter limits on the size of viral genomes, be it rna or dna: at 19.3 kb, the rna genome of ctv (fig. 2) is the largest known plant virus genome. the single most important peculiarity in the infection cycle of plant viruses is that they must rely on direct cell-to-cell movement via pd to successfully colonize a plant (boevink and oparka, 2005; lucas and lee, 2004) . the idea that the pd is a bottleneck for intercellular translocation of virions or genomes has recently gained experimental support (gilbertson et al., 2003) . the corollary of the limited size and translocation capacity of the pd channels is that viral genomes larger than ∼8 kbp for dsdna, ∼3 kb for ssdna, or ∼20 kb for ssrna cannot move between the cells and therefore are eliminated during evolution. what is the driving force behind the increase in viral genome complexity? it is certainly not the host adaptation or virus-host coevolution because both simple prokaryotic and most complex eukaryotic organisms play hosts for the simple and complex viruses alike. a simple and convincing theory of the origins of genome complexity in eukaryotes has been recently advanced by lynch and conery (2003) . this theory states that the increase in genome complexity is a stochastic result of the relieved selection pressure that follows dramatic reductions in population size; in small populations, drift may result in fixation of genomic changes, such as duplication of numerous genes and propagation of selfish elements, which are not tolerated in larger populations with intensive purifying selection. often, the reduction in population size would be caused by local or global environmental catastrophes that lead to abrupt drop in the population size for a few or many species at a time. support for this non-adaptationist concept of genomic complexity comes from extensive analyses of genes, introns, and selfish dna in a large number of complete prokaryotic and eukaryotic genomes (koonin, 2004; lynch and conery, 2003) . although a similar analysis is yet to be attempted for viruses, it is tempting to apply this concept to evolution of viral genetic complexity. what would be considered an 'environmental catastrophe' for a virus? the most obvious answer is the drastic reduction in a population size of the host organisms. another possibility is the emergence of a new antiviral defense system, such as innate or acquired immunity, or rna silencing. it is, probably, the combination of both that has driven acquisition of multiple new genes by viral genomes in certain lineages, followed by the gradual adaptation of these genes for the needs of infection. a corollary of the comparative analysis of closterovirus genomes is that their relative complexity and variability go hand in hand (fig. 2) . the abundance of unique, rare, and novel genes that appear to be picked at random by different closteroviruses is compatible with the idea of genome complexity emerging as a byproduct of increased genome entropy. human and bacterial oxidative demethylases repair alkylation damage in both rna and dna putative 65 kda protein of beet yellows closterovirus is a homologue of hsp70 heat shock proteins beet yellows closterovirus: complete genome structure and identification of a leader papain-like thiol protease rattlesnake" structure of a filamentous plant rna virus built of two capsid proteins further variability within the genus crinivirus, as revealed by determination of the complete rna sequence of cucurbit yellow stunting disorder virus genetic analysis of the cell-to-cell movement of beet yellows 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virus subgenomic rnas the origin and early evolution of plants on land genome structure and phylogenetic analysis of lettuce infectious yellows virus, a whitefly-transmitted, bipartite closterovirus the phylogeny of rna-dependent rna polymerases of positive-strand rna viruses a non-adaptationist perspective on evolution of genomic complexity or the continuing dethroning of man evolution and taxonomy of positivestrand rna viruses: implications of comparative analysis of amino acid sequences on the origin of genomes and cells within inorganic compartments viral class 1 rnase iii involved in suppression of rna silencing complete genome sequence and analyses of the subgenomic rnas of sweet potato chlorotic stunt virus reveal several new features for the genus crinivirus mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment complete nucleotide sequence and genome organization of grapevine leafroll-associated virus 3, type member of the genus ampelovirus analysis of the rna of potato yellow vein virus: evidence for a tripartite genome and conserved 3 -terminal structures among members of the genus crinivirus three distinct suppressors of rna silencing encoded by a 20-kb viral rna genome plasmodesmata as a supracellular control networks in plants the origins of genome complexity the family closteroviridae revised molecular systematics of aphids and their primary endosymbionts subcellular localization of the hsp70-homolog encoded by beet yellows closterovirus nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2 synthesis of subgenomic rnas by positivestrand rna viruses plant virus-derived small interfering rnas originate predominantly from highly structured single-stranded viral rnas the 64-kda capsid protein homolog of beet yellows virus is required for assembly of virion tails interaction between hsp70 homolog and filamentous virions of the beet yellows virus the plant tree of life: and overview and some points of view nucleotide sequence and organization of eight 3 open reading frames of the citrus tristeza c losterovirus genome sequence requirements for rna strand transfer during nidovirus discontinuous subgenomic rna synthesis leader proteinase of the beet yellows virus functions in long-distance transport leader proteinase of the beet yellows closterovirus: mutation analysis of the function in genome amplification functional specialization and evolution of leader proteinases in the family closteroviridae a replicationcompetent chimera of the plant and animal viruses complex molecular architecture of beet yellows virus particles movement protein of a closterovirus is a type iii integral transmembrane protein localized to the endoplasmic reticulum identification of the subgenomic mrnas that encode 6-kda movement protein and hsp70 homolog of beet yellows virus genes required for replication of the 15.5-kilobase rna genome of a plant closterovirus hsp70 homolog functions in cell-to-cell movement of a plant virus actin cytoskeleton is involved in targeting of a viral hsp70 homolog to the cell periphery interaction between long-distance transport factor and hsp70-related movement protein of beet yellows virus the 1.2-megabase genome sequence of mimivirus suppressor of rna silencing encoded by beet yellows virus a heterogeneous population of defective rnas is associated with lettuce infectious yellows virus the neighbor-joining method: a new method for reconstructing phylogenetic trees closterovirus bipolar virion: evidence for initiation of assembly by minor coat protein and its restriction to the genomic rna 5 region closterovirus encoded hsp70 homolog and p61 in addition to both coat proteins function in efficient virion assembly the rna structures engaged in replication and transcription of the a59 strain of mouse hepatitis virus a positive-strand rna virus replication complex parallels form and function of retrovirus capsids effects and side effects of viral rna silencing suppressors on short rnas partial nucleotide sequence and genome organization of a canadian isolate of little cherry virus the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools lettuce infectious yellows virus: in vitro acquisition analysis using partially purified virions and the whitefly, bemisia tabaci complete nucleotide sequence of a strawberry isolate of beet pseudoyellows virus characterization of a novel member of the family closteroviridae from mentha spp nucleotide sequence, genome organization and phylogenetic analysis of strawberry pallidosis associated virus, a new member of the genus crinivirus size selective recognition of sirna by an rna silencing suppressor induction and suppression of rna silencing: insights from viral infections rna silencing suppressor p21 of beet yellows virus forms an rna binding octameric ring structure ribo-gnome: the big world of small rnas nucleotide sequence and genome organization of grapevine leafrollassociated virus-2 are similar to beet yellows virus, the closterovirus type member processing and subcellular localization of the leader papain-like proteinase of beet yellows closterovirus sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis the authors are grateful to eugene v. koonin for stimulating discussions and critical reading of the manuscript. the work in v.v.d. lab is supported by grants from the national institutes of health (gm053190) and bard (is-3784-05), and in jptv lab by the academy of finland (grants 1102003 and 1102134). j.f.k. is junior professional officer funded by sida, sweden. key: cord-281526-7t9e4lgn authors: yin, lijuan; zeng, yuyao; wang, wei; wei, ying; xue, chunyi; cao, yongchang title: immunogenicity and protective efficacy of recombinant fusion proteins containing spike protein of infectious bronchitis virus and hemagglutinin of h3n2 influenza virus in chickens date: 2016-09-02 journal: virus res doi: 10.1016/j.virusres.2016.07.010 sha: doc_id: 281526 cord_uid: 7t9e4lgn infectious bronchitis (ib) is an acute and highly contagious viral respiratory disease of chickens and vaccination is the main method for disease control. the s1 protein, which contains several virus neutralization epitopes, is considered to be a target site of vaccine development. however, although protective immune responses could be induced by recombinant s1 protein, the protection rate in chickens was still low (<50%). here, we generated fused s1 proteins with ha2 protein (rs1-ha2) or transmembrane domain and cytoplasmic tail (rs1-h3(tm)) from hemagglutinin of h3n2 influenza virus. after immunization, animals vaccinated with fusion proteins rs1-ha2 and rs1-h3(tm) demonstrated stronger robust humoral and cellular immune responses than that of rs1 and inactivated m41 vaccine. the protection rates of groups immunized with rs1-ha2 (87%) were significantly higher than the groups inoculated with rs1 (47%) and inactivated m41 vaccine (53%). and chickens injected with rs1-h3(tm) had similar level of protection (73%) comparing to chickens vaccinated with rs1 (47%) (p = 0.07). our data suggest that s1 protein fused to the ha2 or tm proteins from hemagglutinin of h3n2 influenza virus may provide a new strategy for high efficacy recombinant vaccine development against ibv. avain infectious bronchitis (ib) was first described in 1931 as a highly contagious disease and thereafter was found to be caused by an infectious bronchitis virus (ibv), which belongs to coronavirus genus within the coronaviridae (cavanagh, 2007; sjaak et al., 2011) . though both live-attenuated and inactivated vaccines are used worldwide to control the disease, ibv occasionally outbreaks in endemic areas (bijlenga et al., 2004; cavanagh, 2003) . the use of attenuated live-vaccines elicits local, systemic and cell-mediated immunity to the virus (cavanagh, 2003; raj and jones, 1997) , but poses a risk of residual pathogenicity associated with vaccine backpassage in flocks (abro et al., 2012; mckinley et al., 2008; mckinley et al., 2011) . though the inactivated ib vaccines are relatively safe, they are efficacious only when used as boosters after priming vaccination with live vaccines (ladman et al., 2002) . thus to develop a safer, more efficacious and economic vaccine candidate is of great interest for scientific research. infectious bronchitis virus (ibv) is an enveloped virus containing a single-stranded positive-sense rna genome of 27.6 kb (wickramasinghe et al., 2014) . the genome of ibv encodes four structural proteins, including spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins (cavanagh, 2007) . people have found that the spike (s) protein, the largest structural protein, constitutes the characteristic club-shaped 16-21 nm projections that emerge from the virion surface and presents a corona-like appearance under electron microscopy (cook et al., 2012) . in addition, the s protein was suggested to have haemagglutinating activity that the entry of the viruses is mediated by sialic acid binding activity of the s protein (schultze et al., 1992) . the s protein is a class i fusion peptide, in which the variable s1 domain is involved in host cell attachment and the conserved s2 domain mediates fusion of the virion and cellular membranes (bosch et al., 2003) . the s1 protein contains the primary neutralizing epitopes that can induce neutralization, hemagglutination inhibition (hi) and serotype-specific antibodies (cavanagh et al., 1986a,b; ignjatovic and galli, 1994; ignjatovic and galli, 1995 koch et al., 1990) , thus making it a main target when designing new ibv vaccines. the s protein alone is considered to be sufficient to induce good immunity (cavanagh, 2007) . however, when the s1 subunit of ibv was recombined using baculovirus, although protective immune responses were induced with multiple inoculations, the percentage of protected chickens was still less than 50% (song et al., 1998) . thus, to induce better immunogenicity against ibv infections, manipulation of the s protein is considered to be a good strategy when designing new ibv vaccines. our previous research demonstrated that various influenza h1, h5 and h9 hemagglutinins (has) proteins containing replaced h3-wt tm showed increased thermal stability and immunogenicity (liu et al., 2014) . it is intriguing for us to know whether recombinant hemagglutinin and s protein can also induce better immunogenicity and protective efficacy against ibv infections. in this study, we first investigated whether the recombinant s protein could induce better immune responses. therefore, we generated a recombinant s1 (rs1) protein and two recombinant s1 fusion proteins, rs1-h3(tm) (fused with the transmembrane (tm) and cytoplasmic tail (ct) of influenza h3n2 ha protein) and rs1-ha2 (fused with the ha2 domain of influenza h3n2 ha protein). our results suggest that the immunogenicity and protection efficacy of these recombinant fusion proteins have been enhanced in chickens. sf9 insect cells were cultured in serum-free sf900ii medium (gibco, grand island, ny, usa) at 27 • c. the virulent m41 strain (china institute of veterinary drug control, ivdc) was propagated in 10-day-old specific pathogen free (spf) chicken embryos. eid 50 for the ibv m41 was calculated according to the reed-muench method as described previously (reed and muench, 1938) . the a/swine/guangdong/01/1998(h3n2) was isolated and maintained by our laboratory. all primers used in this study were synthesized by invitrogen and summarized in table 1 . a bac-to-bac baculovirus expression system was used for the production of recombinant expression bacmids and baculoviruses. briefly, ibv-m41 s1 gene, h3(tm) and ha2 fragments (genbank accession number fj830855.1) from h3n2 strain (a/swine/guangdong/01/1998) were first amplified and cloned into pmd-18t vector (takara) and then amplified sequentially. s1-h3(tm) and s1-ha2 fusion genes were then generated by overlapped pcr and cloned between the sali and hindш sites of pfastbac1 vector (invitrogen) to generate recombinant shuttle plasmids (fig. 1a) . the shuttle vectors were then chemically transformed into competent dh10bac tm escherichia coli cells (invitrogen). all clones were verified by sequencing (invitrogen). recombinant baculovirus generation and infection were performed as previously described (liu et al., 2013) . briefly, the obtained recombinant bacmids were transfected into sf9 cells and incubated for 3 days. the target recombinant baculoviruses (rbvs) were then harvested from the supernatant. the structure of the recombinant fusion proteins are shown in fig. 1b. cell lysates were separated on 10% sds polyacrylamide gels, and then electrophoretically transferred to polyvinylidene difluoride membranes (millipore, billerica, ma). membranes were blocked and subsequently detected with chicken polyclonal sera (china institute of veterinary drugs control) against ibv virus at a 1:2000 dilution, horseradish peroxidase (hrp)-conjugated anti-chicken secondary antibody at a 1:5000 dilution (ptglab, usa) and commercial ecl kit (pierce). for recombinant proteins expression, sf9 cells were infected with recombinant or wild-type baculoviruses and cultured for 3 days, and the cells were collected, ultrasonicated, and then centrifuged at 12,000 × g for 30 min at 4 • c. the supernatants were further centrifuged at 120,000 × g for 3 h at 4 • c. the resulting precipitates were resuspended in pbs and loaded onto a discontinuous sucrose gradient of 30%, 40%, 50%, and 60% sucrose for recombinant proteins enrichment, and ultracentrifuged at 65,000 × g for 16 h at 4 • c. fractions were collected from the gradient interphases as described previously (liu et al., 2014) , and their recombinant proteins contents were analyzed using coomassie blue stained sds-page electrophoretogram. formalin-inactivated m41 virus was purified and concentrated by ultracentrifugation as previously described (kong et al., 2010) . the overall protein concentrations of recombinant proteins and inactivated ibv were determined using bca protein assay kit (pierce, company); and the protein concentrations of the three recombinant proteins (rs1 protein, rs1-h3(tm) protein and rs1-ha2 protein) and s1 protein concentration of inactivated ibv were determined using sds-page gel electrophoresis by genesnap and genetools from syngene software with bsa as standard. ninety 10-day-old spf chickens were collected from the spf experimental animal center (dahuanong animal health products co., ltd., guangdong, china), and housed in individual isolators under positive pressure, then randomly divided into six groups (n = 15 chickens/group). recombinant proteins, inactivated m41 virus, sf9 cell lysate (infected by wild-type baculoviruses), and pbs were emulsified with montanide tm isa 71 vg at a 3:7 ratio respectively (w:w; antigens:adjuvant) as recommended by the manufacturer (seppic, paris, france) and then were used in intramuscular injection. group 1 chickens were immunized with 5 g rs1 protein per chicken. group 2 chickens were injected with 5 g rs1-h3(tm) protein per chicken. group 3 chickens were vaccinated with 5 g rs1-ha2 protein per chicken. group 4 chickens received 5 g inactivated m41 (s1 protein) per chicken, as a positive control. for negative controls, animals in the remaining two groups were injected with either rwt (sf9 cell lysate infected by wild-type baculoviruses) or pbs. booster immunization, using the same antigen at inoculation, was conducted 2 weeks after the prime. two weeks after booster immunization, chickens from each group were challenged with 2 × 10 4 eid 50 of the ibv-m41 strain via the nasal-ocular route in 200 l pbs per chicken, and were observed daily for clinical symptoms over a week period and euthanised at 7 day after challenge. the trachea and kidney tissues of chickens in all groups were harvested for further detection of virus. all animal experiments were conducted in compliance with the institutional guidelines for animal protection rights in china. sera were collected from chickens at 0, 14 and 28 days after primary immunization for ibv-specific antibody detection. the purified m41 virosomes were made following the procedures as previously described (liu et al., 2013) , and used as antigen to detect the ibv-specific antibodies in an indirect elisa. briefly, the purified virosomes at a concentration of 3.5ug/ml were coated, and incubated with serial dilutions of each serum sample (37 • c for 1 h). the secondary donkey anti-chicken hrp-conjugated antibodies (ptglab, usa) were used at a 1:5000 dilution. the optical density value was 450 nm. the end-point titer was determined as the reciprocal of the final dilution giving a threefold optical absorbance of negative control as described previously (liu et al., 2014) . cellular immune response was assessed by counting the numbers of various types of functional t lymphocytes in vaccinated chickens. two weeks after the boosting immunization and prior to challenge, five chickens were selected randomly from each group and peripheral blood samples were collected from the brachial wing vein using heparinized syringes. peripheral blood mononuclear cells (pbmcs) were isolated and adjusted to 1 × 10 7 cells/ml. the samples (100 l; 1 × 10 6 cells) were incubated for an hour at room temperature with mouse anti-chicken cd4-fitc, cd8-rpe and cd3-sprd (southern biotech, birmingham, al, usa). flow cytometry was performed on a facs calibur instrument (becton dickinson, san jose, ca, usa) and data were analyzed with cel-lquest software (bd bio-sciences). tracheal and renal tissues of chickens were collected for detection of the challenge virus. total rnas were extracted from tissue suspensions using takara minibest viral rna/dna extraction kit (takara biotechnology, dalian, china) and subjected to rt-pcr (primescrip tm rt-pcr kit, takara bio, china) using primers directed at the nucleocapsid as previously described (zhang et al., 2014) . the absence of detectable virus in the tracheal and kidney was considered to have been protected by the vaccine against the challenge. fig. 1 . baculovirus construct for production of proteins. (a) construction of recombinant plasmids. pfbac1 contains polyhedrin promoter (pph), sv40 pa, sv40 polyadenylation signal; gentamicin; tn7r and tn7l. different target genes (s1, s1-h3 and s1-ha2) were inserted between pph and sv40 pa respectively. (b) construction of recombinant fusion proteins rs1, rs1-h3(tm) and rs1-ha2. for rs1-h3(tm) protein, rs1 protein was fused to the ct and tm domain of h3n2 ha protein; for rs1-ha2 protein, rs1 protein was fused to the ha2 domain of the ha protein of h3n2. the data were statistically analyzed by using a student's twotailed t-test when only two groups were compared or by one-way analysis of variance (anova) when more than two groups were compared. p values (p) less than 0.05 were considered statistically significant. different recombinant baculoviruses were constructed to express the target fused protein (fig. 1a) . to construct rs1-h3(tm) protein, recombinant s1 protein was fused to the cytoplasmic tail (ct) and transmembrane (tm) domain of hemagglutinin (ha) from influenza h3n2 subtype. for rs1-ha2 protein, rs1 protein was fused to the ha2 domain of the ha protein (fig. 1b) . to check whether the three recombinant proteins rs1, rs1-h3(tm), rs1-ha2 could be expressed in the sf9 cells, we analyzed protein expression in collected cell lysates from the recombinant baculovirus-infected sf9 cells. western blot under denaturing condition showed that these recombinant proteins were detectable as clearer distinct bands with molecular weight of 100 kda, 110 kda and 130 kda ( fig. 2a) . the molecular weight of the recombinant rs1 protein was comparable to the native protein of 100 kda (cavanagh et al., 1986a,b) . this result was further confirmed by sds-page using concentrated recombinant protein contents utilizing discontinuous sucrose gradient centrifugation (fig. 2b) , and these concentrated recombinant proteins were prepared for vaccination later in our study. to investigate whether the recombinant proteins could induce better immune response against ibv infection, we detected ibvspecific antibody in the sera of vaccinated chicken using indirect elisa. in general, two weeks after primary vaccination (day 14), ibv-specific antibody level increased in all groups besides rwt and pbs. the antibody level in chickens immunized with rs1-h3(tm) and rs1-ha2 increased significantly, comparing to the rs1 group and inactivated m41 group (fig. 3) . two weeks after booster vaccination (day 28), the levels of anti-ibv antibodies increased further in chickens immunized with inactivated m41 virus, rs1, rs1-h3(tm) and rs1-ha2. the inactivated m41 group developed a higher level of antibody than the rs1 group (p < 0.05). moreover, chickens vaccinated with rs1-h3(tm), rs1-ha2 had significantly higher antibody titers than animals in the rs1 group (p < 0.01), while fig. 3 . infectious bronchitis virus (ibv)-specific antibody titers in chickens sera. six groups of chickens (n = 15 chickens per group) were subcutaneously immunized with rs1, rs1-h3(tm), or rs1-ha2, respectively. inactivated m41 was used as a positive control, rwt (sf9 cell lysate infected by wild-type baculoviruses) and pbs were used as negative controls. sera from each chicken were collected and the ibv-specific antibody titers were determined by elisa. data is represented by the mean antibody titer +s.d. ratio of cd3 + cd8 + /cd3 + cd4 + t-lymphocytes 2 weeks after booster immunization a . ratio of cd3 + cd8 + /cd3 + cd4 + t-lymphocytes inactivated vaccine 0.46 ± 0.10a rs1 0.48 ± 0.05a rs1-h3(tm) 0.71 ± 0.03b rs1-ha2 0.73 ± 0.02b the difference between groups with the same letter (a or b) was not significant (p > 0.05). a the test was repeated five times in each group. data is presented by means ± sd. there was no difference between the groups of rs1-h3(tm) and rs1-ha2. these data indicate that the fused s1 proteins can boost better immune responses than inactivated m41 virus and s1 protein alone, resulting in a significantly increased antibody level. as cd3 + cd4 + helper t cells and cd3 + cd8 + cytotoxic t cells function as crucial components in cellular immunity, we counted the number of cd3 + , cd3 + cd4 + and cd3 + cd8 + t lymphocytes utilizing flow cytometry 14 days after the booster to measure immune response. levels of cd3 + and cd3 + cd8 + t lymphocyte increased significantly in group of rs1-ha2 and rs1-h3(tm) comparing to those groups inoculated with rs1, inactivated m41, rwt and pbs (p < 0.01). there was no significant difference between the rs1-h3(tm) and rs1-ha2 groups. and the percentages of the t lymphocytes were comparable between the rs1 and inactivated m41 vaccine groups (fig. 4) . further, the ratio of cd3 + cd8 + to cd3 + cd4 + t lymphcytes in rs1-h3(tm) (0.71 ± 0.03) and rs1-ha2 (0.73 ± 0.02) vaccinated groups were significantly higher than that in chickens immunized with rs1 (0.48 ± 0.05) and inactivated m41 (0.46 ± 0.10) (p < 0.05) ( table 2 ). these results suggest that though rs1 alone can elicit cellular immune responses in chickens, the recombinant rs1-h3(tm) and rs1-ha2 can significantly enhance the cell-mediated immune response. to further confirm our results, chickens were challenged with the virulent m41 strain. morbidity, tracheal infections, renal infections and protection rates after challenge are summarized in table 3 . in general, chickens immunized with inactivated m41 vaccine, rs1, rwt and pbs developed symptoms as dyspnea, coughing and rales on a different level. to further evaluate the level of protection after challenge, rt-pcr was performed to detect viral rna in the collected tracheas and kidneys. chickens vaccinated with rs1 (47%) presented a similar protection level comparing to chickens immunized with the inactivated m41 virus (53%). however, chickens immunized with rs1-ha2 were better protected (87%) comparing to chickens vaccinated with rs1 (47%) and inactivated m41 (53%) (p < 0.05), while chickens injected with rs1-h3(tm) had similar level of protection (73%) comparing to chickens vaccinated with rs1 (47%) (p = 0.07). there was a significant difference between the rs1-ha2-vaccinated group and the rs1-immunized group (p < 0.05) in the number of collected tracheas and kidneys that were positive for the virus. these data further confirm our previous data presenting better immunogenesis of the fused s1 proteins and suggest that these fused proteins, especially rs1-ha2 protein, can provide a new strategy for ibv vaccine development. the oligomeric spike (s) protein is a trans-membrane glycoprotein, usually forms dimers or trimers that extend from coronavirus membranes. this protruding virion protein is considered to be the main antigen and therefore mainly used to induce immunogenicity against coronaviruses (cavanagh, 1983; delmas and laude, 1990; lewicki and gallagher, 2002) . for instance, researches on severe acute respiratory syndrome (sars), which belongs to coronavirus, have suggested that recombinant trimeric full-length s proteins are highly immunogenic and able to induce an efficacious protective immune response (kam et al., 2007; zhou et al., 2006) . more recently, li et al. showed that the fused s protein with the foldon domain derived from t4 bacteriophage presented significantly greater potency in the induction of neutralizing antibodies than the s protein . these researches have suggested that the manipulated s protein could serve as major target for immune therapy and vaccine design against coronaviruses. once this integral protruding membrane protein is assembled, it undergoes endoproteolysis and cleaves to two noncovalently associated s1 and s2 fragments (cavanagh, 1983; cavanagh et al., 1986a,b) . the s1 domain contains the primary neutralizing epitopes for ibv, and thus is considered to be responsible determine its pathogenicity (wickramasinghe et al., 2014) . in 1998, song et al. showed that recombinant s1 (rs1) glycoprotein, expressed in a baculovirus expression system, could induce protective immunity in chickens against challenge with virulent nephropathogenic ibv, though multiple inoculations were required and the rs1 protein induced only 50% protection of the kidney (song et al., 1998) . other researchers displayed the s1 glycoprotein on the bacmam virus-based surface and presented that the bv-dual-s1 protein elicited stronger humoral and cell-mediated immune responses (zhang et al., 2014) . thus, enhancement of the antigenicity of the s1 fig. 4 . flow cytometry analysis of t lymphocyte subsets in chickens after immunization with rs1, rs1-h3(tm), rs1-ha2, inactivated m41, rwt (sf9 cell lysate infected by wild-type baculoviruses) and pbs. this test was performed two weeks after the boosting immunization. data are presented by means + sd. n = 5 chickens per group. the protection rate among the five groups are indicated with different letters. the difference among groups with the same letter (a-c)was not significant (p > 0.05). a morbidity was recorded for each day after challenge and is presented as total number of sick chickens in each group. b virus identified intrachea by rt-pcr detection. c virus identified in kidney by rt-pcr detection. d protection rate was determined by the number of unaffected chickens (without detection in tracheal and kidney)/total number of chickens in each group. glycoprotein is considered to be critical for the development of effective vaccines against ibv. our previous research presented that various influenza h1, h5 and h9 hemagglutinins (has) proteins containing replaced h3-wt tm showed increased immunogenicity (liu et al., 2014) . to improve the immunogenicity and protective efficacy of ibv s1 protein, here we generated two fusion proteins rs1-h3(tm) and rs1-ha2 from s1 protein of ibv and hemagglutinin of influenza virus h3n2. after challenged with virulent ibv m41 strain, our results demonstrated that fusion proteins performed better protection compared with inactivated m41 vaccine and recombinant rs1 protein alone, while the latter groups presented similar protection ratio. thus, an augment of the humoral and cellular immune responses was suggested to be induced by the fused rs1-h3(tm) and rs1-ha2 proteins. people suggested that humoral immunity is critical for disease recovery and virus elimination (cook et al., 1991) . consist with the immunity protective test, in this study, the level of specific antibodies induced by the recombinant rs1-h3(tm) and rs1-ha2 was higher than the rs1 and inactivated m41 group. one interesting thing is that, two weeks after booster vaccination (day 28), the antibody level induced by recombinant s1 protein was lower than that of inactivated virus in our study. it is possible that the inactivated m41 vaccine, which is derived from native viruses, comprised almost all the epitopes of ibv and thus presented better performance in inducing a ibv-specific antibody response after booster vaccination. it is generally known that cd3 + cd4 + helper t cells and cd3 + cd8 + ctls play a major role in inducing t cell-mediated cellular immunity (doherty et al., 1997) . some reports have demonstrated that the cd8 + ctl response are critical in the control of infectious bronchitis (ib) and more efficient than cd3 + cd4 + helper t cells in controlling respiratory virus infections (cook et al., 1991; thompson et al., 1997) . the cd4 + t-cell responses provide help for b-cells to mature and generate specific antibodies as well as directly antiviral cytokines, increase the proliferation, maturation, and functional activity of cd8 + ctl (doherty et al., 1997; kotani et al., 2000) . in this study, we found an significant increase of the cd3 + cd4 + and cd3 + cd8 + t lymphocyte subgroups in chickens immunized with rs1-h3(tm) and rs1-ha2, comparing to the rs1 and inactivated m41 vaccine groups. the ratio of cd3 + cd8 + to cd3 + cd4 + t lymphocytes in chickens immunized with rs1-h3(tm) or rs1-ha2 was greater than that in chickens inoculated with rs1 and inactivated m41 vaccine. these findings indicate the advantage of employing fused s1 protein to tms or ha2 of ha proteins in an effectively stimulating cellular immune responses, especially in promoting the ctl response, which is critical for virus clearance and recovery of chickens from ibv infections (collisson et al., 2000) . in summary, here we have successfully expressed two fused s1 proteins, rs1-ha2 and rs1-h3(tm) proteins, and demonstrated that these fusion proteins are superior to rs1 protein alone in terms of immunogenicity and protective efficacy. accordingly, our results suggest that the two recombinant fusion proteins rs1-ha2 and rs1-h3(tm) are suitable vaccine candidates for the development of vaccines against ibv. emergence of novel strains of avian infectious bronchitis virus in sweden development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex 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 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receptors immunogenicity and protection efficacy of monomeric and trimeric recombinant sars coronavirus spike protein subunit vaccine candidates assembly and immunogenicity of coronavirus-like particles carrying infectious bronchitis virus m and s proteins recombinant influenza h1, h5 and h9 hemagglutinins containing replaced h3 hemagglutinin transmembrane domain showed enhanced heterosubtypic protection in mice avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination attenuated live vaccine usage affects accurate measures of virus diversity and mutation rates in avian coronavirus infectious bronchitis virus infectious bronchitis virus: immunopathogenesis of infection in the chicken a simple method of estimating fifty percent endpoints neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes infectious bronchitis virus variants: a review of the history, current situation and control measures induction of protective immunity in chickens vaccinated with infectious bronchitis virus s1 glycoprotein expressed by a recombinant baculovirus systemic and local antibody responses to infectious bronchitis virus in chickens inoculated with infectious bursal disease virus and control chickens the avian coronavirus spike protein bacmam virus-based surface display of the infectious bronchitis virus (ibv) s1 glycoprotein confers strong protection against virulent ibv challenge in chickens a recombinant baculovirus-expressed s glycoprotein vaccine elicits high titers of sars-associated coronavirus (sars-cov) neutralizing antibodies in mice key: cord-286703-ipoj13va authors: de wilde, adriaan h.; falzarano, darryl; zevenhoven-dobbe, jessika c.; beugeling, corrine; fett, craig; martellaro, cynthia; posthuma, clara c.; feldmann, heinz; perlman, stanley; snijder, eric j. title: alisporivir inhibits mersand sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model date: 2017-01-15 journal: virus res doi: 10.1016/j.virusres.2016.11.011 sha: doc_id: 286703 cord_uid: ipoj13va currently, there is no registered treatment for infections with emerging zoonotic coronaviruses like sarsand mers-coronavirus. we here report that in cultured cells low-micromolar concentrations of alisporivir, a non-immunosuppressive cyclosporin a-analog, inhibit the replication of four different coronaviruses, including mersand sars-coronavirus. ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of sars-cov infection in a mouse model. nevertheless, our data provide a basis to further explore the potential of cyp inhibitors as host-directed, broad-spectrum inhibitors of coronavirus replication. the outbreak of the severe acute respiratory syndromecoronavirus (sars-cov) in 2003 and the continuing circulation of the middle east respiratory syndrome-coronavirus (mers-cov; since 2012) have highlighted the potentially lethal consequences of zoonotic coronavirus infections in humans. approximately 8000 people were infected during the sars-cov epidemic (∼10% mortality; http://www.who.int/csr/sars/en/), while the global mers tally is now over 1800 laboratory-confirmed cases, with a mortality of about 35% (http://www.who.int/csr/disease/coronavirus infections/en/). in mid-2015, an air travel-related outbreak in south korea (186 confirmed cases, 36 deaths, and about 17,000 potentially exposed individuals quarantined) again illustrated the socioeconomic impact of this kind of emerging pathogen, also outside regions in which the virus is endemic (http://www.who.int/ csr/don/07-july-2015-mers-korea/en/). the lack of effective methods to prevent or treat coronavirus infections in humans remains a serious public health concern, especially given increasing evidence that sars-like coronaviruses continue to circulate in bats and may have the potential to readily cross the species barrier and emerge as human pathogens (ge et al., 2013; menachery et al., 2015) . data from cell culture infection models (chan et al., 2013a (chan et al., , 2013b de wilde et al., 2013b; falzarano et al., 2013a; kindler et al., 2013; zielecki et al., 2013) and experiments in rhesus macaques (falzarano et al., 2013b) and marmosets (chan et al., 2015) suggested that interferons (ifns) are potent inhibitors of mers-cov replication. the outcome of the clinical use of interferons was variable, with some reports questioning the long-term survival benefits (al-tawfiq et al., 2014; omrani et al., 2014; shalhoub et al., 2015) , whereas others suggested that further investigation is warranted (khalid et al., 2016) , possibly in combination with the use of lopinavir/ritonavir (kim et al., 2015) . unfortunately, some of the negative outcomes are confounded by the late-stage treatment of critically-ill patients. since the development and registration of novel therapeutic compounds is generally time consuming, the repurposing of approved drugs offers one of the few shortcuts to establishing anticoronavirus therapy. several fda-approved compounds have been reported to inhibit mers-cov and sars-cov replication in cell culture (de wilde et al., 2014; dyall et al., 2014; hart et al., 2013) , but their efficacy in animal models remains to be determined. previously, the fda-approved drug cyclosporin a (csa) was shown to inhibit the replication of a variety of viruses (reviewed in nagy et al., 2011) , including coronaviruses (carbajo-lozoya et al., 2014; de wilde et al., 2013b de wilde et al., , 2011 kim and lee, 2014; pfefferle et al., 2011; http tanaka et al., 2012) . csa targets members of the cyclophilin (cyp) family, which are peptidyl-proline isomerases (ppiases) that act as chaperones in protein folding and function (davis et al., 2010) . since the immuno suppressive properties of csa (schreiber and crabtree, 1992) are an undesirable side-effect in the context of antiviral therapy, numerous non-immunosuppressive cyp inhibitors have been developed. among these, alisporivir (alv) showed increased sustained viral response during treatment of chronic hepatitis c virus (hcv) infection in phase iii clinical trials, when treatment with ribavirin and pegylated interferon was combined with alv (buti et al., 2015; flisiak et al., 2012; pawlotsky et al., 2015; zeuzem et al., 2015) . in this study, we have first investigated whether alv inhibits mers-and sars-cov replication in cell culture. we employed previously described cell-culture based screening assays relying on the rapid cytopathic effect (cpe) observed in coronavirus-infected cells (de wilde et al., 2013b (de wilde et al., , 2014 . first, the viability of mers-cov-infected vero cells (moi 0.005) treated with increasing alv concentrations was assessed at 3 days post infection (p.i.). we found that virus-induced cpe was prevented by alv in a dosedependent manner (ec 50 3.6 m; table 1 . we next established that alv treatment also reduced the yield of infectious mers-cov progeny. vero and huh7 cells were infected with mers-cov (moi 0.01) and treated with increasing but noncytotoxic alv concentrations (for cell viability data, see fig. 1 ; grey lines) from 1 h post infection (p.i.) onward. progeny virus from mers-cov-infected vero cells was harvested at 48 h p.i. and titrated by plaque assay, which revealed a ∼2 log reduction when using 6.3 m alv ( fig. 2a ). this effect was more pronounced in huh7 cells, as a comparable decrease in progeny titer was already observed when using 3.1 m alv (fig. 2b ). similar results were obtained using fully differentiated primary human airway epithelial (hae) cells, of which the non-ciliated cells were shown to be the primary target for mers-cov infection (raj et al., 2013) . first, air-liquid interface cultures derived from two donors were cultured for 14 days on semi-permeable transwell membranes (van wetering et al., 2007) . next, these hae cell cultures were pretreated for 16 h with 25 m alv or 0.13% etoh (vehicle control) and infected with mers-cov isolate emc/2012 (moi of 2; titer determined on vero cells). after a 48-h alv treatment, mers-cov progeny titers were reduced by 1 or 3 log, depending on the donor (fig. 2c-d) . in a second laboratory, the ability of alv to inhibit mers-cov replication was assessed independently in vero, llc-mk2, and huh7 cells. cells were inoculated with mers-cov (emc/2012) at an moi of 0.001 and medium containing between 0.625 and 20 m of alv was added at 1 h p.i. supernatant was collected on day 3 and levels of infectious virus progeny assessed by tcid 50 assay. in parallel, toxicity was assessed using a cell viability assay. on day 3, a 4-to 5-log decrease in infectious progeny was observed at a dose of 10 m alv and ec 50 values of 3.9, 2.8, and 4.0 m were calculated for vero, llc-mk2, and huh7 cells, respectively ( fig. s1 and table 1) . sars-cov infection (moi 0.01) in vero cells was sensitive to 3.1 m alv (fig. 2e ), resulting in a near-complete block when using 6.3 m alv. a ∼1.5 log reduction in virus progeny could be achieved in veroe6 cells using the same dose (fig. 2f) , suggesting that alv treatment in veroe6 cells was twice less effective compared to vero cells. for both sars-cov and mers-cov, differences in alv sensitivity in various cell lines were observed. one explanation for such difference would be that alv uptake differs per cell line. interestingly, we also observed remarkable difference in e.g. sars-cov-induced cell death between veroe6 and vero cells, ranging from severe cpe in the former to minimal cpe in the latter after the same incubation period (unpublished observations). this suggests that these related cell lines can respond quite differently to virus infection. to investigate the potential of alv as a broad-spectrum coronavirus inhibitor, we assessed the effect of alv treatment on the replication of two additional coronaviruses, murine hepatitis virus (mhv; strain a59) and human coronavirus 229e (hcov-229e). following infection with an moi of 0.01, media were harvested at 16 h p.i. for mhv on 17cl1 cells or 48 h p.i. for hcov-229e on huh7 cells ( fig. 2g and h) . when using an alv dose of 6.3 m, mhv progeny titers were decreased by 1 log, while a 25-m dose resulted in a ∼2log reduction without showing any signs of toxicity in uninfected cells (cell viability values >85% of untreated 17cl1 control cells were observed for all alv concentrations tested; data not shown). hcov-229e production was reduced to a similar extent (∼1.5 and ∼2-log reduction at 6.3 and 12.5 m alv, respectively). as ribavirin has previously been reported to inhibit mers-cov replication (falzarano et al., 2013a) and alv and ribavirin have been used together during clinical trials for hepatitis c treatment (pawlotsky et al., 2015) , this combination was tested in llc-mk2 cells. combining ribavirin and alv treatment primarily had an additive effect on antiviral activity, with the exception of the combination of 25 g/ml ribavirin and 5 m alv, which had a synergy value of 1.51 (fig. 3a) as calculated using macsynergy ii (prichard and shipman, 1990) . increasing concentrations of ribavirin gradually lowered the cell viability measured without affecting cellular morphology when assessed microscopically (fig. 3b) . the above findings suggested that a combination approach might have a chance of success in animal models. therefore, we assessed whether similar inhibitory effects could be observed in a mouse model. we employed the mouse-adapted ma15 strain of sars-cov (roberts et al., 2007) and animals were treated daily with alv (60 mg/kg) and/or ribavirin (50 mg/kg). unfortunately, treatment with alv alone did not enhance survival (data not shown) and combined therapy with alv and ribavirin did not prevent weight loss (fig. 4a ) or enhance survival (fig. 4b) . since the alv concentration required for sars-cov inhibition in cell culture was >100-fold higher than that required for inhibition of hcv replication (coelmont et al., 2010; paeshuyse et al., 2006; puyang et al., 2010) , the negative outcome of this animal experiment may not be too surprising. nevertheless, the use of cyp inhibitors remains a promising and innovative antiviral strategy. this class of drugs can potentially inhibit against broad range of pathogenic viruses, including hepatitis b virus (phillips et al., 2015 ; ec 50 of 4.1 m), hcv (coelmont et al., 2010; paeshuyse et al., 2006; puyang et al., 2010; ec 50 values between 0.02 and 0.23 m), and human immunodeficiency virus 1 (ptak et al., 2008; ec 50 values in the low-nanomolar range). we previously reported that also the replication of two arteriviruses, which are distantly related to coronaviruses, can be blocked by the alv-related nonimmunosuppressive csa analog debio-064 (de wilde et al., 2013a). mock-infected cells that did not receive alv or solvent were used as a reference for cell viability (their relative viability was set at 100 %). cells were incubated for 3 days with the exception for mers-cov emc/2012 in huh7 cells (2 days) and cell viability was monitored using the celltiter 96 ® aqueous non-radioactive cell proliferation assay (promega). in addition, the (potential) toxicity of alv treatment was monitored in parallel in mock-infected cell cultures. graphs show the results (average and sd) of a representative experiment that was performed in quadruplicate. all virus-cell combinations were tested at least twice. lines represent relative cell viability in the absence of infection (alv toxicity control); bars represent relative cell viability after infection and alv treatment. i., virus titers in the culture medium were determined by plaque assays as described before (van den worm et al., 2012) . (c,d) hae cells from two different donors were cultured on semi-permeable 12-well transwell membranes for 14 weeks. about 16 h prior to infection, 25 m alv, 0.13% etoh, or medium was added to the basal side of the cell layer. subsequently, cells were apically infected with mers-cov (moi of 2; titer determined on vero cells). after 48 h, virus release from the apical side of the cell layer was determined by harvesting the mucous fluid and subsequent plaque assay. (e, f) sars-cov-infected (e) vero or (f) veroe6 cells (moi 0.01) were treated with various concentrations of alv from 1 h p.i. onwards, and virus titers in the culture medium at 32 h p.i. were determined by plaque assay. (g) 17cl1 cells infected with mhv-a59 (moi 0.01) or (h) huh7 cells infected with hcov-229e (moi 0.01) were treated with alv from 1 h p.i. onwards, and infectious progeny titers were determined at 16 h p.i. and 48 h p.i., respectively. the graphs show the results of one representative experiment (mean ± sd, n = 3). for all experiments, control infections include cells that remained untreated ("-") or are treated with an amount of etoh equalling that present at the highest alv concentration used. two-sided student's t test (graphpad prism 7 software) was used to determine the significance of inhibition of virus replication between etoh-treated and alv-treated samples ( * p < 0.05; ** p < 0.005; *** p < 0.001; n.s. not significant). our study reveals that alv is a broad-spectrum coronavirus inhibitor in cell culture, as it inhibits the replication of both alphaand betacoronaviruses (figs. 1 and 2 and carbajo-lozoya et al., 2014) . further research is needed to elucidate the exact mechanism of action underlying alv's interference with coronavirus replication, as well as the involvement of cyps in the coronavirus replication cycle. for hcv, alv has been shown to disrupt functional interactions between cyclophilin a (cypa) and viral proteins and/or rna (coelmont et al., 2010; garcia-rivera et al., 2012; nag et al., 2012) . interestingly, although alv has a ∼4 times higher affinity for cypa compared to csa (unpublished data), the ec 50 values for both inhibitors are similar, leaving the possibility that alv and other cyp inhibitors target cov replication independently of cypa. in a previous study, a 4-log reduction in hcov-nl63 virus progeny was reported upon treatment of infected caco-2 cells with 10 m alv (ec 50 0.8 m; carbajo-lozoya et al., 2014). the observed variations in ec 50 values suggest that different coronaviruses may not be equally sensitive to the drug, although they may also reflect differences in e.g. experimental design, cyp expression levels, and/or alv uptake or turnover in different cell lines. the lack of alv activity in the sars-cov animal model suggests that the drug itself may not be suited for the treatment of coronavirus infections. nevertheless, cyp inhibitors remain interesting leads for the development of host-directed anti-coronavirus therapy, as well as interesting tools to study the role of cyps in coronavirus replication in more detail. data are from two independent laboratories. ec50 and cc50 values were calculated as described previously (de wilde et al., 2014; falzarano et al., 2013a) . the selectivity index (si), the relative efficacy of a compound in specifically inhibiting virus replication, was calculated as cc50/ec50. statistical analyses were performed using the results of at least two independent experiments. a ec50 and cc50 values are means (± se) from a representative experiment (n = 4) that was repeated at least twice. b si: selectivity index, calculated as cc50/ec50. c data is presented in fig. 1 . d virus yield is determined by tcid50 assay (data is presented in fig. s1 ; falzarano et al., 2013a) . e experiments performed to independently confirm the antiviral effect of alv in a second laboratory. to 100 g/ml ribavirin from 1 h p.i. onwards. virus titers in the culture medium at 3 days p.i. were determined by tcid50 as previously described (falzarano et al., 2013a) . (b) in parallel, control cells were treated with the same compound concentrations to determine cytotoxicity with a celltiter 96 aqueous one solution cell proliferation assay. ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study alisporivir with peginterferon/ribavirin in patients with chronic hepatitis c genotype 1 infection who failed to respond to or relapsed after prior interferon-based therapy: fundamental, a phase ii trial human coronavirus nl63 replication is cyclophilin a-dependent and inhibited by non-immunosuppressive cyclosporine a-derivatives including alisporivir broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus tropism and innate immune responses of the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset deb025 (alisporivir) inhibits hepatitis c virus replication by preventing a cyclophilin a induced cis-trans isomerisation in domain ii of ns5a structural and biochemical characterization of the human cyclophilin family of peptidyl-prolyl isomerases repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques update on alisporivir in treatment of viral hepatitis c multiple mutations in hepatitis c virus ns5a domain ii are required to confer a significant level of resistance to alisporivir isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards porcine epidemic diarrhea virus induces caspase-independent apoptosis through activation of mitochondrial apoptosis-inducing factor combination therapy with lopinavir/ritonavir, ribavirin and interferon-alpha for middle east respiratory syndrome: a case report efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential a sars-like cluster of circulating bat coronaviruses shows potential for human emergence suppression of viral rna binding and the assembly of infectious hepatitis c virus particles in vitro by cyclophilin inhibitors emerging picture of host chaperone and cyclophilin roles in rna virus replication ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study the non-immunosuppressive cyclosporin debio-025 is a potent inhibitor of hepatitis c virus replication in vitro alisporivir plus ribavirin, interferon free or in combination with pegylated interferon, for hepatitis c virus genotype 2 or 3 infection the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors alisporivir inhibition of hepatocyte cyclophilins reduces hbv replication and hepatitis b surface antigen production a three-dimensional model to analyze drug-drug interactions inhibition of human immunodeficiency virus type 1 replication in human cells by debio-025, a novel cyclophilin binding agent mechanism of resistance of hepatitis c virus replicons to structurally distinct cyclophilin inhibitors dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice the mechanism of action of cyclosporin a and fk506 ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study isolation of a novel coronavirus from a man with pneumonia in saudi arabia randomised clinical trial: alisporivir combined with peginterferon and ribavirin in treatment-naive patients with chronic hcv genotype 1 infection (essential ii) human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus cyclosporin a inhibits the replication of diverse coronaviruses cyclophilin inhibitors block arterivirus replication by interfering with viral rna synthesis mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture epithelial differentiation is a determinant in the production of eotaxin-2 and −3 by bronchial epithelial cells in response to il-4 and il-13 we thank dr. frauke fischer, dr. nikolai naoumov (novartis, switzerland) and dr. grégoire vuagniaux (debiopharm, switzerland) for helpful discussions and providing alisporivir. we supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2016.11. 011. key: cord-275016-ij5yaqkx authors: someya, yuichi; takeda, naokazu; miyamura, tatsuo title: characterization of the norovirus 3c-like protease date: 2005-03-08 journal: virus res doi: 10.1016/j.virusres.2005.02.002 sha: doc_id: 275016 cord_uid: ij5yaqkx the recombinant 3c-like protease of chiba virus, a norovirus, expressed in escherichia coli cells was purified and characterized as to effects of ph, temperature, salt contents, and sh reagents on its proteolytic activity. the optimal ph and temperature of the 3c-like protease for the proteolytic activity were 8.6 and 37 °c, respectively. increased concentration (∼100 mm) of monovalent cations such as na(+) and k(+) was inhibitory to the activity. hg(2+) and zn(2+) remarkably inhibited the protease activity, while mg(2+) and ca(2+) had no virtual effect. several sulfhydryl reagents such as p-chloromercuribenzoic acid, methyl methanethiosulfonate, n-ethylmaleimide and n-phenylmaleimide also blocked the activity, confirming the previous result that cysteine residue(s) were responsible for the proteolysis. viral proteases play a central role in the maturation of functional viral proteins, and hence in its genome replication and the formation of virus particles (dougherty and semler, 1993) . the 3c and 3c-like proteases belonging to the chymotrypsin-like protease superfamily are found in several animal, insect, and plant virus families such as picornaviridae, comoviridae, caliciviridae, coronaviridaee, and arteriviridae (dougherty and semler, 1993; gorbalenya et al., 1989) . their three-dimensional structures have been determined by x-ray crystallography in poliovirus , rhinovirus (matthews et al., 1994) , hepatitis a virus (bergmann et al., 1997) , and coronavirus (anand et al., 2002 yang et al., 2003) , tobacco etch virus (phan et al., 2002) and equine arteritis virus (barrette-ng et al., 2002) . these studies allowed us to identify the active-site amino acid residues with a combination of site-directed mutagenesis studies (boniotti et al., 1994; cheah et al., 1990; gosert et al., 1997; hammerle et al., 1991) . several 3c and 3c-like proteases were purified and their biochemical properties were well characterized (baum et al., 1991; chisholm et al., 2001; davis et al., 1997; ziebuhr et al., 1997) . they are characterized by their proteolytic activities being inhibited by both cysteine and serine protease inhibitors. as a result of these studies, therapeutic drugs, especially for rhinovirus, are being developed (hammerle et al., 1991; mckinlay, 2001; turner, 2001) . norovirus (formerly norwalk-like virus) is a genus of the family caliciviridae and is a major causative agent of nonbacterial acute gastroenteritis in humans (clarke et al., 1998; estes et al., 1997) . norovirus cannot be propagated in cell cultures, and animal models have not been developed, which hampers the progress of biochemical and molecular biological studies of norovirus. we cloned and sequenced the whole genome of a chiba strain of norovirus (chiba virus), and its 3c-like protease has been heterologously expressed in escherichia coli in an active form (someya et al., 2000) . furthermore, we identified the active-site amino acid residues and concluded that norovirus 3c-like protease had a catalytic dyad consisting of his30 and cys139 (someya et al., 2002) . this is a unique feature since most of the 3c and 3c-like proteases have a catalytic triad consisting of his, asp/glu, and cys/ser, similar to chymotrypsin (dougherty and semler, 1993; gorbalenya et al., 1989 shown that the active site of coronavirus 3c-like protease was also composed of two residues, his and cys (anand et al., 2002) . to obtain information regarding the biochemical properties of norovirus 3c-like protease, we purified the bacterially expressed recombinant 3c-like protease from chiba virus and characterized as to effects of ph, temperature, salt contents, and sh reagents on its activity. the dna fragment encoding all residues (ala1 to glu181) of the chiba virus 3c-like protease was amplified by pcr, in that ndei and aor51hi restriction sites were introduced at the 5 and 3 ends, respectively. the ndei-aor51hi fragment was placed downstream of the t7 promoter with a linker encoding a sequence containing six consecutive his residues. the resultant plasmid, pt7prohis, encodes a 3clike protease with a met residue in the n-terminal and with a saghhhhhhg sequence in the c-terminal. the ndei-sphi fragment from puchis3cd (someya et al., 2000) encoding the 3cd region was placed downstream of the t7 promoter to construct pt7his3cd. next, the c139a mutation of the 3c-like protease was introduced and designated pt7his3cd-c139a. the dna fragment encoding glutathione s-transferase (gst) was amplified by pcr, in that apai and sphi restriction sites were introduced at the 5 and 3 ends, respectively. the apai-sphi fragment encoding gst was ligated to the apai-sphi fragment of pt7his3cd-c139a. the resultant plasmid was designated pt7his3cd-gst. this plasmid encodes the n-terminal his-tag sequence (mgghhhhhhgasa), followed by the whole sequence of the c139a mutant 3c-like protease and gly1 to pro19 of 3d rna-dependent rna polymerase, and then followed by gst. e. coli bl21-codonplus (de3)-ril cells (stratagene, la jolla, ca) were transformed with pt7prohis or pt7his3cd-gst. cells were grown on medium a (mcmurry et al., 1980) supplemented with 0.2% glucose and 0.1% casamino acids at 37 • c. when the absorbance at 530 nm reached 0.5, iptg was added to a final concentration of 1 mm for induction of gene expression. after 2 h, cells were harvested, then washed and resuspended with 20 mm tris-hcl (ph 8.0) buffer containing 0.1 m nacl. cells were disrupted by sonication. after the removal of cell debris, the supernatant was subjected to ultracentrifugation (150,000 × g, 1 h). the resultant supernatant was mixed with talon metal affinity resin (clontech, palo alto, ca) equilibrated with 20 mm tris-hcl (ph 8.0) buffer containing 0.1 m nacl. the resin was washed with 20 mm tris-hcl (ph 8.0) buffer containing 0.1 m nacl and 0.1 m imidazole for removal of non-specifically bound proteins. the proteins of interest were eluted with 20 mm tris-hcl (ph 8.0) buffer containing 0.1 m nacl and 0.5 m imidazole and concentrated using ultrafree-15 unit with biomax-10 membrane (millipore, bedford, ma). proteins were determined by the method of bradford (bradford, 1976 ). usually, 5 m enzyme and 5 m substrate were reacted in 20 l of the mixture containing 50 mm bis-trispropane-hcl buffer (ph 8.62) for 16 h. details of reaction mixtures were described in the respective sections. the reaction was stopped by the addition of an equal volume of 2× sample buffer, and the proteins were then analyzed by sds-polyacrylamide (13%) gel electrophoresis. gels were stained with coomassie brilliant blue r-250. in order to estimate the efficiency of the proteolytic cleavage, density of the respective stained bands was scanned and calculated using nih image for macintosh version 1.62. the viral non-structural proteins and the proteolytic cleavage sites within the orf1 polyprotein are depicted in fig. 1a . in order to purify the chiba virus 3c-like protease, we constructed pt7prohis with the gene fragment encoding the protease with his-tag in its c-terminus (fig. 1b) . the prohis protein (calculated molecular mass of 20.6 kda) expressed from pt7prohis was used as an enzyme in this study. in order to observe proteolysis at the cleavage site between the 3c and 3d, we at first used the his-3cd-c139a mutant protein expressed from pt7his3cd-c139a as a substrate, which was the n-terminal his-tagged 3cd fragment containing the ala mutation of active-site cys139 of the 3c-like protease (fig. 1b) . however, this protein tended to aggregate at higher ph after prolonged incubation. other parts from the native chiba virus orf1 polyprotein could not be effectively produced in e. coli cells. therefore, the his3cd-gst protein (calculated molecular mass of 48.0 kda) was used as a substrate, which retains the cleavage site between the 3c and 3d. the region covering the entire 3c and the n-terminal 19 amino acids of 3d was placed under the control of t7 promoter with the n-terminal his-tag and the c-terminal glutathione s-transferase (gst), the resultant plasmid being designated pt7his3cd-gst (fig. 1b) . although this protein itself has the 3c-like protease moiety, it never undergoes autocatalytic cleavage because the c139a mutation is introduced. if his3cd-gst is cleaved at the 3c/3d junction (le/gg) by the active prohis, the his3c-c139a moiety of 20.7 kda and the 3d -gst moiety of 27.3 kda are produced. enzymatic reaction was initiated by mixing enzyme (prohis) and substrate (his3cd-gst). we could not obtain clear results when the reaction was performed in a short period (2-4 h) (data not shown). however, even if a substrate was mixed with an enzyme for a long period (more than 24 h), complete cleavage could not be achieved. one possibility for the incomplete cleavage is that the cleavage site of the substrate (his3cd-gst) could not be accessible by the protease because the substrate was not properly folded. alternatively, part of the cleavage site in the substrate might be masked by its own catalytic site or the catalytic site of other substrate molecule, so that the enzyme might have competed with the substrate for the cleavage site. very recently, belliot et al. (2003) reported trans cleavage activity of the 3c-like protease from the genogroup ii md145 norovirus (belliot et al., 2003) . they used bacterially expressed 3c-like protease with its c-terminus his-tagged as an enzyme and the entire orf1 protein or 3cd fragment containing the active-site cys mutation as a substrate which was expressed in the in vitro transcription/translation reaction. cleavage reaction was performed in the ph 7.4 buffer at 30 • c. data showed that the 3c-like protease cleaved not only the 3c/3d junction but also other proposed junctions, and that substrate protein appeared to be cleaved completely after 24-h incubation. however, consistent with our experiment, a long time incubation was required for the result being obvious. five micromolar enzyme and 5 m substrate were mixed in 50 mm bis-tris-propane-hcl buffer of various ph containing 25 mm nacl, and reacted at 37 • c for 16 h. fig. 2 shows that maximum cleavage was obtained at ph 8.62, although the proteolytic cleavage occurred over a wide range of ph. faint bands of 80 and 60 kda were thought to be dimers of the substrate and its cleaved product (3d -gst), respectively. when the efficiency of cleavage at ph 8.62 is 100%, those at ph 7.22, 7.66, 8.12, and 9.08 were estimated as 70, 86, 89, and 79%, respectively. a slope of relative activity was gentle at acidic ph below 8.62, while it was sharp at alkaline ph above 8.62. in the subsequent experiments, a buffer of ph 8.62 was used. five micromolar enzyme and 5 m substrate were mixed in 50 mm bis-tris-propane-hcl (ph 8.62) containing 25 mm nacl, and reacted for 16 h at 4, 15, 26, 37, or 48 • c. as shown in fig. 3 , optimal temperature for the activity was 37 • c. the activity at 4, 15, 26, and 48 • c was estimated as 23, 31, 55, and 80% of that at 37 • c, respectively. we next examined the effects of monovalent and divalent cations on the proteolytic activity. the activity was compared with that under the condition where 50 mm bis-tris-propane-hcl (ph 8.62) containing 5 mm edta fig. 3 . effects of temperature on protease activity. five micromolar enzyme (prohis) and 5 m substrate (his3cd-gst) were reacted in the 50 mm bis-tris-propane-hcl (ph 8.62) buffer containing 25 mm nacl for 16 h at the indicated temperature. without any cations was used. the addition of 25, 50, or 100 mm nacl decreased the proteolytic activity to 83, 52, or 36%, respectively (fig. 4a) . kcl also had an inhibitory effect on the activity (data not shown). as for the divalent cations, 5 mm mgcl 2 or 5 mm cacl 2 did not affect the activity (108 and 105%) (fig. 4b ). in contrast, 5 mm zncl 2 or 5 mm hgcl 2 completely inhibited the activity (fig. 4b ). this result was consistent with cys being a critical residue of the active site (someya et al., 2002) , and inhibition by zn 2+ indicated that norovirus 3c-like protease was different from metalloprotease. when hg 2+ was used, two faint bands of 30 and 15 kda slightly appeared (lane hgcl 2 , fig. 4b ). it was possible that high concentrations of hg 2+ allowed the enzyme to alternatively cleave the substrate and/or the enzyme itself. with the addition of 5 mm mncl 2 , part of the proteins used for reaction appeared to be aggregated since coomassiestained materials stayed in the well (fig. 4b ). because mn 2+ oxidized the proteins, the part may have remained in the well and the other part may be flown from the gel after degradation into small molecular weight peptides. most of the 3c and 3c-like proteases have a cysteine residue as a nucleophile of the active site (dougherty and semler, 1993; gorbalenya et al., 1989) . these proteases should therefore be sensitive to sulfhydryl reagents. in fact, as shown in fig. 4b , high concentrations (5 mm) of hg 2+ and zn 2+ blocked the activity of the 3c-like protease. in fig. 5 , the effects of several sulfhydryl reagents were tested. five micromolar substrate was reacted with 5 m enzyme in the presence of 10 m sulfhydryl reagents. ten micromolar hg 2+ or zn 2+ almost completely inhibited the activity. p-chloromercuribenzoate (pcmb), which includes an hg(ii) within the molecule, was also a good inhibitor of the 3c-like protease (82% inhibition). in contrast, p-chloromercuribenzenesulfonate (pcmbs) slightly inhibited the activity (27% inhibition), despite pcmbs also having an hg(ii) similarly to pcmb. methyl methanethiosulfonate (mmts), n-ethylmaleimide (nem), and n-phenylmaleimide (npm), which form a covalent bond with a sulfur atom of the cysteine residue, almost completely inhibited the activity. the reaction with nem and npm slightly altered the mobility of the enzyme molecules, possibly because a bulkier modified side chain of the active-site cysteine residue affected the structure of the enzyme, or because more than one molecule of the inhibitors attached to an enzyme. neither 10 m iodoacetate (iaa) nor iodoacetamide (iam) inhibited the activity. these two reagents may be less sensitive to an sh group than other modifying reagents. phenyl methylsulfonylfluoride (pmsf) is well known as an irreversible inhibitor of serine proteases. because the 3c and 3c-like proteases have a structural architecture similar to chymotrypsin, and with essentially the same proteolysis mechanism (dougherty and semler, 1993; gorbalenya et al., 1989) , pmsf may inhibit the activity of the 3c and 3c-like proteases. ten micromolar pmsf was less effective but significantly inhibited the activity of the chiba virus 3c-like protease (20% inhibition). the effect of ␤-mercaptoethanol (␤-me) was also examined here. the residual activity of the 3c-like protease was approximately 80% of the control in the presence of 10 m ␤-me (fig. 5) . even in the presence of much higher concentrations (5 mm) of ␤-me, approximately 60% activity remained (data not shown). these results suggest that a disulfide bond is not essential to the activity. however, we cannot exclude the possibility that a disulfide bond is buried inside the folded protein and cannot be reduced by high concentrations of ␤-me. the optimal ph for the proteolytic reaction by the chiba virus 3c-like protease was around 8.6, which is much more alkaline than physiological ph (fig. 2) . the 3c-like protease was sensitive to increased concentrations of na + (fig. 4a ). in addition, high concentrations of k + , which is abundant inside the cell in a normal state, were also inhibitory to the activity (data not shown). the presence of mg 2+ or ca 2+ did not affect the activity at all (fig. 4b) . these features might suggest that the 3c-like protease is localized and works in a limited environment inside the infected cells, rather than spreading throughout the cells. more information is necessary to elucidate when and where the 3c-like protease does work in the infected cell. we have previously shown that the active site of the chiba virus 3c-like protease is composed of cys139 and his30 (someya et al., 2002) . as expected, sulfhydryl reagents as well as hg 2+ and zn 2+ inhibited the activity of the 3c-like protease, although the degree of inhibition varied dependent on the sulfhydryl reagents used. small (hg 2+ , zn 2+ , and mmts) and hydrophobic (nem and npm) reagents tended to inhibit the activity more strongly than hydrophilic reagents (pcmb and pcmbs) (fig. 5) . this tendency may reflect the environment of the active site. the present results provide the basic biochemical features of the norovirus 3c-like protease. structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs structure of arterivirus nsp4. the smallest chymotrypsinlike proteinase with an alpha/beta c-terminal extension and alternate conformations of the oxyanion hole purification, properties, and mutagenesis of poliovirus 3c protease in vitro proteolytic processing of the md145 norovirus orf1 non-structural polyprotein yields stable precursors and products similar to those detected in calicivirus-infected cells the refined crystal structure of the 3c gene product from hepatitis a virus: specific proteinase activity and rna recognition identification and characterization of a 3c-like protease from rabbit hemorrhagic disease virus, a calicivirus a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3c cysteine protease and cellular trypsin-like serine proteases expression and partial purification of recombinant tomato ringspot nepovirus 3c-like proteinase: comparison of the activity of the mature proteinase and the vpg-proteinase precursor human enteric rna viruses: caliciviruses and astroviruses expression and purification of recombinant rhinovirus 14 3cd proteinase and its comparison to the 3c proteinase expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes norwalk and related diarrhea viruses cysteine proteases of positive strand rna viruses and chymotrypsinlike serine proteases a distinct protein superfamily with a common structural fold identification of active-site residues in protease 3c of hepatitis a virus by site-directed mutagenesis site-directed mutagenesis of the putative catalytic triad of poliovirus 3c proteinase structure of human rhinovirus 3c protease reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein recent advances in the treatment of rhinovirus infections active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in escherichia coli refined x-ray crystallographic structure of the poliovirus 3c gene product structural basis for the substrate specificity of tobacco etch virus protease complete nucleotide sequence of the chiba virus genome and functional expression of the 3c-like protease in escherichia coli identification of active-site amino acid residues in the chiba virus 3c-like protease the treatment of rhinovirus infections: progress and potential the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor biosynthesis, purification, and characterization of the human coronavirus 229e 3c-like proteinase we thank t. mizoguchi for her secretarial work. this work was supported in part by a grant for research on emerging and re-emerging infectious diseases from the ministry of health, labor, and welfare. key: cord-330260-xuw31zfn authors: chen, hui-wen; huang, yuan-pin; wang, ching-ho title: identification of taiwan and china-like recombinant avian infectious bronchitis viruses in taiwan date: 2009-01-20 journal: virus res doi: 10.1016/j.virusres.2008.11.012 sha: doc_id: 330260 cord_uid: xuw31zfn infectious bronchitis virus (ibv) infections in poultry cause great economic losses to the poultry industry worldwide. the emergence of viral variants complicates disease control. the ibv strains in taiwan were clustered into two groups, taiwan group i and taiwan group ii, based on the s1 gene. a variant was previously identified and showed a distinct s1 gene homology with other local strains. this study investigated the 3′ 7.3 kb genome of eight taiwan strains isolated from 1992 to 2007. the genes of interest were directly sequenced. sequence analyses were performed to detect any recombination event among ibvs. the results demonstrated that all of the examined viruses maintained the typical ibv genome organization as 5′-s-3a-3b-e-m-5a-5b-n-utr-3′. in the phylogenetic analyses, various genes from one strain were clustered into separate groups. moreover, frequent recombination events were identified in the simplot analyses among the taiwan and china ck/ch/ldl/97i-type strains. putative crossover sites were located in the s1, s2, 3b, m genes and the intergenic region between the m and 5a genes. all of the recombinants showed chimeric ibv genome arrangements originated from taiwan and china-like parental strains. field ibvs in taiwan undergo genetic recombination and evolution. avian infectious bronchitis virus (ibv), with a large infectious rna genome (27.6 kb), belongs to the family coronaviridae, the group 3 coronavirus. the rna molecule of the ibv is linear, singlestranded, positive sense and possesses a 5 cap and a 3 poly-a tail. ibvs can replicate in respiratory, alimentary and urogenital tracts in chickens of all ages, resulting in great economic losses to the poultry industry worldwide (cavanagh and naqi, 2003) . clinical signs associated with respiratory and enteric tracts and renal damage might be found in infected chickens (cavanagh, 2007) . during infection, ibv possesses a unique discontinuous transcription system, i.e. a nested set of 3 co-terminal subgenomic mrnas sharing a common leader sequence in the 5 end are transcribed in the presence of negative-stranded rna intermediates. the viral polymerase "jumping" possibly contributes to the high rna recombination frequency in coronaviruses (lai, 1992; lai and holmes, 2001) . the ibv genome encodes four structural proteins: spike glycoprotein (s), envelope protein (e; also known as sm), membrane glycoprotein (m), and nucleocapsid protein (n) (cavanagh and naqi, 2003) . the spike glycoprotein is post-translationally cleaved into s1 * corresponding author. tel.: +886 2 23690628; fax: +886 2 23631542. e-mail address: chingho@ntu.edu.tw (c.-h. wang) . and s2 subunits. the s1 subunit anchors onto the viral outer membrane using the s2 subunit to form a club-shaped projection on the mature virion (cavanagh, 1983) . the s1 glycoprotein is involved in cell attachment and carries epitopes for serotype-specific hemagglutination-inhibition and virus-neutralization antibodies (cavanagh and davis, 1986; hodgson et al., 2004; ignjatovic and sapats, 2005; koch et al., 1990) . the s2 glycoprotein, in which two antigenic determinants were identified (koch et al., 1990) , may possess different secondary structures that affect the s1 specific antibody binding (callison et al., 1999) . the membrane glycoprotein is associated with virus assembling and budding (lai and holmes, 2001) . the nucleocapsid protein interacts with genomic rna to form the viral nucleocapsid, playing a role in viral rna synthesis and cell immunity (lai and holmes, 2001) . in addition, four non-structural proteins of unknown function are expressed by the polycistronic genes, gene 3 and 5 . virus mutants carrying truncated 3b genes demonstrate increased virulence and growth advantages in vitro and in ovo (shen et al., 2003) . the open reading frames (orfs) of 3a, 3b, 5a, and 5b encode accessory proteins not essential for ibv replication (casais et al., 2005; hodgson et al., 2006; youn et al., 2005) . as a signature of avian coronaviruses, ibv strains are continuously evolving through point mutations and recombination of their genomes. those variants have better adaptation or increased virulence advantageous to ib outbreaks. to date, a large number of ibv sero-or genotypes have been identified worldwide (cavanagh, 2007 1171/92 1992 broiler 3 taoyuan nephropathogenic tw-i 2296/95 1995 broiler 2 taoyuan nephropathogenic tw-ii 2575/98 1998 broiler 4 changhua nephropathogenic tw-i 2992/ spike glycoprotein gene. (bochkov et al., 2006; dolz et al., 2006; dolz et al., 2008; jackwood et al., 2007) . it was reported that slight sequence differences in the s1 gene probably lead to poor crossprotection (cavanagh et al., 1997) . viruses of different types can co-circulate within a region (capua et al., 1999; liu et al., 2006) , raising the inter-strain rna recombination frequency (bochkov et al., 2007; jia et al., 1995; lee and jackwood, 2000) . the wide use of live virus vaccine may also critically contribute to the genetic evolution of ibvs by acting as a heterologous rna donor template (kusters et al., 1990; wang et al., 1993) . the emergence of viral variants has complicated disease control requiring persistent ibv molecular surveys. ibv strains in taiwan were previously clustered into two groups, taiwan group i (tw-i) and taiwan group ii (tw-ii), on the basis of the s1 gene (wang and tsai, 1996) . however, a variant isolated in 2002 showed an unusually high s1 gene homology with china strains, but not in the n gene, suggesting an inter-strain recombination event (huang et al., 2004) . in this study, the 3 7.3 kb genomes from taiwan ibv strains were investigated to elucidate the genetic diversity of viruses. eight ibvs isolated in taiwan from 1992 to 2007 (huang et al., 2004) and the vaccine strain h120 (abic biological laboratories teva ltd., israel) were recovered for this study. the case histories of local strains are listed in table 1 . viruses were propagated in the allantoic cavity of 9-11-day-old specific pathogen free embryonated eggs (animal health research institute, council of agriculture, tamsui, taiwan). each egg received 0.1-0.2 ml inoculation. after 48-72 h incubation, allantoic fluid was collected and frozen at −80 • c until use. viral rna was extracted from 200 l of virus-infected allantoic fluid using a viral nucleic acid extraction kit (geneaid biotech ltd., taipei, taiwan) following the manufacturer's protocol. previously published primers (huang and wang, 2007) were employed in this study to amplify the gene fragments. for sequencing the 3 untranslated region (utr) of h120, one additional forward primer was designed as 5 -ggaacaatgcacagctggaa-3 from strain h120-gd (genbank accession no. ay028296). reverse transcriptase-polymerase chain reaction (rt-pcr) was performed with one step in a reaction volume of 50 l containing 0.5 l of 5 u/l realtaq dna polymerase (real biotech, taipei, taiwan dna sequencing in both strands from separate rt-pcr products was conducted by a commercial service (mission biotech). each nucleotide was determined from at least four identical results. the obtained nucleotide sequences were compiled and the amino acid sequences were deduced using dnastar software (dnastar, madison, wi). the ibv reference strains were retrieved from the genbank database with the accession number listed in table 2 . the alignx program of the vector nti suite 8 software table 2 ibv reference strains included in this study. country accession numbers armidale australia dq490205 (s1-n) a vic australia dq490221 (s1-n) bj china ay319651 (s1-n) taiwan dq646406 (s1-n) 2296/95 taiwan dq646404 (s1-n) 2575/98 taiwan dq646405 (s1-n) beaudette usa nc 001451 (s1-n) cal99 usa ay514485 (s1-n) cu-t2 usa u49858 (s1-n) de072 usa af274435 (s1), ay024337 (s2) (informax, north bethesda, ml) was used to generate multiple sequence alignments and determine the nucleotide identity. phylogenetic analyses were performed with the neighbor-joining method using mega version 4 (tamura et al., 2007) . the bootstrap values were determined from 1000 replicates of the original data. the consecutive ibv nucleotide sequences from the s to n genes (6.8 kb) based on the multiple alignment results were introduced into similarity plots with simplot version 3.5.1 (lole et al., 1999) . the nucleotide identity was calculated using the kimura 2-parameter method with a transition-transversion ratio of 2 in each window of 500 bp. the window was successively furthered along the alignment using a 20-bp increment. at least four sequences were required to initiate an analysis. the ibv sequences resulting from this study were submitted to the genbank database. the accession numbers are eu822336 (strain 3468/07), eu822337 (strain 3374/05), eu822338 (strain 3263/04), eu822339 (strain 3071/03), eu822340 (strain 2992/02), and eu822341 (strain h120). fig. 1 . phylogenetic analyses of the taiwan strains (᭹), h120 ( ) and reference strains for structural and non-structural protein genes of ibvs. the phylogenetic trees were constructed using the mega version 4 by the neighbor-joining method (bootstrapping for 1000 replicates with its value >70%). sequences that covered the 3 7.3 kb genome were determined from the taiwan ibvs and strain h120. all of the examined viruses were found to maintain the typical ibv genome organization as 5 -s-3a-3b-e-m-5a-5b-n-3 (data not shown). the s gene size ranged from a minimum of 3471 nucleotides (strain 3071/03) to a maximum of 3501 nucleotides (strains 2992/02 and 3374/05). compared with other local strains, single base mutations in the 3 end of the s and 3b genes from the strain 3071/03 changed the genetic code from gaa to taa (glutamine → stop codon), resulting in 27-and 3-base truncated orfs, respectively. similarly, in the 3374/05, a single base t-insertion in the 3b gene created an early stop codon, leading to a 48-base truncation. however, the 3a, 5b, and n gene orf sizes were conserved among ibvs. all of the virus genomes carried an intergenic (ig) region located between the m and 5a genes with a size of 351-362 nucleotides. the ig region of the strain h120 was 55 bases longer than that of the strain beaudette. the 3 utr, downstream of the stop codon in the n gene, a region of 475 and 500 nucleotides was sequenced from local strains and h120, respectively. all of the local strains shared a high sequence identity (96-100%) in this region. however, only 52% and 73% identity were observed between h120 and its closely related m41 and beaudette strains, respectively. phylogenetic analyses were performed based on the nucleotide sequence alignment using each orf from the s to n genes among eight taiwan and reference strains (fig. 1) . in the s1 gene, the taiwan strains showed the highest identity (84%) with the beaudette reference strain and the lowest (60%) with the de072. the taiwan strains (except for 2992/02 and 3374/05) could be clustered into two groups, tw-i and tw-ii, based on the s1 gene. strains 2992/02 and 3374/05 were closely related (>95% identity) to the china genotype vii strains (ck/ch/ldl/97i, ck/ch/ldl/98i, q1, j2, and t3) (liu et al., 2006) . in the s2 gene, all of the local strains were grouped with ck/ch/ldl/97i and ck/ch/ldl/98i except for the two viruses isolated before 1995 (strains 1171/92 and 2296/95). strains 2575/98 and 2992/02 were classified with ck/ch/ldl/97i and ck/ch/ldl/98i in the analyses of the 3a and 3b genes. the e gene analysis segregated the 2992/02 into the china group. strains 2992/02 and 3468/07 were distributed with the china strains in the m gene analysis. the 5a, 5b and n genes analyses revealed that all of the local strains were in the same group. nucleotide sequences from the three orfs were conserved among the taiwan strains with >92% homology. the 3 6.8 kb genome sequence (s-n gene) of six taiwan ibvs were queried in the simplot analysis. strains 1171/92, h120 and the china strain ck/ch/ldl/97i were used as putative parental strains when strains 2575/98, 2992/02, 3071/03, 3374/05, 3468/07 (tw-i) were queried. the parental strain 1171/92 was replaced with 2296/95 when strain 3263/04 (tw-ii) was queried. the similarity plot displays the consecutive nucleotide identity (%) among the queried strain and parental strains. strains were considered as recombinants if any crossover event took place between two putative parental strains. the breakpoint in which the parental strains have equal identity to the query strain is the predicted recom-bination site. as fig. 2 demonstrates, crossover events between parental 1171/92 or 2296/95 and ck/ch/ldl/97i were detected in each plot. the recombination sites were located in the s1, s2, 3b, m genes, and the ig region between the m and 5a genes. each putative recombinant was schematically assembled using taiwan and china-like sequence fragments. the genomic positions of those crossover sites were indicated within the plot (numbers in red). in taiwan, field outbreaks are frequently reported despite routine vaccine use. there were originally two ibv genotypes (tw-i and tw-ii) circulating in the field wang and tsai, 1996) until genetic variants 2992/02 (huang et al., 2004) and 3374/05 emerged. both variants showed a high s1 gene homology with the proventriculus pathogenic strains j1, q2, t3, ck/ch/ldl/97i and ck/ch/ldl/98i, belonging to the china genotype vii (liu et al., 2006; yu et al., 2001) . the 3 6.8 kb gene of ck/ch/ldl/97i was used as a putative parental strain. mass type viruses have been widely used as vaccine strains in taiwan. our primary concern was the possible viral recombination resulting from the co-circulation of heterologous vaccine strains in flocks. the consecutive sequence of 3 7.3 kb genome in strain h120 was obtained first in this study and served as another putative parental strain. to our surprise, six local strains experienced crossover events with the strain ck/ch/ldl/97i instead of h120. thus, inter-strain recombination events had occurred between the ibvs from taiwan and china. taiwan and china are geographically separate. neither live poultry nor processed poultry products from china have been allowed to import into taiwan for years. how those recombinants arose remains unknown. a recently identified ibv isolate in korea (kr/d64/05) also revealed a close relationship to the china ck/ch/ldl/97i-type strains (lee et al., 2008) . furthermore, the appearance of the chinalike strains in taiwan is reminiscent of the spread of the china qxibv strain in european countries (beato et al., 2005; domanska-blicharz et al., 2006) . in this case, it could be speculated that migrating birds provide the genetic sources of ibv variants in taiwan. a chicken-nephropathogenic ibv strain was identified from a non-diseased teal (anas sp.) in china (liu et al., 2005) . thus, the transport of ibvs over long distances by other avian species appears to be possible (cavanagh, 2005) . however, we still cannot overlook illegal trafficking or unapproved vaccine use in the field. all six recombinants defined in this study were inter-genotypic recombination and the "china-like" sequence substitutions took place in multiple genes. in particular, partial s gene replacement was observed in every recombinant. since coronaviruses possess different host range or cell tropism through the variance in the s gene (casais et al., 2003; kuo et al., 2000) , alterations in the antigenic characteristic in those variants could be expected. in this study, however, nearly all of the strains showed nephropathogenicity (lesions of kidney) in chickens, rather than the proventricular lesions caused by the ck/ch/ldl/97i-type strains. how a variant with a chimeric genome arrangement from heterologous strains demonstrates its tissue tropism or pathogenicity in a host is not clear. in addition, it was found that chicks challenged with virulent ck/ch/ldl/97i were incompletely protected by commercial vaccines and other heterologous strains (liu et al., 2007) . to effectively control the ib disease in taiwan, the protective effect of vaccines against challenges from those recombinants awaits to be investigated. to our knowledge, this is the first use of simplot for genetic analyses of ibv strains. the similarity plot can depict the genetic distance among the aligned sequences in a graphical window. in this study ibv recombinants could be defined directly from the simplot analyses, and the crossover events and corresponding genome positions were readily observed. in phylogenetic analyses, strains were deduced as recombinants if different genes from the genomes were clustered into separate phylogenetic groups. parallel results were obtained from both analyses. the emergence of ibv variants through rna recombination was previously described (brooks et al., 2004; jia et al., 1995; lee and jackwood, 2000; mondal and cardona, 2007; wang et al., 1993) . recombination events occurred in multiple genes. the consensus ig sequences ct(t/g)aacaa or the conserved regions around were assumed as the recombination "hot spots" in ibvs (lee and jackwood, 2000) . in addition, the cttttg sequence was observed adjacent to the putative recombinant junction (brooks et al., 2004; wang et al., 1993) . in this study, t-rich motifs (a/t/g)ttttg, consensus among a recombinant and its parental strains, were located upstream of several crossover sites. for instance, the motifs were found in the s2 gene of the 3263/04, the 3b gene of the 2575/98, and the ig region of the 2992/02 and 3468/07. all of the recombinants experienced crossover events in the 3 end of the s gene, indicating a possible region for template switching in viral rna synthesis. similar results were obtained from the ibv strain cu-t2 and cal99, whose recombination sites were deduced in the 3 751 and 700 nucleotide of the s gene, respectively (jia et al., 1995; mondal and cardona, 2007) . in addition to viral recombination, point mutation is a way of virus evolution. the single base nonsense mutations resulted in truncations of the s and 3b genes in the taiwan strains 3071/03 and 3374/05. the naturally occurring ibv strains with mutations or complete deletions in non-structural orfs 3a, 3b, 5a, and 5b revealed lower virus growth rate or titer in ovo (liu et al., 2008) and in vivo (mardani et al., 2008) . this study describes the dual viral genomes incorporation in the separated areas having no known interactions between poultry. the molecular investigation in the 3 7.3 kb of the ibv genome demonstrates that inter-strain recombination events contribute to the genetic diversity of the taiwan strains. field ibvs in taiwan undergo genetic recombination and evolution, which might lead to disease control difficulty. the biological and antigenic characteristics of the ibv variants await further studies. evidence of circulation of a chinese strain of infectious bronchitis virus (qxibv) in italy molecular epizootiology of avian infectious bronchitis in russia phylogenetic analysis of partial s1 and n gene sequences of infectious bronchitis virus isolates from italy revealed genetic diversity and recombination genes 3 and 5 of infectious bronchitis virus are accessory protein genes comparisons of envelope through 5b sequences of infectious bronchitis coronaviruses indicates recombination occurs in the envelope and membrane genes infectious bronchitis virus s2 gene sequence variability may affect s1 subunit specific antibody 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characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens the financial support of the national science council and council of agriculture, executive yuan, taiwan, is greatly appreciated. key: cord-338602-6n309bnp authors: gadotti, ana carolina; de castro deus, marina; telles, joao paulo; wind, rafael; goes, marina; garcia charello ossoski, roberta; de padua, alessandra michalski; noronha, lucia; moreno-amaral, andrea; baena, cristina pellegrino; tuon, felipe francisco title: ifn-γ is an independent risk factor associated with mortality in patients with moderate and severe covid-19 infection date: 2020-09-23 journal: virus res doi: 10.1016/j.virusres.2020.198171 sha: doc_id: 338602 cord_uid: 6n309bnp background: innate and adaptive immune responses have been evaluated in infected patients with covid-19. the severity of the disease has been supposed to be associated with some profile not reported with other bacterial and viral pneumonia. we proposed a study in patients with moderate to severe covid-19 infection to evaluate the interleukin patterns and its role as prognosis factors. methods: a prospective cohort with moderate and severe cases of covid-19 infection from june to july 2020. blood samples from patients were collected regularly to evaluate ifn-γ, tnf-α, il-4, il-6, and il-10. clinical, laboratory, radiological data, and outcomes were recorded. the outcome variable was in-hospital death, survival, mechanical ventilation, and admission at the intensive care unit. data are presented in median and interquartile range [iqr]. results: we evaluated the th1 and th2 responses according to evolution, distinguishing possible predictive markers. the ifn-γ median of 323 pg/ml [iqr 166-570] was found in patients who died and 208 pg/ml [iqr 155-392] in the survival group (p = 0.017). ifn-γ was also higher in the early stages of the disease (394 pg/ml [iqr 229-575] against 162 pg/ml [iqr 117-259], p < 0.001). il-4 that was increased in late-stage (182 pg/ml [iqr 162-199] against 131 pg/ml [iqr 124-152], p < 0.001) but not associated with mortality. also, death was also related to male gender (relative risk = 1.5 [95% confidence interval = 1.1-2.0]). conclusion: our results suggest that the activation of the host immune response between th1 or th2 in covid-19 infection may be related to the final result between discharge or death. this implies an attempt to control cytokines, such as ifn-γ, with combined therapies for clinical treatment. in early 2020, the world health organization declared pandemic due to coronavirus disease 2019 . since then, a large number of researches have been published, from epidemiological to experimental studies, trying better to understand immunological pathways of covid-19 and possible treatments. cd3+, cd4+, and cd8+ lymphocytes count are usually decreased according to disease stages 1 . besides it, cytokine storm is also present in severe patients due to elevation of interleukins such as tnf-α, il-6, il-8, and il-10 2, 3 . thus, the differences among host immune responses play a major role in covid-19 severity. older age, elevated c-reactive protein, serum lactate dehydrogenase, bilirubin, blood urea nitrogen, and decreased albumin are described as prognostic factors of severe covid-19 4 . comorbidities are also described as prognosis factors and the higher the number of them, the poorer the prognosis (i.e., hypertension, diabetes, chronic pulmonary disease, coronary artery disease, malignancies) 5 . interestingly, in an italian and spanish cohort, genetic factors related to abo blood-group system were reported as susceptibility to covid-19 respiratory failure 6 . different interleukins are described as prognostic factors. a reasonable hypothesis is that (i) pro-inflammatory innate immunity and (ii) anti-inflammatory system are related to disease severity or death once il-6, il-8, and il10 are closely described as prognostic factors in patients diagnosed with covid-19 1, 3, 7 . nevertheless, few studies have focused on the adaptive immune system to evaluate the balancing between th1 and th2 response. consequently, il-4 is usually not studied concomitant with ifn-γ and il-6 to better characterize th2, th1, and innate immune response. ifn-γ is the type ii ifn produced by nk cells and t lymphocytes, although cells of different phases of the immune response (innate and adaptative, respectively), this cytokine is important in all phases of the immune response 8 . j o u r n a l p r e -p r o o f ifn-γ system is essential for antiviral defense. ifn-γ downregulate virus replication and it activates cytokine production by t cells, augmenting the cytotoxic t lymphocyte killing activity 9 . however, persistent high levels of ifn-γ worsens the systemic inflammation, and increasing tissue injury and organ failure 10 . considering this ambiguous role of ifn-γ in the outcome, it is important to understand the serum pattern of this and other cytokines in patients with covid-19. the aim of this study was to investigate the interleukins patterns in patients admitted due to covid-19 and its role as prognosis factors. j o u r n a l p r e -p r o o f this was a prospective cohort of hospitalized patients diagnosed with covid-19 conducted in the south of brazil, curitiba, paraná, from june to july 2020. patients were admitted only after approval of the research ethics committee and an approved consent form. blood samples from patients were collected to evaluate ifn-γ, tnf-α, il-4, il-6, and il-10. clinical, laboratory, radiological data, and outcomes were recorded. covid-19 infection was defined by clinical-radiological presentation plus a nasopharyngeal swab polymerase chain reaction (pcr) positive to covid-19. inclusion criteria were hospitalized patients with moderate or severe confirmed covid-19 infection. the moderate disease was defined as an adult with clinical signs of pneumonia (fever, cough, dyspnea, fast breathing) but no signs of severe pneumonia, including spo2 ≥ 90% on room air; and severe disease as an adult with clinical signs of pneumonia (fever, cough, dyspnea, fast breathing) plus one of the following: respiratory rate > 30 breaths/min; severe respiratory distress; or spo2 < 90% on room air 11 . patients diagnosed with other viral infections, such as hiv, hcv, hbv, or another common respiratory virus, were excluded, as well as solid organ or hematological transplantation patients. patients who used tocilizumab were also excluded. j o u r n a l p r e -p r o o f patients were classified according to days of symptoms: 0-10 days and >10 days. blood samples were collected using a standard coagulation tube (sst ii advance, bd biosciences) to obtain the serum, which was aliquoted and stored at -80 ° c until analysis. the cytokines were measured using commercially available elisa kits for tnf-α, ifn-γ, il-6, il-4, and il-10 (immunotools, friesoythe, germany), according to the manufacture instructions. several clinical and laboratory data were evaluated. the outcome variable was in-hospital death, survival, mechanical ventilation, and admission to the intensive care unit (icu). continuous variables were expressed as median values and interquartile range (iqr) and analyzed by mann whitney test. categorical variables were expressed as absolute frequencies with proportions and analyzed by chi-square or fisher exact test. a p-value < 0.05 was considered significant. all variables in the univariate model meeting a cut-off of p <0.1 were included in the multivariable model. spss v23.0 (ibm, chicago, il) and graphpad prism v7 (graphpad, san diego, ca) were used for statistical analysis. the variable days of symptoms was split in >10 days or ≤ 10 days using the optimal binning procedure on spss for all cytokines included in the analysis. fifty-six patients were included in all samples. median age was 61 [47-73] years, and 69.6% (n=39) were male. most common comorbidities were systemic arterial hypertension (50%, n=28), diabetes (25%, n=14) and chronic obstructive pulmonary disease (16.1%, n=9). at hospital admission, moderate and severe patients were composed of 85.7% (n=48) and 14.3% (n=8) from the sample, respectively. general clinical characteristics are in table 1 . steroids were used in 58.9% (n=33) and hydroxychloroquine in 12.5% (n=7). the median length of hospitalization was 13 [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] days. specific characteristics from each patient group (0-10d vs. >10d) are demonstrated in table 2 . at admission, median leukocytes, lymph cells and platelets counts were 7,600 [ figure 1 . ifn-γ levels were higher in patients from group 0-10d of symptoms (p<0.001); while il-4 levels were higher in patients from group >10d of symptoms (p<0.001). thus, ifnγ and il-4 was inversely proportional related (p=0.03) ( table 3 ). there was no relation between use of steroids during treatment of covid-19 and citokines levels (p>0.05). table 2 . in univariate analysis, death was related to gender (p=0.029), icu (p<0.001), oti (p=0.002), ifn-γ (p=0.017) and length of hospitalization (p=0.025); while survival was related to fever (p=0.036). cytokines analysis is shown in figure 2 . nevertheless, in multivariate analysis, death remained related to age, gender, and ifn-γ (p<0.05). in this prospective cohort of patients, higher levels of ifn-γ were related to a poorer prognosis. these results demonstrate that an effective th1 response is not sustained and probable it is followed by the development of th2 immune adaptive response. il-6 and il-10 did not differ when compared to days of symptoms, evolution, nor outcomes. previous studies elucidated the capacity of nk and nk t cells to produce ifn-γ before the specific th1 adaptive immune response 12, 13 . our results converge with this hypothesis once higher ifn-γ levels were detected in early covid-19 infection than healthy populations 14 . nevertheless, these levels were not sustained after ten days of symptoms. in those with sustained ifn-γ levels, the mortality increased. th2 profile cytokine il-4 presents an upward trend in disease progression in critically ill patients 15 . il-4 levels may (i) inhibit naive cd4+ cells from proceeding to th1 maturation or in our cohort, gender, and ifn-γ levels were related to poorer prognosis. besides it, since the covid-19 beginning, it was demonstrated that hospital admission was more likely in the male gender with comorbidities 18, 19 . hypertension, cardiovascular diseases, and diabetes were the most frequent comorbidities in our patients. these factors may explain the high icu admission rate presented in this study. furthermore, the death rate found was 32%, which is higher than j o u r n a l p r e -p r o o f other general cohorts 20 . nevertheless, given the high icu admission rate, it is expected that outcomes results become closer to cohorts of critically-ill patients 21 . previous studies have not reported the association between ifn-γ and death, even evaluating the covid-19-reactive cd69+ expressing ifn-γ producing cd8+ t in 25 patients with severe and moderate disease 22 . most studies have described the evident immunological dysfunction in the moderate and severe disease, with reduced expression of ifn-γ by cd4+ t, cd8+ t, and nk cells 23 . the same author found that ifn-γ by cd4+ t cells tended to be lower in severe cases than moderate cases. however, none of these studies evaluated the levels of ifnγ with death. some subsets of t lymphocytes correlated with in-hospital death and severity of illness, but only the innate immune cytokines were evaluated 24 . it was recently demonstrated that levels of the ifn-γ secreted by cd4+ and cd8+ t cells from patients with moderate disease were compatible with those in critically ill patients. in this case, ifn-γ increased over time in critically ill patients, but with decreased levels in moderate patients, contrasting with the results presented here. however, ifn-γ was highly correlated with viral load, suggesting that the virus can boost the secretion of these cytokines 15 . in a previous report, the proportion of memory and naïve helper t cells decreased in severe cases 25 . patients with covid-19 also have a lower level of regulatory t cells, suggesting that the adaptive immune response is prejudiced. however, another study showed a higher number of cd4 t cells producing ifn-γ in comparison with a control group (without disease) 26 . in children with covid-19, ifn-γ is increased, but with inferior levels than we found, suggesting a less severe disease in the pediatric population 27 . in cardiomyocytes, there is an up-regulation of genes responsible for ifn-γ suppression, also suggesting an effect ifn-γ in the cardiac function, in a sepsis-like pattern 28 . furthermore, the relationship of ifn-γ disruption is not j o u r n a l p r e -p r o o f only associated with covid-19 and the immune system, but there is a relationship of microbiome modification, altering the cell transcriptome with gene overexpression, which can be associated with the cytokine storm 29, 30 . interleukin-4 plays a critical role in the th2 pathway, being predominantly associated with fibrogenic inflammatory remodeling, while th1 cells exert anti-fibrotic activity by secreting gamma interferon (ifn-γ) and interleukin 2. il-4 also induce alternative activation of m2 macrophages, promoting the release of tgf-β and platelet-derived factor. this phase is characterized by the expansion of resident fibroblasts and the matrix remodelling 20 . since severe covid-19 can lead to diffuse alveolar damage, which has a potential of developing septal fibrosis; recovering patients with high levels of il-4 may might progress to pulmonary fibrosis aggravating impairment of the lung functions. our study has some limitations and strengths that merit consideration. as limitations, our study evaluated the first patients admitted during the outbreak in our city. therefore, changes in clinical management during the evolving epidemics might have a differential impact on our studied outcomes. our limited sample size might have decreased our power; however, also because of the pandemic, the findings of this study offer new, potentially useful information for this patient population. on the other hand, our grouping by symptom days may have standardized the disease's different stages when patients were admitted. another concern is memory bias for onset of symptoms, because some patients could be in the wrong group (more or less 10 days). we evaluated the th1 and th2 responses according to the time of evolution, so we were able to identify possible predictive markers such as (i) ifn-γ in early-stage and (ii) il-4 in late-stage for the outcomes of discharge or death. our results suggest that the moderate to the severe progression of covid-19 may have been one of the causes of the immune responses developed by the host at the beginning of the covid-19 infection, influencing the need for combination therapy to block these inflammatory mediators. the activation of the host immune response between th1 or th2 in covid-19 infection may be related to the final result between discharge or death. any source to control cytokines, such as ifn-γ, with combined therapies for clinical treatment will be important in the future for covid-19 infection. viral and host factors related to the clinical outcome of covid-19 clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet profiling serum cytokines in covid-19 patients reveals il-6 and il-10 are disease severity predictors. emerg microbes infect a tool for early prediction of severe coronavirus disease 2019 (covid-19): a multicenter study using the risk nomogram in wuhan and guangdong, china development and validation of a clinical risk score to predict the occurrence of critical illness in 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in patients with covid-19 pneumonia haematological and immunological data of chinese children infected with coronavirus disease 2019. data brief single-cell transcriptome analysis indicates new potential regulation mechanism of ace2 and nps signaling among heart failure patients infected with sars-cov-2. medrxiv metatranscriptomic characterization of covid-19 identified a host transcriptional classifier associated with immune signaling the society for immunotherapy of cancer perspective on regulation of interleukin-6 signaling in covid-19-related systemic inflammatory response figure 2. levels of cytokines from patients with moderate and severe covid-19 infection according to the outcome (data in the median with iqr) key: cord-314415-yr0uxok2 authors: guo, zijing; he, qifu; tang, cheng; zhang, bin; yue, hua title: identification and genomic characterization of a novel cress dna virus from a calf with severe hemorrhagic enteritis in china date: 2018-08-15 journal: virus res doi: 10.1016/j.virusres.2018.07.015 sha: doc_id: 314415 cord_uid: yr0uxok2 in this study, a novel circular replication-associated protein (rep)-encoding single stranded (cress) dna virus was discovered in diarrheic sample of a calf with severe hemorrhagic enteritis. the virus, named bo-circo-like virus ch, has a circular genome with 3909 nucleotides (nt). six putative open reading frames (orfs) were identified, including rep, capsid (cap) and four proteins of unknown function. both the genome size and the number as well as the organization of encoded orfs, bo-circo-like virus ch is most closely related to po-circo-like virus 21 detected in pig faeces. a preliminary survey using specific primers for the rep region showed that 5.3% (4/75) of diarrheic samples were positive for bo-circo-like virus, and all 42 healthy samples were negative. in conclusion, our results indicate that bo-circo-like virus ch may represent a new virus in bovine. further investigation is needed to determine the relationship between the virus infection and diarrhea. viruses with a circular ssdna genome are found in archaea, bacteria, and eukaryotic organisms (shulman and davidson, 2017) . until recently, the cress dna viruses have been classified into six families by the international committee on taxonomy of viruses (ictv), namely geminiviridae, nanoviridae, circoviridae, genomoviridae, bacilladnaviridae and smacoviridae. viruses of families geminiviridae nanoviridae and bacilladnaviridae were known to infect plants (ramesh et al., 2017; rybicki and martin, 2014; kazlauskas et al., 2017) . viruses of the new families smacoviridae and genomoviridae had been identified in faecal samples of various vertebrates, including humans and bovine krupovic et al., 2016; steel et al., 2016; kim et al., 2012) . the family circoviridae includes the genera circovirus and cyclovirus, which widely infected humans and certain animals (fu et al., 2018; hattermann et al., 2003; li et al., 2011; lorincz et al., 2011; phan et al., 2014) . the genomes of circoviruses and cycloviruses range in size from 1.7 to 2.1 kb and contain two major orfs, which encode rep and cap proteins (breitbart et al., 2017) . the taxonomic classification for the family circoviridae suggested that rep identities and genome organization should be used to identify a genome as belonging to either the circovirus or cyclovirus genus (rosario et al., 2017) . furthermore, a new species demarcation threshold of 80% genome-wide pairwise identity for members of the family circoviridae (breitbart et al., 2017; rosario et al., 2017) . in addition to six recognized cress dna viral families, a large number of novel cress dna viruses, waiting to be classified by ictv, have been rapidly discovered in faeces of humans and animals through viral metagenomics in recent years. the hosts include chimpanzees (blinkova et al., 2010) , pigs zhang et al., 2014) , rodents (phan et al., 2011) , bats (ge et al., 2011) , humans (castrignano et al., 2013) , dromedaries (woo et al., 2014) , duck (steel et al., 2016) , deer (steel et al., 2016) , llama (steel et al., 2016) , chamois (steel et al., 2016) , bovine (steel et al., 2016) , flying fox (male et al., 2016) , chickens (lima et al., 2017) and macaques (kapusinszky et al., 2017) . among these novel cress dna viruses, a group of viruses with large genomes have been proposed family kirkoviridae according to genomic and rep-phylogenetic characteristics (li et al., 2015; zhao et al., 2017) . until now, seven reported cress-dna viruses genomes can be classified as the proposed family kirkoviridae (li et al., 2015; shan et al., 2011; zhao et al., 2017) , including four genomes determined in the feces of healthy and diarrhoeic pigs , one genome determined in the liver and spleen of a horse with fatal idiopathic hepatopathy (li et al., 2015) and two genomes determined in patients feces with type 1 diabetes (zhao et al., 2017) , successively. a significant characteristic of these viruses in the proposed family kirkoviridae shows large genomes ranging in size from 2833 to 3923 nt with three to six orfs. additional, all the rep proteins of these viruses show significant genetic distance with that of the viruses within family circoviridae. although one member named kirkovirus equ1 in the proposed family kirkoviridae was suggested that it may be potentially related to disease (li et al., 2015) , the pathogenesis of these viruses is still unknown. in this study, a novel bovine-derived cress dna virus was found in diarrheic faecal sample through viral metagenomics. the fecal sample was collected from a 4-month-old simmental cross calf with severe hemorrhagic enteritis accompanied by dyspnea and fever in october 2016 in sichuan province, china. the sample was suspended in phosphate-buffered saline (pbs) to create a 10% suspension and centrifuged at 12 000g for 10 min at 4°c. the supernatant was then filtered through a 0.22 mm filter (millipore) to remove intact bacteria and other large cellular debris. the filtrates was treated with 10 u dnase and 1.5 mg rnase enzymes (takara) at 37°c for 90 min to remove unprotected nucleic acids. total nucleic acid from the sample was extracted using a qiaamp viral rna mini kit (qiagen) following the manufacturer's instructions. reverse transcription was performed using superscript iii reverse transcriptase (invitrogen) and random hexamers (invitrogen). the quantity of cdna was determined by a qubit fluorometer (life technologies). the sample of 230 ng cdna was used to construct a library according to the manufacturer's instructions (truseq rna sample preparation kit) and sequenced on hiseq 4000 (illumina), as described in the previous studies (chen et al., 2015) . raw sequence reads were trimmed to remove the reads of adaptors, duplicate reads and bovine genomic sequences, and a minimum length of 150 bp was selected. the reads that passed the data processing were considered the useful sequences. in parallel, all useful reads were subjected to de novo contig assembly using the idba_ud (peng et al., 2012) , and evaluate assembly results using soap2 software (http:// soap.genomics.org.cn/). the assembly data were aligned with sequences in the ncbi nonredundant nucleic database (nt) and the nonredundant protein database (nr) using blastn and blastx, respectively. the taxonomies of the aligned read with the best blast value (e value < 10 −4 ) were selected and used for further grouping analysis. the abundance of viruses was scanned by soap aligner software to further analyse the diversity of the species identified from the calf diarrheic sample. a total of 5 211 770 bases were generated from the illumina sequencing run, and approximately 2.1% of the sequence data matched those of mammalian viruses. the raw sequence reads from the viral metagenomic libraries were deposited in genbank database under the accession number srp108885. seven konwn mammalian viruses consist of bovine enterovirus (8785 reads), bovine kobuvirus (549 reads), bovine herpesvirus 1 (199 reads), nebovirus (107 reads), bovine coronavirus (50 reads), bovine adenovirus type 3 (5 reads) and bocaparvovirus (2 reads) were found, and the length of each read was 150 bp. notably, 122 reads reveal significant sequence similarity with po-circo-like viruses (genbank accession no. nc_025682.1 and jf713717. 1), and the reads were assembly into two large contigs. one contig consists of 2277 nt (position: 3904-3912-0-2291 nt) and the other contig consists of 1231 nt (position: 2728-3912-0-48 nt), referencing the genome (3912 nt) of strain po-circo-like virus 21 (genbank accession no. nc_025682.1). to acquire the full-length genome of the cress-dna virus and further verify the sequences of viral metagenomic, four set of primers were designed based on the viral metagenomic sequencing data (genbank accession no. srp108885). the primers sequences were shown in table 1 . pcr was performed with premix taq™ (takara, china), following the manufacturer's protocol. the pcr amplification products were gel-purified using a gel extraction kit (omega, usa) and then cloned into the pmd19-t vector and transformed into escherichia coli dh5αcompetent cells (fig. 1) . the recombinant plasmids were extracted using a plasmid extraction kit (omega, usa) and then sequencing was performed by sangon biotech (china, chengdu). the sequences were assembled using seqman software (version 7.0; dnastar, usa). putative orfs in the circular genomes were identified using the orf finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf. html). the hairpin and stem-loop structures were identified in mfold webserver (zuker, 2003) . nucleotide and deduced amino acid sequences were analyzed using the megalign program of dnastar7.0 software (dnastar inc., wi, usa) to determine sequence homology. mega 7.0 was used to perform the multiple sequence alignment and subsequently to build a maximum likelihood phylogenetic tree using the lg model with 1000 bootstrap support (kumar et al., 2016) . the novel circular dna virus has a complete genome of 3909 nt with g+c content of 38.76% (genbank accession no. mh316857). six putative orfs (> 100aa) were identified, including rep protein of 307 aa, cap protein of 180 aa and four proteins of unknown function of 185, 129, 106 and 104 aa. a stem-loop structure was predicted to be located close to the putative rep gene stop codon in the intergenic region (fig. 2) . both the genome size and the number as well as the organization of encoded orfs of the virus were similar to those of the viruses of recently proposed kirkoviridae (li et al., 2015; shan et al., 2011; zhao et al., 2017) . (li et al., 2015; zhao et al., 2017; wang et al., 2018) . due to the remarkably diversity in the genome size, number of orfs and genome sequences of these cress dna viruses, it is only possible to construct a phylogenetic tree for them based on the rep gene, which is the most conserved gene in the genomes of these novel viruses (castrignano et al., 2017; varsani and krupovic, 2017) . both the genome size and the number as well as the organization of encoded orfs of strain bo-circo-like virus ch indicated that it showed closest genetic relationship with strain po-circo-like virus 21. however, two orfs (129 residues and 104 residues) of the virus did not have significant similarity to any orfs in genbank, and a recombination event seems occurred in a orf (185 residues). generally, recombination and horizontal acquisition of non-homologous genes were significant features in ssdna virus (krupovic and koonin, 2014; lefeuvre et al., 2009; martin et al., 2011) . this phenomenon was also observed in the cress dna virus (kazlauskas et al., 2017 (kazlauskas et al., , 2018 lefeuvre and moriones, 2015) , which may contribute to evolution of the cress dna virus. the putative rep protein (307 residues) of bo-circo-like virus ch shares 93.9% aa identity and 88.1% aa identity with that of po-circolike virus 21 and po-circo-like virus 22, respectively, but shares 35.7-51.5% aa identity with that of other five reported members of the proposed family kirkoviridae. the rep-representative members of the families circoviridae, nanoviridae, geminiviridae genomoviridae, bacilladnaviridae and smacoviridae, and still unassigned novel ssdna viruses available in genbank database were used constructed a maximum-likelihood phylogenetic tree. the result showed that bo-circo-like virus ch is clustered into a independent branch with seven reported strains of proposed family kirkoviridae and eight cress-dna virus strains recently submitted to genbank database; bo-circo-like virus ch is more closely related to po-circo-like virus and shows significant genetic differences with viruses in the families circoviridae, nanoviridae, geminiviridae genomoviridae, bacilladnaviridae and smacoviridae (fig. 3) . further analysis of 16 strains in the new clade suggested that these strains display large genomes ranging in size from 2833 to 4905 nt with three to six orfs. the hosts includs human, macaca, bovine, porcine, horse, dromedary and rodent. although the classification criteria of the proposed family kirkoviridae is not yet clear (li et al., 2015; zhao et al., 2017) , it seems that these viruses may be classified into the family proposed kirkoviridae. the third-largest orf (180 residues) of bo-circo-like virus ch was predicted to encode cap protein since high percentage (8/19) of the basic amino acids arginine and lysine near the n-terminus of the orf was observed, which is characteristic of circovirus cap proteins (stewart et al., 2006) . additional, a blastp search using the orf showed that it hits the putative cap protein of macaca mulatta cg10456 (genbank accession no. ku043416.1; qc = 81%, i = 48%, ev = 2e-28). the putative cap protein of bo-circo-like virus ch also matches hypothetical protein of six strains of the proposed family kirkoviridae except for kirkovirus equ1. the putative cap protein of bo-circo-like virus ch shares 80.6% aa identity and 80.1% aa identity with that of po-circo-like virus 21 and po-circo-like virus 22, respectively, and shares 38.9-46.4% aa identity with that of other four members of the proposed family kirkoviridae. four orfs of unknown function were also identified in bo-circo-like virus ch. among these orfs, two orfs (129 residues and 104 residues) did not have significant similarity to any orfs in genbank database, including orfs of the viruses belong to family kirkoviridae. one orf (106 residues) of other two orfs showed that it only hits a putative protein of po-circo-like virus 21 (qc = 74%, i = 90%, ev = 1e-18) using a blastp search. interestingly, for another orf (185 residues), partial aa fragment (1-70aa) in the n-terminus showed 71% aa identity with the strain po-circo-like virus 22, but the region was deleted in strain po-circo-like virus 21, and the second region (71-185aa) only showed 21.4% aa identity with two-component system sensor histidine kinase bass of pectobacterium atrosepticum (genbank accession no. wp_011095521.1), which seems a recombination event in the orf. due to the lack of the parental nt sequences in genbank database, recombination analysis could not be further performed. in fact, it is not surpring that high rates of recombination in cress dna genomes had been reported (kazlauskas et al., 2018; lefeuvre and moriones, 2015) . to investigate the presence of this newly identified virus in other cattles, 75 diarrheic and 42 healthy faecal samples were collected from the provinces of sichuan (two farms), henan (three farms), liaoning (three farms) and shandong (three farms), china. a primer set was designed based on conserved rep sequences. the primer sequences were as follows; f: 5′-aagaacacctggatgaaggaacgc-3′; r: 5′-gccagtcatcaatcacaaccctct-3′, the amplified fragment was 540 bp. viral dna was extracted from individual calf fecal samples using phenol-chloroform. the pcr amplification products were gelpurified using a gel extraction kit (omega, usa) and then cloned into the pmd19-t vector and transformed into escherichia coli dh5αcompetent cells. the recombinant plasmids were extracted using a plasmid extraction kit (omega, usa) and then sequencing was performed by sangon biotech (china, chengdu). in the case of bovine fecal samples, approximately 5.3% (4/75) of diarrheic faecal samples were positive for bo-circo-like virus, and all 42 healthy samples were negative by pcr assays. four positive samples were detected from two farms (detection rate: 2/14 and 2/23, fig. 2 . predicted genome organization of strain bo-circo-like virus ch. six orfs (> 100aa) were predicted in strain bo-circo-like virus ch, including cap, rep and four proteins of unknown function. the potential stem-loop was predicted to be located close to the putative rep gene stop codon in the intergenic region; detailed nucleotides sequences are also presented. respectively), and the geographical distance between two farms is more than 1000 km. the 540 bp of the four bo-circo-like virus sequences shared 98.2%-99.3% nt identity with bo-circo-like virus ch, and shares 90.2%-90.4% nt identity with strains po-cicro-like virus 21. phylogenetic analysis of these partial rep nt sequences showed that all bo-circo-like virus were clustered into a dependent branch and were more related to po-cicro-like virus 21 (fig. 4) . the pcr screening sequence fragments were deposited in genbank under the following fig. 3 . phylogenetic analysis was performed based on the amino acid sequence of orf1 presumable encodes rep protein. the sequence alignments included strain bo-circo-like virus ch in this study, representative members of the circoviridae, geminiviridae, nanoviridae, genomoviridae, bacilladnaviridae and smacoviridae families, the proposed new genera of krikoviruses, and still unassigned novel cress-dna viruses with the best blastp matchs in genbank database. the tree was constructed by the maximum-likelihood method with lg model and 1000 bootstrap replicates in mega 7.0 software. only bootstrap values > 50% are shown. the strain in this study is marked with red box, and seven reported strains of the proposed family kirkoviridae are marked with black boxes. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article). accession numbers mh316858-mh973561. although the number of fecal samples was limited, all bo-circo-like virus in this study were detected from diarrheic faecal samples. recently, one member named kirkovirus equ1 in the proposed family kirkoviridae was suggested that it may be potentially related to disease, and the virus was found in the liver and spleen of a horse with fatal idiopathic hepatopathy (li et al., 2015) . thus, further investigation is needed to determine the pathogenesis of the bo-circo-like virus. in conclusion, a novel bovine-derived cress dna virus, named bo-circo-like virus ch, was identified from a calf with fatal hemorrhagic enteritis in china. it shows a circular genome of 3909 nt with six putative orfs, including rep protein, cap protein and four proteins of unknown function. based genome organization and phylogenetic analysis, the bo-circo-like virus ch may represent a new virus in the proposed family kirkoviridae. further investigation is needed to determine the pathogenesis of the virus. the raw sequence reads from the viral metagenomic libraries have been deposited into as bioproject, biosample and sra at ncbi, the accession number were prjna389792, samn07208491 and srp108885, respectively. the complete genome sequence of bo-circolike virus ch has been deposited in genbank under accession no. mh316857; the 4 partial rep sequences of bo-circo-like virus have been deposited in genbank under accession nos. mh316858-mf973561. this work was funded by sichuan province applied foundation project (grant number 2017jy0066), the innovation team for animal epidemic diseases prevention and control on qinghai-tibet plateau, state ethnic affairs commission (grant number 13td0057) and the innovative research project of graduate students in southwest university for nationalities (grant number cx2017sz057). the authors declare that they have no conflicts of interest. the study does not contain any studies with human participants or animals performed by any of the authors, and all experiments in this study are in compliance with ethical standards for research. fig. 4 . phylogenetic analysis based on partial orf1 nucleotides sequences (540-nt) of presumable encodes rep gene. the sequence alignments included five bo-circo-like virus strains detected in this study and seven reported strains of the proposed family kirkoviridae. the tree was constructed by the maximum likelihood method with hasegawa-kishino-yano model and 1000 bootstrap replicates in mega 7.0 software. only bootstrap values > 50% are shown. the strains in this study are marked with red boxes. 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fricke, markus; marz, manja; ziebuhr, john title: rna structure analysis of alphacoronavirus terminal genome regions date: 2014-12-19 journal: virus res doi: 10.1016/j.virusres.2014.10.001 sha: doc_id: 259671 cord_uid: 7de21oaq coronavirus genome replication is mediated by a multi-subunit protein complex that is comprised of more than a dozen virally encoded and several cellular proteins. interactions of the viral replicase complex with cis-acting rna elements located in the 5′ and 3′-terminal genome regions ensure the specific replication of viral rna. over the past years, boundaries and structures of cis-acting rna elements required for coronavirus genome replication have been extensively characterized in betacoronaviruses and, to a lesser extent, other coronavirus genera. here, we review our current understanding of coronavirus cis-acting elements located in the terminal genome regions and use a combination of bioinformatic and rna structure probing studies to identify and characterize putative cis-acting rna elements in alphacoronaviruses. the study suggests significant rna structure conservation among members of the genus alphacoronavirus but also across genus boundaries. overall, the conservation pattern identified for 5′ and 3′-terminal rna structural elements in the genomes of alphaand betacoronaviruses is in agreement with the widely used replicase polyprotein-based classification of the coronavirinae, suggesting co-evolution of the coronavirus replication machinery with cognate cis-acting rna elements. coronaviruses are enveloped, positive-strand rna viruses with exceptionally large genomes of approximately 30 kb. they have been assigned to the subfamily coronavirinae within the family coronaviridae (de groot et al., 2012a; masters and perlman, 2013) . together with the families arteriviridae, roniviridae, and mesoniviridae, the coronaviridae form the order nidovirales (de groot et al., 2012b) . the family coronaviridae is currently comprised of four genera called alpha-, beta-, gammaand deltacoronavirus. closely related virus species in these genera are grouped together in specific lineages. coronaviruses infect mammals and birds and include pathogens of major medical, veterinary and economic interest (de groot et al., 2012a) , with severe acute respiratory syndrome (sars) coronavirus (sars-cov) and middle east respiratory syndrome (mers) coronavirus (mers-cov) providing two prominent examples of newly emerging, highly pathogenic coronaviruses in humans (drosten et al., 2003; ksiazek et al., 2003; zaki et al., 2012) . besides their large genome sizes, coronaviruses and related nidoviruses stick out from other plus-strand rna viruses by the large number of virally encoded nonstructural proteins that are either involved in viral rna synthesis or interact with host cell functions (reviewed in masters and perlman, 2013; ziebuhr, 2008) . most of the nonstructural proteins (nsp) are expressed as subdomains of two large replicase gene-encoded polyproteins called pp1a (∼450 kda) and pp1ab (∼750 kda). co-and posttranslational cleavage of pp1a/1ab by two types of virus-encoded proteases gives rise to a total of 15-16 mature proteins that (together with the nucleocapsid protein and cellular proteins) form the viral replication-transcription complex (rtc) (almazán et al., 2004; schelle et al., 2005; ziebuhr, 2008; ziebuhr et al., 2000) . this multi-protein complex replicates the viral genome and produces an extensive set of 3 -coterminal subgenomic messenger rnas (sg mrnas), the latter representing a hallmark of corona-and other nidoviruses (pasternak et al., 2006; sawicki et al., 2007; ziebuhr and snijder, 2007) . the sg mrnas are used to express open reading frames located in the 3 -proximal third of the genome. they essentially encode the viral structural proteins, such as the nucleocapsid (n), membrane (m), spike (s) and envelope (e) proteins, and a varying number of accessory proteins, the latter often involved in functions that counteract antiviral host responses (masters and perlman, 2013; narayanan et al., 2008b) . coronavirus sg mrnas contain a common 5 leader sequence (approximately 60-95 nt) that is identical to the 5 end of the genome. the complement of this sequence is attached to the 3 end of nascent negative strands in a complex process called "discontinuous extension of negative strands" sawicki, 1995, 1998; sawicki et al., 2007; sethna et al., 1991) . this process involves attenuation of negativestrand rna synthesis at transcription-regulating sequences (trs) located upstream of the individual orfs in the 3 -proximal genome region ("body trss", trs-b). guided by basepairing interactions between the negative-strand complement of a trs-b and the trs located downstream of the 5 leader on the viral genome ("leader trs", trs-l), the nascent minus strand may be transferred from its downstream position on the template (at the trs-b) to the trs-l, where negative-strand rna synthesis is then resumed and completed by copying the 5 leader sequence. the set of 3 antileader-containing sg minus-strand rnas is subsequently used as templates for the production of the characteristic nested set of 5 capped, 5 leader-containing and 3 -polyadenylated sg mrnas in coronavirus-infected cells (lai et al., 1983; sawicki et al., 2001; sawicki and sawicki, 1995; sethna et al., 1989; spaan et al., 1983) . sg minus-strand rnas contain a u-stretch at their 5 end, thus providing a template for 3 polyadenylation of sg mrnas wu et al., 2013) . similar to other rna viruses, coronavirus genomes contain important rna signals in their 5 and 3 -terminal genome regions, mainly (but not exclusively) in the untranslated regions (utr). these signals are required for viral rna synthesis (replication and/or transcription) and are collectively referred to as cis-acting rna elements (chang et al., 1994; dalton et al., 2001; izeta et al., 1999; kim et al., 1993; liao and lai, 1994; lin et al., 1994 lin et al., , 1996 zhang et al., 1994) . as indicated above, coronaviruses also contain cis-acting elements at internal positions in the genome, the best documented ones being the leader and body trss which play a vital role in the transfer of the nascent minus-strand rna to an upstream position on the template (see above). other internal cis-acting elements include specific rna signals required for genome packing, which have been characterized in a small number of coronaviruses escors et al., 2003; morales et al., 2013; penzes et al., 1994) , and a complex rna pseudoknot structure located in the orf1a-orf1b overlap region that mediates a (−1) ribosomal frameshift event and thus controls the expression of the second large orf on the coronavirus genome rna (orf1b) (brierley et al., 1987 (brierley et al., , 1989 de haan et al., 2002; namy et al., 2006) . both viral and cellular proteins, including the viral n protein, heterogeneous ribonucleoprotein (hnrnp) family members, polypyrimidine tract-binding protein, and poly(a)-binding protein (pabp), have been shown to bind to specific coronavirus cis-acting elements and there is evidence to support the biological significance of some of these protein-rna interactions (reviewed in shi and lai, 2005; sola et al., 2011b) . the coronavirus rtc is a multi-subunit assembly comprised of more than a dozen viral and an unknown number of cellular proteins. the complex is anchored through transmembrane domains present in nsps 3, 4 and 6 to intracellular membranous structures that provide a specialized membrane-shielded compartment in (or at) which viral rna synthesis takes place (den boon and ahlquist, 2010; gosert et al., 2002; kanjanahaluethai et al., 2007; knoops et al., 2008; oostra et al., 2008; oostra et al., 2007; snijder et al., 2006; van hemert et al., 2008) . over the past years, viral components of the coronavirus rtc have been characterized in considerable detail, providing a wealth of functional and structural information (reviewed in imbert et al., 2010; masters, 2006; ulferts et al., 2010; ulferts and ziebuhr, 2011; ziebuhr, 2008) . a large number of virally encoded enzymes, including protease, adp-ribose-1 -phosphatase, ntpase, 5 -to-3 helicase, rna 5 -triphosphatase, rna polymerase, guanosine-n7 and ribose 2 -o methyltransferases, 3 -to-5 exoribonuclease and uridylatespecific endoribonuclease, have been identified (baker et al., 1989; bhardwaj et al., 2004; chen et al., 2009 chen et al., , 2011 decroly et al., 2008 decroly et al., , 2011 eckerle et al., 2010; ivanov et al., 2004; kanjanahaluethai and baker, 2000; minskaia et al., 2006; putics et al., 2005; saikatendu et al., 2005; seybert et al., 2000; te velthuis et al., 2010; ziebuhr et al., 1995) . in some cases, these activities could be linked to specific steps of viral rna synthesis and/or rna processing or were shown to interfere with cellular functions (reviewed in masters and perlman, 2013; ziebuhr, 2008) . there are multiple interactions between the individual replicase gene-encoded nsps and the structural basis and functional implications of these interactions have been studied in a few cases. for example, it has been shown that the exoribonuclease and ribose 2 o-methyltransferase activities associated with nsp14 and nsp16, respectively, are each stimulated by specific interactions with nsp10 (bouvet et al., 2014; decroly et al., 2011) . also, there is evidence that a heteromultimeric complex formed by nsp7 and nsp8 interacts with (and serves as a processivity factor for) the rna-dependent rna polymerase (rdrp, nsp12) (zhai et al., 2005) . additional interactions between individual subunits of the rtc have been suggested on the basis of two-hybrid screening data (pan et al., 2008; von brunn et al., 2007) and there is evidence that a substantial number of coronavirus nsps form homo-and/or heterooligomeric complexes (anand et al., 2002 (anand et al., , 2003 bouvet et al., 2014; chen et al., 2011; ricagno et al., 2006; su et al., 2006; xiao et al., 2012; zhai et al., 2005) . despite major progress in the characterization of proteins and cis-acting rna elements involved in coronavirus rna synthesis, the molecular mechanisms that mediate specific steps of coronavirus rna replication and transcription are far from being understood. important information on cis-acting rna elements has been obtained from studies using defective interfering (di) rnas and, more recently, genetically engineered coronavirus mutants (reviewed in brian and baric, 2005; masters, 2007; sola et al., 2011b) . in this article, we will summarize previous work on (beta)coronavirus genomic cis-acting rna elements and will then move on to present conclusions from our recent bioinformatic and rna structure probing studies on cis-acting rna elements conserved in alphacoronaviruses. historically, cis-acting rna elements required for coronavirus rna synthesis have mainly been studied by using naturally occurring and genetically engineered defective interfering rnas (di rnas) (reviewed in brian and baric, 2005; brian and spaan, 1997; masters, 2007; sola et al., 2011b) . di rnas are replicationcompetent genome-derived rna molecules that contain extensive internal deletions but retained all the cis-acting rna signals required for replication by functional replicase complexes provided by a helper virus that replicates in the same cell (levis et al., 1986; weiss et al., 1983) . essentially, these cis-acting elements comprise the untranslated 5 -and 3 -terminal genome regions but, in some cases, may also extend into coding regions. in some cases, they also contain noncontiguous sequences derived from internal genome regions. coronavirus di rnas were first described for the betacoronaviruses mhv and bcov (chang et al., 1994; de groot et al., 1992; hofmann et al., 1990; luytjes et al., 1996; makino et al., 1985 makino et al., , 1988a makino et al., , 1988b makino et al., , 1984 . subsequently, these studies were extended to alpha-and gammacoronaviruses (izeta et al., 1999; mendez et al., 1996; penzes et al., 1994 penzes et al., , 1996 . over the years, studies of defective genomes proved to be very useful for identifying coronavirus rna elements required for replication (and packaging). however, di rnas also have disadvantages, a major one being homologous recombination between the rna replicon and the helper virus genome. thus, for example, artificial di rnas containing mutant 5 leader sequences rapidly acquire the leader sequence of the helper virus, a process commonly referred to as "leader switching" (chang et al., 1994 (chang et al., , 1996 . the latter occurs very often if poorly replicating mutant di rnas are to be characterized which generally require amplification steps by serial passaging to determine their phenotype. with the development of reversegenetic systems suitable to produce and manipulate full-length coronavirus cdna copies, an attractive alternative for studying cisacting rna elements at the genome level (including long range rna-rna interactions) is now available that overcomes some of the limitations of di rna-based systems (almazán et al., 2000; casais et al., 2001; scobey et al., 2013; tekes et al., 2008; thiel et al., 2001; van den worm et al., 2012; yount et al., 2000 yount et al., , 2003 . di rnas studies on representative betacoronaviruses revealed that less then 500 nt from the 5 end of the genome (466 nt in mhv and 498 nt in bcov) are required for replication (chang et al., 1994; kim et al., 1993; luytjes et al., 1996) . in subsequent studies on alphaand gammacoronaviruses, minimal 5 cis-acting signals of 649 and 544 nt were determined for tgev (escors et al., 2003) and ibv, respectively (dalton et al., 2001) . the 5 -terminal genome regions of 466 nt (mhv) to 649 nt (tgev) comprise the entire 5 utr ranging in size from 210 nt (mhv, bcov and hcov-oc43) to 314 nt (tgev) and thus extend into the nsp1-coding region of orf 1a (see below). by contrast, the gammacoronavirus ibv features a much larger 5 utr (528 nt) and lacks a counterpart of nsp1 (ziebuhr et al., 2001) . it therefore appears that the gammacoronavirus 5 utr (alone) contains all the 5 rna signals required for genome replication. cis-acting rna structures in the 5 -terminal region of the coronavirus genome have first been studied for bcov using di rnabased systems (brown et al., 2007; chang et al., 1994 chang et al., , 1996 gustin et al., 2009; raman et al., 2003; raman and brian, 2005) . in the 5terminal 215 nts of the bcov genome, four stem-loops (designated i [comprised of ia and ib], ii, iii, and iv) were defined. enzymatic probing and mutational analysis of both naturally occurring and genetically engineered di rnas were used to (i) corroborate the predicted rna secondary structures and (ii) determine their functional significance in di rna replication. more recently, two further stem-loops (called sl-v and sl-vi) were identified in the bcov nsp1-coding region of which sl-vi was confirmed to be essential for di rna replication (brown et al., 2007) . subsequent studies suggested (a varying degree of) conservation of 5 cis-acting elements among betacoronaviruses and even the more distantly related alpha-and gammacoronaviruses (chen and olsthoorn, 2010; kang et al., 2006) . to facilitate the discussion of data obtained in studies of different viruses by different laboratories, we will use in this article a uniform nomenclature for the main rna structural elements in the 5 -proximal genome region (sl1, sl2, [sl3 if present], sl4 and sl5). the nomenclature is based on sl designations used by the leibowitz and giedroc laboratories and in predictions of genus-and lineage-specific conservation patterns of 5 cis-acting elements in alpha-, beta-and gammacoronaviruses (chen and olsthoorn, 2010; kang et al., 2006; liu et al., 2006 liu et al., , 2007 . the proposed functional and structural conservation of 5 cis-acting elements among betacoronaviruses received strong support by reverse-genetic data demonstrating that sars-cov sl1, sl2, and sl4 can functionally replace their counterparts in the mhv genome when introduced individually (kang et al., 2006) . by contrast, replacement of the entire mhv 5 utr with that of sars-cov did not produce a viable mhv mutant, suggesting a requirement for additional stable or transient long-range rna-rna interactions of the 5 utr with other genome regions. evidence to support this hypothesis was obtained in subsequent studies. for example, the energetically unstable lower part of mhv sl1 was found to be involved in long-range rna interactions with the 3 utr ) (see below). also, in a study using mhv/bcov chimera, a region downstream of sl4 was revealed to be engaged in long-range interactions with the nsp1-coding region, thus possibly forming an extensive higher-order rna structure (guan et al., 2012) . a subsequent bcov di rna mutagenesis study further suggested that this multipartite rna structure may involve several stem-loop (sub)structures identified in earlier studies (gustin et al., 2009; raman and brian, 2005) but require refolding of other rna structures suggested earlier to be essential for di rna replication (brown et al., 2007) . the study by su et al. (2014) also identified an intriguing requirement in cis of an oligopeptide sequence in the nproximal part of nsp1, suggesting that nsp1 may be an essential cis-acting protein factor in betacoronavirus replication, in addition to its multiple other functions (brockway and denison, 2005; huang et al., 2011a,b; kamitani et al., 2006 kamitani et al., , 2009 lei et al., 2013; lokugamage et al., 2012; narayanan et al., 2008a; tanaka et al., 2012; tohya et al., 2009; wathelet et al., 2007; züst et al., 2007) . possible interacting partners for nsp1 remain to be identified. the 5 -proximal sl1 and sl2 are predicted to be conserved across all genera of the coronavirinae (chen and olsthoorn, 2010; liu et al., 2007) . nuclear magnetic resonance (nmr) spectroscopy studies of mhv and hcov-oc43 sl1 rnas supported the predicted stem-loop and revealed 2-3 noncanonical base pairs in the middle of the stem. the fully base-paired sl1 was proposed to exist in equilibrium with higher-energy (partially unfolded) conformers. characterization of mhv mutants containing specific replacements in sl1 and sequence analysis of second-site revertants supports a "dynamic sl1" model in which sl1 is structurally and functionally bipartite . while the upper part of sl1 is (required to be) stable, the lower part is (required to be) unstable, possibly indicating the requirement for an optimized stability to permit or fine-tune transient long-range (rna-or protein-mediated) interactions between the 5 and 3 utrs required for sgrna transcription and genome replication, respectively. sl2 is the most conserved structure in the coronavirus 5 utr (chen and olsthoorn, 2010; liu et al., 2007) . it is comprised of a 5-bp stem and a highly conserved loop sequence, 5 -cuugy-3 , that was shown to adopt a 5 -uynmg(u)a or ucuyg(u)a-like tetraloop structure (lee et al., 2011; liu et al., 2009) . reverse genetics data confirmed that sl2 is required for mhv replication and may have a specific role in sgrna synthesis. within certain structural constraints, nucleotide replacements were found to be tolerated or could be rescued by increasing the stem stability, suggesting a limited plasticity of this important cis-acting rna element . the predicted sl3 (named sl-ii in bcov di rna studies) appears to be conserved only in a subset of beta-and gammacoronaviruses (chen and olsthoorn, 2010) . for bcov and closely related viruses, the trs-l core sequence (cs) has been proposed to be exposed in the sl3 loop region, a structure similar to the trs-l hairpin structure reported for the related arterivirus equine arteritis virus (eav) (chang et al., 1996; van den born et al., 2004 van den born et al., , 2005 . by contrast, in most other coronaviruses, the trs-l cs and flanking regions were suggested to be located in nonstructured regions (chen and olsthoorn, 2010; stirrups et al., 2000; wang and zhang, 2000) . sl4 is a long hairpin located downstream of the trs-l cs and suggested to be conserved in all coronavirus genera (chen and olsthoorn, 2010; raman et al., 2003; raman and brian, 2005) . in the vast majority of coronaviruses, sl4 contains a short orf comprised of only a few codons. because of its position in the colors are used to indicate conserved base pairs: from red (conservation of only one base-pair type) to purple (all six base-pair types are found); from dark (all sequences contain this base pair) to light colors (1 or 2 sequences are unable to form this base pair). the gray bars below the alignment indicate the extent of sequence conservation. gray shadows are used to link rna structures with the corresponding dot-bracket notations above the alignment. to refine the alignment, an anchor at the highly conserved apical loop of sl2 was used. genome upstream of orf1a it is generally referred to as the uorf. recent reverse genetics work in the mhv system yang et al., 2011) showed that disruption of the uorf yields viable mutants that, however, acquire alternate uorfs upon serial passaging in cell culture. in vitro, uorf-disrupted rnas showed enhanced translation of the downstream orf. the available data suggest that the uorf represses orf1a/1b translation and has a beneficial but non-essential role in coronavirus replication in cell culture. sl4 may be further subdivided into sl4a and sl4b. interestingly, despite its conservation in coronaviruses, sl4 tolerates extensive mutations. thus, for mhv, it was shown that base pairing in sl4a is not required for replication and separate deletions of sl4a and sl4b are tolerated. by contrast, complete deletion of sl4 and a 3-nt deletion immediately downstream of sl4 abolished or profoundly impaired viral rna synthesis. the characterization of second-site mutations and a viable mhv mutant in which sl4 was replaced with a shorter sequence-unrelated stem-loop led to a model in which sl4 was proposed to function as a spacer element that controls the orientation of sl1, sl2, and trs-l and thereby directs subgenomic rna synthesis . the sl4 sequence overlaps with the 'hotspot' of the 5 -proximal genomic acceptor required for bcov discontinuous transcription (wu et al., 2006) , thus further supporting a role of the region immediately downstream of trs-l in subgenomic rna synthesis. chen & osthoorn (2010) predicted that variations of another rna structure called sl5 may be conserved in specific coronaviruses genera and/or lineages. in alpha-and betacoronaviruses, sl5 extends into orf1a. depending on the lineage studied, conserved loop sequences could be identified in substructural hairpins of sl5. sequence conservation was found to be more pronounced in alpha-compared to betacoronaviruses. thus, for example, in alphacoronaviruses, three hairpins, called sl5a, 5b, and 5c, each containing a 5 -uuccgu-3 loop sequence, were identified. similar structures were only partly conserved in betacoronaviruses and significant lineage-specific variations in the substructural hairpins and their loop sequences were identified. a possible sl5 equivalent in gammacoronaviruses was predicted to adopt a rod-like structure that lacks conserved loop sequences (chen and olsthoorn, 2010). as outlined above, possible betacoronavirus sl5 substructures located within (or extending into) the nsp1-coding region (termed sls iv, v, vi, and vii) have been characterized structurally and functionally using bcov di rna and mhv reverse genetics systems (brown et al., 2007; guan et al., 2011 guan et al., , 2012 raman and brian, 2005) . to a large extent, previous work on coronavirus cis-acting elements has focused on only two species of closely related lineage a betacoronaviruses (represented by bcov and mhv), while there is limited information on functionally and structurally related elements in other coronaviruses. we therefore decided to embark on a more detailed characterization of putative alphacoronavirus cis-acting elements located in the 5 and 3 genome regions. as a starting point, we used 20 coronavirus genomes representing all the currently approved species from the four genera of the coronavirinae and the newly identified mers-cov (de groot et al., 2013) and calculated alignment-based secondary structures using locarna (v.1.7.2) . although locarna considers both sequence and secondary structure to calculate these multiple alignments, we failed to detect rna secondary structures conserved across all coronavirus genera when using these highly diverse sequences. we therefore resorted to producing separate genus-wide alignment-based secondary structure predictions for coronaviruses. to do this, we used complete 5 utr regions and 20 nts from the orf1a 5 end to calculate consensus structures with rnaalifold -r -p --color --nolp --mea (v.2.0.7) (lorenz et al., 2011) and -c, respectively, if constraints were used. the subgroups (lineages) obtained in these sequence alignments were consistent with the previously recognized subgroups of alpha-and betacoronaviruses (de groot et al., 2012a, 2013) (not shown). as shown in fig. 1 , all the functionally relevant rna secondary structure elements identified previously in the betacoronavirus 5 genome regions, sl1, sl2 and sl4, could be identified. in line with previous predictions and despite the pronounced sequence diversity in the 5 -terminal genome regions, these rna structures are suggested to be conserved among all currently approved betacoronavirus lineages and species. this conclusion is also supported by the large number of covariations, suggesting a strong selection pressure to retain these base-pairing interactions. fig. 1 also shows the lack of conservation of a stable hairpin structure containing the trs-l. it should be noted that, for several betacoronaviruses, it is possible to force a stem-loop containing the trs-l, but this stem-loop is only supported by two conserved base pairs. in line with previous reports, bovine coronavirus (and other viruses belonging to the species betacoronavirus 1) appear(s) to be an exception in that a more stable sl3 containing the 5 -ucuaaac-3 sequence in the loop region can be predicted in this case. overall, the structure prediction shown in fig. 1 turned out to be in perfect agreement with previous studies (see chapter 2.2). we therefore used this approach in subsequent studies of conserved rna structure elements located in the 5 genome regions of alphacoronaviruses. predictions were verified and refined by rna structure probing analyses (ehresmann et al., 1987; qu et al., 1983) using in vitro-transcribed rnas with sequences corresponding to the 5 -terminal genome regions of hcov-229e and hcov-nl63, respectively (to be published elsewhere). for the calculation of secondary structures of single sequences, we used rnafold --nolp (v.2.0.7) (lorenz et al., 2011) and -c, respectively, if constraints were used. using multiple alignments calculated with locarna, we were able to identify rna secondary structures conserved among (all) alpha-and betacoronaviruses, thus confirming and extending earlier studies (figs. 1 and 2) (chen and olsthoorn, 2010; raman et al., 2003) . these include (i) sl1, (ii) sl2 (with its short stem and highly conserved loop region [5 -uuugu-3 in alphacoronaviruses]), (iii) a poorly structured region containing the trs-l cs and some flanking sequence, (iv) sl4 (containing the uorf) and (v) sl5, the latter being conserved very well among all alphacoronavirus species (fig. 2) and significantly more diverse in betacoronaviruses (chen and olsthoorn, 2010) . the large number of covariant base pairs in the alphacoronavirus sl5 (fig. 2) suggest significant constraints and a major functional role for this structure in alphacoronavirus (and, possibly, betacoronavirus) replication and there is indeed some experimental evidence to support this hypothesis (brown et al., 2007; su et al., 2014) . using rna structure probing information obtained for the 5 -terminal 600 nts of hcov-229e and hcov-nl63, we confirmed and refined our rna secondary structure predictions for two b-lineage alphacoronaviruses (figs. 3 and 4) . the data obtained in these studies (details to be published elsewhere) support a model in which the ∼310-nt 5 genome regions consistently fold into 4 major rna structures called sl1, sl2, sl4, and sl5. the latter contains 3 hairpin substructures, sl5a, 5b, and 5c, featuring highly conserved 5(6)-nt loop sequences. the consensus secondary structure predicted for alphacoronaviruses ( fig. 2 ) was found to fit very well the individual structure predictions for hcov-229e and hcov-nl63 ( fig. 3a and 4a ) and the inclusion of structure probing information as additional constraints required only very few minor adjustments in our structure predictions (figs. 3b and 4b). most importantly, the basal part of the predicted sl4 was now predicted to be unpaired, thereby extending the single-stranded region downstream of the trs-l and also affecting the spacing between sl4 and sl5. the (predicted) basal stem of sl4 contains the most conserved sequence within the alphacoronavirus 5 -terminal rna structural elements (fig. 2 , see the red base pairs). it is therefore reasonable to think that this structurally flexible region is involved in long-range rna-rna interactions. in line with this idea, a previous tgev reverse genetic study showed that mutants permitting additional base-pairing interactions of the copy trs-b upstream of a reporter sgrna with the 5 -gaaa-3 sequence immediately downstream of the tgev trs-l cs (5 -acuaaac-3 ) (see also fig. 2 ) enhance the production of this specific reporter sgrna (zúñiga et al., 2004) . these functional data and our structural analyses of alphacoronavirus 5 -terminal genome regions lead us to suggest that the basal part of sl4 exists in a flexible state, thereby possibly facilitating strand transfer during sg minus-strand rna synthesis. both this rna structural flexibility and the role of proteins that bind in this region and thereby likely affect the sl4 structure remain to be further investigated. of note, hnrnp family members along with the viral n protein have been shown to bind in this region and the n protein has been suggested to have chaperone and trs-l/trs-b unwinding activities (galán et al., 2009; grossoehme et al., 2009; huang and lai, 1999; keane et al., 2012; li et al., 1997 li et al., , 1999 shi and lai, 2005; sola et al., 2011a,b; zúñiga et al., 2007 zúñiga et al., , 2010 . it is therefore tempting to speculate that cellular and/or viral proteins bind and unwind the energetically labile sl4 substructure to facilitate the strand transfer during sg minus-strand rna synthesis. initial information on 3 cis-acting elements required for rna replication was (again) derived from betacoronavirus di rna studies (kim et al., 1993; lin and lai, 1993; luytjes et al., 1996) . deletion mutagenesis data suggested that 3 cis-acting elements encompass the entire 301-nt 3 utr plus poly(a) tail, along with a portion of the nucleocapsid (n) protein gene. however, subsequent studies showed that the structural protein genes (including the n protein coding region) tolerate major changes including combinations of single-site mutations and rearrangements of entire genes, suggesting that the 3 -proximal coding regions do not form part of the 3 cis-acting element (de haan et al., 2002; goebel et al., 2004b; lorenz et al., 2011) . similarly, for members of the species alphacoronavirus 1 (tgev, fcov), it was shown that the n gene was not required for replication (izeta et al., 1999) and even deletions of the accessory protein genes 7a and 7b were tolerated in fcov, suggesting that the 3 cis-acting replication signals do not exceed 283 nts plus poly(a) tail (haijema et al., 2004) . for the gammacoronavirus ibv, a minimal 3 -terminal sequence of 338 nts was reported to be required for di rna replication (dalton et al., 2001) , supporting the idea that, across all coronavirus genera, the 3 utr contains all the cis-acting elements required for replication. for mhv, it was shown that a significantly smaller fragment of no more than 55 nts suffices for the initiation of negative-strand rna synthesis (lin et al., 1994) , suggesting differential requirements for plus-versus minus-strand rna synthesis. furthermore, using betacoronavirus di rna systems, a short poly(a) tract of at least 5-10 nts was found to be required for replication (spagnolo and hogue, 2000) . cis-acting rna elements present in the 3 -terminal genome regions have been studied most extensively in betacoronaviruses. the first essential rna structural element, called bulged stemloop (bsl), was discovered in mhv (hsue and masters, 1997) . it comprises 68 nts immediately downstream of the mhv n gene stop codon and was discovered as a result of attempts to replace the mhv 3 utr with that of bcov (hsue and masters, 1997) . despite limited sequence identity in the 3 utrs of the two viruses, replacements of the entire 3 utr (and specific parts of it) were tolerated, suggesting the presence of conserved structures (rather than sequences) that perform cis-acting functions in betacoronavirus replication (hsue and masters, 1997) . the conservation of functionally equivalent elements in the 3 (and 5 ) utr(s) among betacoronavirus genomes was further supported by a study showing that a bcov-derived reporter di rna was efficiently replicated by a range of bcov-and mhv-related betacoronaviruses (wu et al., 2003) . interestingly, a possible bsl equivalent was also identified in ibv and other gammacoronaviruses and its functional significance was demonstrated using ibv di rna constructs (dalton et al., 2001) . the nearly perfect stem-loop structure in ibv comprises 42 nts and ((((... ) )))))).. )))))))...........))))))).. (((....(((((((((..... .) ))))))) )....))))).....))). (......)))))..)....)))))).))..)) ))))))))) )...))))))......... fig. 2 . alignment-based secondary structure prediction of 5 genome regions of alphacoronaviruses. the viruses included in this analysis represent all currently recognized species in the genus alphacoronavirus. the alignment was calculated by locarna, the structure by rnaalifold. the consensus sequence is represented using the iupac code. colors are used to indicate conserved base pairs: from red (conservation of only one base-pair type) to purple (all six base-pair types are found); from dark (all sequences contain this base pair) to light colors (1 or 2 sequences are unable to form this base pair). the gray bars below the alignment indicate the extent of sequence conservation at a given position. gray shadows are used to link rna structures with the corresponding dot-bracket notations above the alignment. to refine the alignment, an anchor at the highly conserved core trs-l was used. is located at the upstream end of region ii, a conserved region in the gammacoronavirus 3 utr. the second essential rna element within the (betacoronavirus) 3 utr is a classical hairpin-type rna pseudoknot (pk) structure that was first discovered in bcov and shown to be required for di rna replication (williams et al., 1999) . in bcov, the pk comprises 54 nts and is located immediately downstream of the bsl. equivalent pk structures were predicted to be conserved in beta-and alphacoronaviruses while gammacoronaviruses were proposed to retain only a few features of this pk or to lack this structure altogether (williams et al., 1999) . in a subsequent study using a reverse genetics approach, the functional significance of the pk in genome replication was demonstrated for mhv (goebel et al., 2004a) . also the more distantly related betacoronaviruses hcov-hku1 (woo et al., 2005) and sars-cov were confirmed to contain this pk structure (goebel et al., 2004b) . the 3 utr regions of minimum free ene rgy = -82.80 kcal/ mol mhv and sars-cov, which only share 38% sequence identity, were shown to be interchangeable, again supporting the conservation of functionally equivalent structures among different betacoronavirus lineages. apparently, this conservation does not extend to alpha-and gammacoronaviruses because replacements of the mhv 3 utr with that of tgev and ibv, respectively, did not give rise to viable mhv mutants (goebel et al., 2004b) . together, these data suggest that coronaviruses evolved several genus-specific cisacting rna elements. for example, the presence of a bsl followed by a pk structure is limited to betacoronaviruses, while other genera appear to contain only one of these elements, with the pk being conserved in alphacoronaviruses and the bsl in gammacoronaviruses (dalton et al., 2001; hsue and masters, 1997; williams et al., 1999 ) (see section 2.6). the structures and functionally important substructures of both the bsl and pk have been characterized in significant detail for bcov and mhv (goebel et al., 2004a; hsue et al., 2000; williams et al., 1999) . in the primary structure, the bsl and pk regions overlap by several nucleotides. formation of the first stem of the pk structure requires base-pairing interactions with the downstream segment f of the bsl, thereby destabilizing the latter structure. in an extensive mhv mutagenesis study, the functional significance of both structures was demonstrated conclusively. because the two structures cannot exist simultaneously and, yet, each of them is essential for viral replication, it was proposed that the two elements may adopt alternate structures that act as a 'molecular switch' controlling the transition between different steps of the viral replication cycle (goebel et al., 2004a) . initial mechanistic insight into how this 'molecular switch' might work was obtained in a subsequent study that provided evidence for a direct interaction between loop 1 of the pk with the extreme 3 end of the mhv genome (züst et al., 2008) . the characterization of second-site revertants arising from mhv mutants with genetically engineered oligonucleotide insertions in loop 1 revealed distinct replacements at the extreme 3 end, thereby retaining specific base-pairing interactions with the loop 1 region and thus precluding the formation of stem 1 of the pk. another set of mutants contained second-site mutations that suggested specific interactions of the pk region with nsp8 and nsp9. based on this study, a model was proposed in which the formation and disruption of the pk by differential base-pairing interactions with the bsl and 3 -terminal genome sequences, respectively, may lead to alternate structures that govern different steps of the minimum free energy = -58.60 kcal/mol initiation and continuation of negative-strand rna synthesis (züst et al., 2008) . further evidence to support this model was obtained in a subsequent mhv reverse genetics study by liu et al. (liu et al., 2013) . thermodynamic investigations revealed a limited stability of the pk structure (stammler et al., 2011) , further supporting the structural flexibility of this cis-acting element and, thus, its proposed role as a 'molecular switch'. the region downstream of the pk is less well conserved among betacoronaviruses. it is generally referred to as the "hypervariable region (hvr)" and is not identical to the hvr identified at the 5 end of the 3 utr in ibv (dalton et al., 2001; williams et al., 1993) . the betacoronavirus hvr was predicted to contain a complex rna structure whose existence and functional significance was supported by enzymatic probing and mhv di rna mutagenesis studies (liu et al., 2001) . by contrast, more recent studies showed that large parts or even the entire hvr region can be deleted without causing major defects in mhv replication, arguing against an important role of this genome region in viral replication (goebel et al., 2007; züst et al., 2008) . interestingly however, mhv hvr mutants proved to be highly attenuated in vivo, suggesting a possible role in pathogenesis (goebel et al., 2007) . about 70-80 nts from the 3 end of the coronavirus genome, there is a conserved octanucleotide sequence, 5 -ggaagagc-3 , which was identified in early coronavirus sequence analyses performed in the late 1980s (boursnell et al., 1985; lapps et al., 1987; schreiber et al., 1989) and subsequently found to be universally conserved across all coronavirus genera, with only very few viruses containing single-site replacements in this sequence (goebel et al., 2007) . this strict conservation suggests an important functional role for the octanucleotide sequence. to date, however, the function of the sequence has not been identified. as mentioned above, the entire hvr including the octanucleotide sequence can be deleted from the mhv genome without causing major defects in viral replication in vitro (goebel et al., 2007) . in line with this, replacements of single nucleotides within the octanucleotide motif were tolerated although, in most cases, the octanucleotide mutants exhibited small-plaque phenotypes and/or delayed single-step growth kinetics. in both high-and low-multiplicity-of-infection experiments, octanucleotide and hvr deletion mutants lagged behind the wildtype virus but reached near-wildtype titers at later time points and had no detectable defect in grna or sgrna synthesis (goebel et al., 2007) . mhv and bcov di rna studies provided evidence that the 3 poly(a) tail present at the 3 end of coronavirus genomes is another essential component of the coronavirus 3 cis-acting signals, with a minimum of 5 to 10 adenylate residues being required for di rna replication (spagnolo and hogue, 2000) . this requirement corresponds well to the minimal binding site of the poly(a)-binding protein (pabp) on di rnas poly(a) sequences (spagnolo and hogue, 2000) . recent studies further suggest that, in the course of bcov infection, 3 poly(a) tail lengths vary between 30 and 65 nts (wu et al., 2013) . this poly(a) tail length variation was confirmed to occur in beta-and gammacoronavirus infections and in a range of cell types, both in vitro and in vivo (shien et al., 2014) . the biological significance of these observations remains to be determined. to identify putative cis-acting elements in the alphacoronavirus 3 utr, we used a range of rna folding algorithms to identify rna structural elements in the hcov-229e and hcov-nl63 3 -terminal genome regions encompassing the last 20 nts from the n gene and the entire 3 -utr. because of the length of these sequences (300 nts), many local secondary structures with similar free energies and base pair probabilities were identified and, thus, it proved to be difficult to make reliable predictions on stable rna secondary structures in this region. as described above for the 5 utr, we therefore decided to use a combination of sequence and structural alignments of all currently recognized alphacoronavirus species to identify conserved rna structures. the predictions were then further refined using structure probing data obtained for hcov-229e and hcov-nl63. the validity of the approach was first tested by analyzing conserved rna structural elements in the betacoronavirus 3 utr for which a large body of information has been obtained in previous structural and functional studies (see above). as shown in fig. 5 , we were able to detect conserved rna structural elements in the betacoronavirus 3 utr, including the bsl and the two sl structures that form the pk immediately downstream of the bsl. consistent with previous studies (goebel et al., 2004a) , our predictions suggest that the formation of the pk requires structural rearrangements at the base of the bsl to permit the base-pairing interactions required to form pk stem 1, the latter involving the pk-sl2 loop sequence and the bsl 3 -terminal sequence ( fig. 5a and b) . interestingly, our analyses also revealed another conserved structural element, a short hairpin, immediately upstream of the pk-sl2. formation of this hairpin would compete with base-pairing interactions required to form the basal part of the bsl and the pk stem 1, respectively (fig. 5b) . furthermore, the hairpin overlaps partly with the pk loop 1 region that, in a previous study, was suggested to interact with the extreme 3 end of the genome (züst et al., 2008) . the conservation of both structure and sequence of this hairpin suggest a biological function for this element. in this context, it may be worth mentioning that the hairpin structure is predicted to be disrupted by the 6-nt insertion in loop 1 that was reported previously to cause a poorly replicating and unstable phenotype in mhv (goebel et al., 2004a) . it remains to be seen if the small hairpin represents yet another element in the intricate network of basepairing interactions between the bsl, the pk, and the 3 end that together constitute the complex molecular switch proposed by the masters laboratory (goebel et al., 2004a) . consistent with previous studies, we identified only one conserved rna structural element in the hvr downstream of pk-sl2. this stem-loop contained the conserved octanucleotide sequence in a single-stranded region. as pointed out above, the role of this element is currently unclear as both the hvr and octanucleotide sequence proved to dispensable for betacoronavirus (mhv) replication in vitro (goebel et al., 2007; liu et al., 2001) . overall, the alignment-based structure prediction algorithms used in our analysis led to conclusions that were consistent with results obtained in previous studies on betacoronavirus 3 utrs, suggesting that the approach might also be suitable to make reliable predictions on conserved rna structural elements in the highly variable alphacoronavirus 3 utrs. our alignment-based secondary structure predictions using representative viruses from all currently recognized alphacoronavirus species revealed the conservation of several rna structural elements in the alphacoronavirus 3 utr. a counterpart of the betacoronavirus bsl structure (goebel et al., 2004a; hsue and masters, 1997) could not be identified in the alphacoronavirus 3 utr while structural elements suitable to form a pk structure could be identified in all alphacoronaviruses (fig. 6a) . interestingly, despite the absence of an upstream bsl in alphacoronaviruses, the formation of this putative pk structure is predicted to require the disruption of a short hairpin immediately upstream of pk-sl2, a scenario that is similar but less complex compared to the situation described above for betacoronaviruses. it remains to be investigated in further studies whether or not alphacoronaviruses employ a molecular switch mechanism similar to what has been described for betacoronaviruses (goebel et al., 2004a) . the predicted sl2 structure (fig. 6 ) could be confirmed by structure probing data obtained for hcov-229e and hcov-nl63 (((.....) )).)))....... (((((((((((...........) )))))))))).. ... ((((((..(((((((((((..((((...((.....) )...))))....))))))))))))))))).. 3' pk-sl2 bulged stem-loop hvr octanucleotide a b c fig. 5 . alignment-based secondary structure prediction of betacoronavirus 3 genome regions. the viruses included in this analysis represent all currently recognized lineages and species in the genus betacoronavirus. the alignment was generated using locarna and the structure was calculated using rnaalifold. the consensus sequence is represented using the iupac code. colors are used to indicate conserved base pairs: from red (conservation of only one base-pair type) to purple (all six base-pair types are found); from dark (all sequences contain this base pair) to light colors (1 or 2 sequences are unable to form this base pair). gray bars below the alignment indicate the extent of sequence conservation. gray shadows are used to link rna structures with the corresponding dot-bracket notations above the alignment. (a) alignment-based secondary structure prediction of the bulged stem-loop (bsl) in the 3 utr. (b) alignment-based secondary structure prediction of the pseudoknot (pk) region in the 3 utr. note that pk-sl1 is an alternate structure that requires base-pairing interactions between the loop region of pk-sl2 and the basal part of the bsl shown in (a). formation of the bsl basal part and pk structure, respectively, are mutually exclusive (see text for details). (c) alignment-based secondary structure prediction of the hypervariable region (hvr) in the 3 utr. to refine the alignment, an anchor at the highly conserved octanucleotide sequence was used. ( fig. 7 , detailed structure probing data to be published elsewhere). furthermore, our structure probing data indicated base-pairing interactions upstream of sl2 in hcov-nl63, supporting the formation of the predicted small hairpin in this region ( fig. 7a and b). by contrast, no such interactions were seen in hcov-229e. also, the structure probing data did not support the formation of a stable pk structure, possibly reflecting a similarly low thermodynamic stability as determined for the equivalent pk in betacoronaviruses (stammler et al., 2011) . further studies including reverse genetics experiments are required to confirm the existence and biological significance of the predicted alphacoronavirus pk structure. with respect to the hvr downstream of pk-sl2, an extensive stem-loop structure was predicted in our analyses of alphacoronavirus 3 utrs (fig. 6b) . the structure is supported by a large number of covariant base pairs and contains the conserved octanucleotide sequence in a single-stranded region. the large distal part of the stem-loop structure was further corroborated by structure probing data (fig. 7 , details to be published elsewhere). in both hcov-229e and hcov-nl63, the octanucleotide sequence was found to be located in the loop region. of note, the cell cultureadapted hcov-nl63 isolate used for our structure probing analysis contained a short deletion apparently acquired upon serial passaging in cell culture, resulting in a significantly smaller loop but retaining the octanucleotide sequence (with one g-to-a replacement) in an identical position in the loop when compared to hcov-229e (see fig. 7a and b ). this serendipitous deletion shows that the distal part of the extended stem-loop structure is not essential for hcov-nl63 replication in cell culture. the data also indicate that, despite the deletion, the octanucleotide sequence retains a position in a loop region of the stem-loop structure and tolerates (.((...) ))))))... (((((((((((...........) ))))))))))... 3' pk-sl1 ..) ))) ......)) ).....))) )).)))). ..))))).. . )))))..))...... )))...... )))..))) ))...)))) .. fig. 6 . alignment-based secondary structure prediction of alphacoronavirus 3 -terminal genome regions. the viruses included in this analysis represent all currently recognized species in the genus alphacoronavirus. the alignment was calculated by locarna, the structure by rnaalifold. the consensus sequence is represented using the iupac code. colors are used to indicate conserved base pairs: from red (conservation of only one base-pair type) to purple (all six base-pair types are found); from dark (all sequences contain this base pair) to light colors (1 or 2 sequences are unable to form this base pair). gray bars below the alignment indicate the extent of sequence conservation at a given position. gray shadows are used to link rna structures with the corresponding dot-bracket notations above the alignment. (a) alignment-based secondary structure prediction of the pseudoknot (pk) region in the 3 utr. formation of the stem of pk-sl1 requires base-pairing interactions with the loop region of sl2. formation of the pk and the two sl structures shown above the alignment are mutually exclusive (see text for details). (b) alignment-based secondary structure prediction of the hypervariable region (hvr) in the 3 utr. to refine the alignment, an anchor at the highly conserved octanucleotide sequence was used. pk-s1 fig. 7 . rna secondary structure predictions of 3 -terminal genome regions of hcov-229e (a) and hcov-nl63 (b). predictions were generated using rnafold --nolp -c. as constraints, structure probing data were used. formation of the predicted pseudoknot (pk) requires base-pairing interactions between the loop region of sl2 and an upstream sequence (and, possibly, structural rearrangements), resulting in the formation of pk stem 1 (pk-s1) as indicated. also shown is the octanucleotide sequence that is conserved across all genera of the coronavirinae. minimal changes, the latter being consistent with mhv reverse genetics data obtained for the hvr/octanucleotide region (goebel et al., 2007) . based on numerous studies on betacoronaviruses including structural, biochemical, and reverse genetic work (di rna and replication-competent virus), a picture of putative cis-acting elements is beginning to emerge (reviewed in masters, 2007; sola et al., 2011b) . previous work also identified a growing number of cellular and viral proteins that bind to these structures and likely have important functions at different steps of genomic and/or subgenomic rna synthesis, genome packaging, genome expression or intracellular targeting of structures engaged in viral rna synthesis (reviewed in narayanan and makino, 2007; sola et al., 2011b) . using a combination of bioinformatic and biochemical methods, the present study confirms and extends this previous work. our study suggests that rna secondary structure elements may be more conserved than previously thought, both within individual coronavirus genera and across different genera as shown here for the genera alphacoronavirus and betacoronavirus. although significantly more work is needed to further characterize the structures identified in this and previous studies and understand their functional role, the available data suggest a cross-genus conservation of a number of rna structural elements among alpha-and betacoronaviruses. the conservation pattern is consistent with the replicase gene-based classification of genera within the subfamily coronavirinae (de groot et al., 2012a) . conserved elements include stem-loops 1, 2, 4, and, possibly, 5 in the 5 -terminal genome region and a putative pk in the 3 utr. the data further suggest that, in both alpha-and betacoronaviruses, the formation of the pk may require structural rearrangements in other regions (upstream of the sl2) and it remains an attractive idea to suggest specific functions for these alternative structures whose mechanistic and functional details remain to be investigated (goebel et al., 2004a; züst et al., 2008) . finally, in line with previous observations (brian and baric, 2005; masters, 2007; sola et al., 2011b) , the study confirms a significant degree of variation in the 3 -terminal region of the 3 utr, with the conserved octanucleotide sequence being consistently located in a single-stranded region of a stem-loop structure. interestingly, specific lineages of beta-, gamma-and deltacoronaviruses (as well as other plus-strand rna viruses) may contain another structural element, called s2m, in the 3 utr, thus further adding to the variability of this genome region in coronaviruses (jonassen et al., 1998; robertson et al., 2005; tengs et al., 2013) . the conservation pattern of the various lineage-specific structural elements argues against a conserved function in viral rna synthesis but rather suggests that the 3 utr may contain elements that are involved in specific virushost interactions and/or pathogenesis as has been shown for the mhv hvr (goebel et al., 2007) . the nucleoprotein is required for efficient coronavirus genome replication engineering the largest rna virus genome as an infectious bacterial artificial chromosome structure 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nsp16/nsp10 protein complex cis-acting sequences required for coronavirus infectious bronchitis virus defective-rna replication and packaging family coronaviridae middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group order nidovirales the fitness of defective interfering murine coronavirus di-a and its derivatives is decreased by nonsense and frameshift mutations coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization crystal structure and functional analysis of the sars-coronavirus rna cap 2 -o-methyltransferase nsp10/nsp16 complex coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2 o)-methyltransferase activity organelle-like membrane compartmentalization of positive-strand rna virus replication factories identification of a novel coronavirus in patients with severe acute respiratory syndrome infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing probing the structure of rnas in solution transmissible gastroenteritis coronavirus packaging signal is located at the 5 end of the virus genome host cell proteins interacting with the 3 end of tgev coronavirus genome influence virus replication characterization of the rna components of a putative molecular switch in the 3 untranslated region of the murine coronavirus genome a hypervariable region within the 3 cis-acting element of the murine coronavirus genome is nonessential for rna synthesis but affects pathogenesis the 3 cis-acting genomic replication element of the severe acute respiratory syndrome coronavirus can function in the murine coronavirus genome rna replication of mouse hepatitis virus takes place at double-membrane vesicles coronavirus n protein n-terminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes genetic evidence of a long-range rna-rna interaction between the genomic 5 untranslated region and the nonstructural protein 1 coding region in murine and bovine coronaviruses an optimal cis-replication stem-loop iv in the 5 untranslated region of the mouse coronavirus genome extends 16 nucleotides into open reading frame 1 bovine coronavirus nonstructural protein 1 (p28) is an rna binding protein that binds terminal genomic cis-replication elements live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis the 5 end of coronavirus minus-strand rnas contains a short poly(u) tract bovine coronavirus mrna replication continues throughout persistent infection in cell culture characterization of an essential rna secondary structure in the 3 untranslated region of the murine coronavirus genome a bulged stem-loop structure in the 3 untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication alphacoronavirus 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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) 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and respiratory syndrome virus virus-encoded proteinases and proteolytic processing in the nidovirales this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology (2010-0002318). key: cord-258546-1tf5ggfo authors: chung, hee-chun; lee, jee-hoon; nguyen, van giap; huynh, thi my le; lee, ga-eun; moon, hyoung-joon; park, seong-jun; kim, hye-kwon; park, bong kyun title: new emergence pattern with variant porcine epidemic diarrhea viruses, south korea, 2012–2015 date: 2016-12-02 journal: virus res doi: 10.1016/j.virusres.2016.06.013 sha: doc_id: 258546 cord_uid: 1tf5ggfo since outbreaks of porcine epidemic diarrhea virus (pedv) in the united states in 2013, explosive outbreaks of ped in south korea have infected all age groups of pigs in 2014–2015 year. this study analyzed a large collection of the spike protein coding gene to infer the spatial-temporal diffusion history of pedv. the studying results suggested that pedvs in korea belonged to different genogroups. while classical g1 was continuingly circulating between provinces of korea, the pandemic g2a were recently introduced from china and usa. by the application of bayesian phylogeographical analysis, this study demonstrated the spatial-temporal transmission of pedvs within korea. of the recent emerged g2a viruses, j3142 strains showed potential recombination breakpoint (376–2,143nt) of s1 gene between knu1303_korea strain_g2a (kj451046) and 45rwvcf0712_thailand strain_g2b (kf724935). the pandemic g2a virus was partial neutralized by the antibodies invoked by the g1based ped vaccine virus. porcine epidemic diarrhea virus (pedv) is an acute contagious diarrhea disease caused in pigs (song and park, 2012; jung et al., 2014) . the virus belongs to genus alpha-coronavirus, family coronaviridae, which includes other genera; beta-, gamma-, and delta-coronavirus, and has positive-sense single stranded rna genome with envelope (woo et al., 2012) . pedv is transmitted mainly through the fecal-oral route, infecting all age groups of pigs but the most severe form of diseases occurs in suckling piglets (song and park, 2012) . in 2013, pedv was reported for the first time in north american and now pedvs are present in all of swine producing countries (song and park, 2012; huang et al., 2013 ; mole, 2013) . in korea, pedv was first reported in 1992 (kweon et al., 1993) , and continuingly circulated and exhibited significant genetic diversity * corresponding author. e-mail address: parkx026@snu.ac.kr (b.k. park). 1 these authors have contributed equally to this study. (choi et al., 2014; lee and lee, 2014) . ped outbreaks re-occurred in korea in 2013, however, it was demonstrated that the emerging pedvs were not variants of old korean isolates or attenuated vaccine strains (chung et al., 2015) . though a recent publication characterized the genetic evolution of pedvs in korea from 1998 to 2013, to our knowledge, the transmission patterns of the virus in korea are largely unknown. using a large dataset collected globally and locally, this study aimed to trace the spatial-temporal dynamics of pedvs in korea. the spike protein coding gene (s gene) was selected for genetic analysis. aimed at provide a detail about the evolution of pedv in korea, we obtained from the previous study 31 partial sequences of the s gene which were collected in years 2002-2005. additionally, this study sequenced complete s genes from pedv positive samples which were stored in our laboratory from october 2012 fig. 1 . bayesian time-scaled phylogeny of pedv with inferred geographical location states. the branches of maximum clade credibility tree were colored according to the most probable location state of their descendent nodes. the color codes are defined in the insert legend. the s gene-based phylogeny clearly divided pedvs into genogroup 1 (g1) and two subgroups of genogroup 2 (g2a, g2b). for clarity, only the leaves of korean pedvs were highlighted. it was obvious that pedvs circulating in korea were within g1. to march 2015. to infer the spatial-temporal diffusion history of pedv, sequences of the s gene which had known collection date and country of origin were achieved from genbank. overall, the final dataset contained 805 sequences originating from asia (china, korea, vietnam, thailand, japan, taiwan), europe (belgium, slovenia, france), and american (usa, canada, mexico, colombia), and covering a sampling period from 1986 to 2015. the details of the dataset are summarized in supplementary table s1. a bayesian framework (lemey et al., 2009 ) was applied to reconstruct the spatial-temporal diffusion history of pedvs. in brief, the spatial diffusion of the time-scaled genealogy is modeled as a standard continuous-time markov chain (ctmc) process over discrete sampling locations. a bayesian stochastic search variable selection (bssvs) approach, which allows the exchange rates in the ctmc to be zero with some prior probability, was used to find a parsimonious set of rates explaining the diffusions in the phylogeny. the analysis was performed using beast package v1.8.2 under assumptions of (i) a codon based srd06 nucleotide substitution model, (ii) a constant population size for the coalescent prior, and (iii) the molecular clock model of uncorrelated lognormal distribution. the analysis was run for 100 million chains, sampling every 10,000 generations. pedv 411 s complete gene sequences aligned were used by recombination detection program v.4.46 (tian et al., 2014 and vlasova et al., 2014) ; x-over automated rdp analysis was used to identify recombination points within the pedv genome. in the previous study, it was noted that mutation occurred at ss6 neutralizing epitope of the s gene of an isolate collected from 2013 to 2014 outbreak. this study tested whether pedv isolated recently was crossed neutralized by serum of pigs which were vaccinated with korean ped oral vaccine (attenuated dr13 strain, green cross veterinary product co., ltd., yong-in, korea). the serum neutralizing test (sn test) was conducted using the previous method (song et al., 2007) , with some modifications. in brief, sera of pigs (n = 25) were heat inactivated at 56 • c for 30 min and stored at −20 • c until use. the sera were serially two-fold diluted. subsequently either bm3 (a recent pedv isolate) or dr13 (ped vaccine strain) of 200 tcid 50 /0.1 ml were mixed at equal volume of the diluted sera. the mixtures were incubated for 1 h at 37 • c. after incubation, 0.1 ml of each virus-serum mixture was infected to monolayer of vero cells. the presence/absence of cpe were monitored daily for 5 days. the sn titers were expressed as the reciprocal of the highest serum dilution which resulted in the inhibition of cpe. the result of the bayesian phylogeographic analysis is shown in fig. 1 . it was obvious that pedvs are circulating in korea from the earliest to the latest sequences (years 1997-2015) , the virus were classified into genogroup 1 (g1) and genogroup 2 (subgroups 2a and 2b). for the korean pedvs of g1, the viruses were found spanning from 1997 until 2012. meanwhile, the viruses of g2 (g2b and g2a) were detected from 2008 until 2015. as shown in fig. 1 , it was clear that the root of korean g1 and g2b viruses were inferred to be originated from korea, while the recent korean pedvs were derived from china and usa. the result implied that the transmission histories of pedvs in korea were diverse, which reflecting both the continuingly circulation of classical g1 and the introduction of the recent pandemic g2a. the detail of spatial-temporal dynamics of pedvs between provinces of korea is shown in figs. 2-4 . among the korean g1 viruses, the result of fig. 2 suggested the dispersal of the virus between different provinces, of which gyeonggi was the main source in the viral migration network. among the korean g2b viruses (fig. 3) , another province (chungnam) was predicted to be the initial source for the spreading of virus into gyeongbuk, and then from gyeongbuk to jeonnam. the picture was totally different from the korean g2a viruses, of which the sources were predicted to be china and usa (fig. 4) . in order to detect potential recombinant strains of j3142 and bm3, we used recombination detection program with 411 references pedv strains in this study. it has not been found that bm3 has recombinant region. interestingly, two potential strains of j3142 had recombination which had breakpoint from 376nt (aligned; 391nt) to 2143nt (aligned; 2206nt). one had major patent similarity of 95.3% with knu1303 korea strain (kj451046) which belongs to the subgroup g2a, and the other had minor patent similarity of 96.4% with 45rwvcf0712 thailand strain (kf724935) which belongs to the subgroup g2b. in this study, j3142 had a variant s1 (duarte and laude, 1994; gallagher and buchmeier, 2001; lee et al., 2010) region, suggesting that the recombination between south korea and thailand strains had occurred. in fig. 5 , we used only 411 references for the recombination analysis; thus, it might be possible that in south korea, there were strains, which are similar to those of thailand such as 45rwvcf0712, leading to the recombinant event. fig. 3 . bayesian time-scaled phylogeny of sub-genogroup 2b of pedvs. the branches of maximum clade credibility tree were colored according to the most probable location state of their descendent nodes. for clarity, the annotations of leaves were omitted. shown in every internal nodes of the phylogeny were the inferred locations. it was observed that chungnam was predicted to be the initial source for the spreading of virus into several provinces. in the sn test (table 1) , 52% (13/25) of the bm3 strain were positive and 88% (22/25) of the dr13 strain were positive. the average titer of the dr13 (14.53 value) was 5 times more than that of the bm3 (2.9 value). the results implied that, the specific antibodies invoked by ped vaccine of g1 inefficiently conferred cross neutralizing activities to the pandemic g2a isolate. our result was inline with the previous report that suggests that limited cross-reactivity was detected between ped vaccine (of genogroup 1) and field viruses (of genogroup 2). the attenuated pedv vaccines based on the cv777 strains or dr13-derived strains might be antigenically less related to the newly emergent pedv strains; thus, the development of new vaccines based on current strains are needed (chen et al., 2014; chung et al., 2015; song and park, 2012; . in summary, this study suggested that pedvs in korea belonged to different genogroups. while classical g1 was continuingly circulating between provinces of korea, the pandemic g2a were recently introduced from china and usa. by the application of bayesian phylogeographical analysis, this study demonstrated the spatial-temporal transmission of pedvs within korea. of the recent emerged g2a viruses, j3142 strains showed potential recombination breakpoint (376-2,143nt) of s1 gene between knu1303 korea strain g2a (kj451046) and 45rwvcf0712 thailand strain g2b (kf724935). the pandemic g2a virus was partial neutralized by the antibodies invoked by the g1-based ped vaccine virus. isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states comparative genome analysis and molecular epidemiology of the reemerging porcine epidemic diarrhea virus strains isolated in korea isolation of porcine epidemic diarrhea virus during outbreaks in south korea sequence of the spike protein of the porcine epidemic diarrhoea virus coronavirus spike proteins in viral entry and pathogenesis origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs isolation of porcine epidemic diarrhea virus (pedv) in korea genetic characterization of porcine epidemic diarrhea virus in korea from outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea suchard. bayesian phylogeography finds its roots novel porcine epidemic diarrhea virus variant with large genomic deletion porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in gansu evidence of recombinant strains of porcine epidemic diarrhea virus distinct characteristics and complex evolution of pedv strains discovery of seven novel mammalian and avian coronaviruses in deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus the authors would like to thank hye jung yang, and jung ah kim for excellent technical assistance. this study was supported by the biogreen 21 program, rural development administration (grant no. pj011184), and by the bio-industry technology development program (grant no. 114055031sb010), ministry of agriculture, food and rural affairs, south korea. the authors declare that there are no conflicts of interest. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2016.06. 013. key: cord-299904-i5c6nf18 authors: cornelissen, e.; dewerchin, h.l.; van hamme, e.; nauwynck, h.j. title: absence of antibody-dependent, complement-mediated lysis of feline infectious peritonitis virus-infected cells date: 2009-04-07 journal: virus res doi: 10.1016/j.virusres.2009.03.017 sha: doc_id: 299904 cord_uid: i5c6nf18 cats infected with virulent feline coronavirus which causes feline infectious peritonitis (fip) usually succumb to disease despite high antibody concentrations. one of the mechanisms that can help resolving infection is antibody-dependent, complement-mediated lysis (adcml) of infected cells. adcml consists of virus-specific antibodies that bind to cell surface expressed viral proteins which result in complement activation and cell lysis. the objective of this study was to determine the sensitivity of fip-virus (fipv) infected cells towards adcml and to examine the role of the accessory proteins 3abc and 7ab in this process. adcml assays, using fipv strain 79-1146 and its deletion mutant strain δ3abc/δ7ab, were performed on: (i) crfk cells that show surface-expressed viral antigens, (ii) monocytes without surface-expressed viral proteins due to retention and (iii) monocytes with surface-expressed viral proteins since the antibody-mediated internalization of these proteins was blocked. as expected, no adcml was detected of the monocytes without surface-expressed viral antigens. surprisingly, no lysis was observed in the crfk cells and the monocytes that do show surface-expressed viral proteins, while controls showed that the adcml assay was functional. these experiments proof that fipv can employ another immune evasion strategy against adcml (besides preventing surface expression): the inhibition of complement-mediated lysis. this new evasion strategy is not attributed to the group-specific proteins since lysis of cells infected with fipv δ3abc/δ7ab was not detected. cats infected with virulent feline coronavirus which causes feline infectious peritonitis (fip) usually succumb to disease despite high antibody concentrations. one of the mechanisms that can help resolving infection is antibody-dependent, complement-mediated lysis (adcml) of infected cells. adcml consists of virus-specific antibodies that bind to cell surface expressed viral proteins which result in complement activation and cell lysis. the objective of this study was to determine the sensitivity of fip-virus (fipv) infected cells towards adcml and to examine the role of the accessory proteins 3abc and 7ab in this process. adcml assays, using fipv strain 79-1146 and its deletion mutant strain 3abc/ 7ab, were performed on: (i) crfk cells that show surface-expressed viral antigens, (ii) monocytes without surface-expressed viral proteins due to retention and (iii) monocytes with surface-expressed viral proteins since the antibody-mediated internalization of these proteins was blocked. as expected, no adcml was detected of the monocytes without surface-expressed viral antigens. surprisingly, no lysis was observed in the crfk cells and the monocytes that do show surface-expressed viral proteins, while controls showed that the adcml assay was functional. these experiments proof that fipv can employ another immune evasion strategy against adcml (besides preventing surface expression): the inhibition of complementmediated lysis. this new evasion strategy is not attributed to the group-specific proteins since lysis of cells infected with fipv 3abc/ 7ab was not detected. © 2009 elsevier b.v. all rights reserved. feline infectious peritonitis (fip) is a fatal disease, characterized by fibrinous-granulomatous serositis often with protein-rich effusions in body cavities, granulomatous-necrotising phlebitis and periphlebitis and granulomatous inflammatory lesions in several organs (weiss and scott, 1981a,b; kipar et al., 1998 kipar et al., , 2005 . the causative agent is a virulent form of the feline coronaviruses (fcovs) belonging to the family coronaviridae, order nidovirales. in vivo, monocytes and tissue macrophages are the target cells and play a central role in the development of the lesions (kipar et al., 2005) . these infected cells should be excellent targets for the immune system to fight the infection. however, in most cases the immune response is not protective and the cat succumbs to the infection. the cell-mediated immunity is believed to be important in control and clearance of the fip-virus (fipv) infection if there is an efficient first response to the infection. the humoral immune response is believed to be not protective. high concentrations of neutralizing antibodies are present in cats with end-stage fip and no difference is seen in the antibody concentration and fluctuations between survivors and non-survivors after a fipv infection (de groot-mijnes et al., 2005) . in general, virus-specific antibodies can help to resolve infection by antibody-mediated lysis of infected cells via cytolytic immune cells with fc receptor (like nk cells, macrophages or neutrophils) or via complement (sissons and oldstone, 1980 ). the complement system is an immunological defense system and plays a role in both the innate and the adaptive immune response against invading pathogens. complement consists of serum and membrane-bound proteins which, once activated, can trigger a biochemical cascade of reactions contributing to the eradication of pathogens (blue et al., 2004) . important complement effector functions are opsonization of pathogens, cytolysis and promoting host inflammatory responses (anaphylatoxin and chemotaxin production) (janeway et al., 2005) . in viral infections, the complement system can be activated by free virus particles and virus-infected cells. complement can inactivate free virus in the presence or absence of antibodies. opsonization of the virus with complement proteins can promote phagocytosis, virolysis and interference with attachment, internalization or uncoating of the virions (hirsch, 1982) . cells infected with enveloped viruses can be lysed by complement in the presence of antibodies if newly synthesized viral glycoproteins are expressed at the plasma membrane of the infected cell. virus-specific antibodies can then bind to these surface-expressed proteins and thereby activate the complement system. eventually, this results in cell death (sissons and oldstone, 1980 process is called antibody-dependent, complement-mediated lysis (adcml). recently, we described for fipv two processes that inhibit the expression of viral proteins at the plasma membrane of in vitro infected monocytes. namely, the retention of viral proteins in infected cells and the antibody-mediated internalization of surfaceexpressed viral proteins. both processes result in the clearance of all detectable viral antigens from the plasma membrane of infected cells (dewerchin et al., 2005; dewerchin et al., 2006) . fipvinfected monocytes/macrophages isolated from naturally infected cats do not express viral proteins at their plasma membrane either (cornelissen et al., 2007) . absence of viral proteins in the plasma membrane of infected monocytes can protect the infected cells from efficient adcml. this has been described for pseudorabies virus (prv), equine herpesvirus-1 (ehv-1) and porcine reproductive and respiratory syndrome virus (prrsv) (van der meulen et al., 2003; van de walle et al., 2003; costers et al., 2006) . the objective of this study was to determine if there is efficient adcml of fipv-infected cells that show surface-expressed viral antigens, since this would open treatment possibilities based on inhibiting antibody-mediated internalization of surface-expressed viral antigens. furthermore, the role of the accessory proteins 3abc and 7ab was assessed in this context. adcml assays with crandell feline kidney cells (crfks) and peripheral blood monocytes were performed. crfks were seeded in six-well plates (nunc) and cultivated in mem-medium containing 5% fetal bovine serum (fbs), 2% lactalbumine, 0.3 mg/ml glutamine, 100 u/ml penicillin, 0.1 mg/ml streptomycin and 0.1 mg/ml kanamycin. peripheral blood monocytes were isolated from feline coronavirus, feline leukemia virus and feline immunodeficiency virus negative cats as described previously (dewerchin et al., 2005) . they were cultivated in six-well plates in rpmi 1640-medium containing 10% fbs, 0.3 mg/ml glutamine, 100 u/ml penicillin, 0.1 mg/ml streptomycin, 0.1 mg/ml kanamycin, 10 u/ml heparin, 1 mm sodium pyruvate and 1% non-essential amino acids 100× (gibco brl). cells were infected with fipv type ii strain 79-1146 or with its attenuated deletion mutant virus strain fipv 3abc/ 7ab at a multiplicity of infection of 1. both viruses were kindly provided by dr rottier (faculty of veterinary medicine, utrecht university, the netherlands). the deletion mutant strain is the fipv strain 79-1146 from which the open reading frames 3abc and 7ab were deleted, using reverse genetics (haijema et al., 2004) . the crfks and the monocytes were mechanically detached (by gently pipetting up and down) from the wells at 18 h postinoculation (hpi) and 12 hpi, respectively, to perform the assays in suspension. the cells were incubated for 1 h with fipv-specific polyclonal antibodies (pabs) (0, 0.9 and 1.8 mg/ml) or antibodies purified from fipv-negative serum (1.8 mg/ml). the fipv-specific pabs originated from cats infected with fipv 79-1146 and were provided by dr rottier. the fipv-negative serum was derived from an fcov-negative cat (ipma antibody titer <20). both pabs had been purified using protein a-sepharose (amersham biosciences). the cells were washed and incubated with 5% complement (noninactivated serum of a fcov-negative cat). then, the cells were stained with ethidium monoazide bromide (ema) (molecular probes) to label dead cells, fixed with 3% paraformaldehyde (vwr), permeabilized with 0.1% saponin (sigma) and stained with specific monoclonal antibodies (mabs) against fipv nucleocapsid (n) protein and membrane (m) protein, followed by fitc-labeled goat anti-mouse igg (molecular probes) to identify fipv-infected cells. the mab recognizing the m and n protein were produced and characterized in our laboratory. nuclei were stained with hoechst 33342 (molecular probes). dead infected cells were counted using fluorescence microscopy. for the monocytes, the antibody-induced internalization was inhibited by pre-treatment for 30 min with myosin light chain kinase inhibitor and inclusion of the inhibitor during antibody and complement incubation (dewerchin, 2008) . since antibodies must be bound to the cell before adcml can occur, an immunofluorescent staining was performed to determine the presence of the antibodies on the surface of the infected cells. cells were fixed with 3% paraformaldehyde after incubation with fipv-specific pabs. antibodies were stained with fitc-labeled goat anti-cat igg (sigma). after permeabilization with 0.1% saponin, infected cells were stained with n-and m-specific mabs and texas red-labeled goat anti-mouse igg (molecular probes). nuclei were stained with hoechst 33342 (molecular probes). two control assays were performed to verify the functionality of the adcml assay: (i) a fipv neutralization assay to detect a higher neutralization in the presence of complement in order to confirm the activity of the feline complement in combination with the fipv-specific pabs and (ii) an adcml assay on pseudorabies virus (prv)-infected feline monocytes and crfks to confirm the activity of the feline complement, the sensitivity of the cells to adcml and to exclude possible interference of the used media. the adcml assay was performed as described above using prv strain kaplan, feline pabs against prv (derived from a geskypur (merial) vaccinated fcov-negative cat, according to manufacturer's instructions), feline complement and fitc-labeled prv-specific pabs to identify infected cells. it has been described that prv-infected porcine macrophages with surface-expressed viral antigens are sensitive to adcml (van de walle et al., 2003). the results of the adcml with fipv 79-1146 and the deletion mutant virus strain fipv 3abc/ 7ab are given in fig. 1 . with both viruses and both cell types, the percentages of dead fipv-infected cells in the adcml assay with virus-specific antibodies were not significantly different from those in the assay without antibodies (p ≤ 0.05). also no significant difference was seen with the adcml assay with fipv-negative antibodies. triplicate assays were performed and results were compared using the friedman test from the spss software package (version 12.0; spss inc.). the presence of fipv-specific antibodies on the surface of infected cells was similar for fipv 79-1146 and fipv 3abc/ 7ab. of the infected crfks, 99 ± 0.3% showed surface-bound antibodies. formation of small aggregates was seen in approximately onefourth of these cells ( fig. 2a) while the other cells showed larger aggregates (patches) (fig. 2b) . of the infected monocytes, 53 ± 1% showed surface-bound antibodies. approximately two thirds of these cells showed small antibody aggregates (fig. 2c) while the remaining cells showed a more homogeneous antibody distribution (fig. 2d ). this means that cells are protected against adcml, irrespective of the expression of viral proteins at their surface. the performed controls showed that the combination of antibodies and complement was effective in neutralizing fipv and that the cells were sensitive to adcml. in the presence of complement the virus neutralization titer was two times and 64 times higher for 1000 and 10 000 tcid 50 of fipv 79-1146, respectively. the adcml assay with prv showed that the adcml was effective for both crfks and feline monocytes. for both cell types there was a significant rise in the percentage of dead cells with higher antibody concentrations (p ≤ 0.05; friedman test) (fig. 1) . the humoral immune system is activated during a fipv infection, but the antibodies formed seem to be ineffective in eliminating virus and virus-infected cells. for fipv-infected cells, antibodymediated internalization of plasma membrane-expressed viral proteins has been described (dewerchin et al., 2006) . this inter-nalization, resulting in the absence of viral proteins in the plasma membrane, can protect the infected cells from efficient adcml. inhibiting this internalization process, resulting in infected cells being recognizable by antibodies, could be a part of a treatment protocol for fip cats. unfortunately, results of this study show that even if there are viral proteins present on the plasma membrane, no lysis does occur through adcml. however, if antibodies bind to the surface-expressed viral proteins, cell lysis may still occur via other cell lysis mechanisms e.g. via antibody-dependent, cell-mediated cytotoxicity. in this mechanism, lysis is performed by activated natural killer cells, neutrophils, monocytes or macrophages. lysis of virus-infected cells by antibodies and complement requires viral proteins that are expressed in a form and configuration recognizable by antibodies that can bind complement (hirsch, 1982) . the fipv-specific antibodies used are able to bind complement since virus-neutralization was higher in the presence of complement. this higher neutralization can be due to virolysis, agglutination of virus-antibody-complement complexes or coating of the virus with complement components which can interfere with the binding of the virus to target cells or alter the surface charge of the virus (hirsch, 1982; lachmann and davies, 1997) . for example, it could be possible that binding of complement to the fc portion of the antibody inhibits antibody-dependent enhancement of infectivity (adei), a mechanism which is described in vitro (hohdatsu et al., 1991) . the fact that neutralizing antibodies are present in a cat with fip, together with the observed enhancement of neutralization by complement in this study, indicates that cell free virus does not play an important role in the pathogenesis of fip (de groot-mijnes et al., 2005) . for the effectiveness of the adcml, the amount of bound antibodies is also important. the more antibodies that are bound on the surface of infected cells, the higher the percentage of lysed cells is . all fipv-infected crfks showed bound antibodies on their surface. the formation of patches is not likely to have an influence on the adcml assay. it has been described for measles virus-infected cells that redistribution of viral antigens has no influence on the effectiveness of the adcml . half of the infected monocytes showed bound antibodies on their surface, which are consistent with previously published results (dewerchin et al., 2005) . as expected, the monocytes without surface-expressed viral proteins were protected against adcml, but also no adcml was seen of the monocytes with surface-expressed viral proteins. the possibility that insufficient amounts of antibodies were bound on infected crfks and monocytes to have a detectable effect of adcml is unlikely, but cannot be completely excluded. it seems that fipv can employ another virus complement evasion strategy in addition to the already described retention of viral proteins in the cytoplasm of infected cells and the antibodymediated internalization of plasma membrane-expressed viral proteins. this additional evasion strategy cannot be attributed to the accessory or group-specific proteins 3abc and 7ab since lysis of cells infected with the double mutant strain fipv 3abc/ 7ab was not detected. these genes encode for proteins that are not necessary for virus growth and infection. their deletion has an attenuating effect on the virulence of the virus in cats (haijema et al., 2004) . the basis of the attenuation is not known, but there is no correlation with the efficacy of the adcml, as shown in the present study. this new immune evasion strategy has to be attributed to the structural proteins or the non-structural proteins of orf 1ab and can be direct via viral proteins or indirect by means of cellular proteins that regulate the complement cascade. various virus complement evasion strategies have been described for other viruses. viruses can express proteins with fc receptor activity that can inhibit the binding of complement to the antibody-antigen complex. the glycoprotein ge expressed by herpes simplex virus (hsv) has an fc receptor activity and protects against antibody and complement-mediated lysis, both in vitro and in vivo (adler et al., 1978; lubinski et al., 2002) . for fipv, the presence of an fc receptor activity has never been studied but the spike proteins of other coronaviruses, namely mouse hepatitis virus (mhv), bovine coronavirus (bcv) and transmissible gastroenteritis virus (tgev) display fc receptor activity (oleszak et al., 1995) . another possibility is that the virus encodes proteins with functional similarities to complement control proteins, inhibitors of the complement cascade. no viral complement control proteins have been described for coronaviruses but several have been described for poxviruses and herpesviruses bernet et al., 2003) . for fipv, this would imply that the spike, membrane and envelope protein or the non-structural proteins encoded by orf 1 should exhibit complement control protein activity, like binding to or accelerating the decay of certain complement factors. finally, it is possible that the virus induces an upregulation of host complement control factors in the infected cell. this has been described for human cytomegalovirus (hcmv) where upregulation of complement regulator cd55 protected infected cells from complement-mediated lysis (spiller et al., 1996) . fipv occurs in two types (types i and ii) with type i prevailing in the field (pedersen, 2009 ). both types can cause clinical fip and evade the immune system of the cat. the results in this study were obtained with a type ii strain. whether cells infected with a type i strain also show inhibition of complement-mediated lysis has to be determined. a different outcome is possible since this evasion strategy could be attributed to the s protein and the main differences between types i and ii are found in this protein (herrewegh et al., 1998) . in conclusion, it can be stated that fipv-infected cells are protected against adcml, both the cells with and without surfaceexpressed viral proteins. it appears that during evolution, fipv has become a master in disguise, exhibiting several immune evasion mechanisms to avoid clearance of infected cells by the humoral immune response. possible role of fc receptors on cells infected and transformed by herpesvirus: escape from immune cytolysis viral mimicry of the complement system the relevance of complement to virus biology absence of surface expression of feline infectious peritonitis virus (fipv) antigens on infected cells isolated from cats with fip porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibodydependent, complement-mediated cell lysis natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease characterization of putative immune evasion mechanisms of feline infectious peritonitis virus replication of feline coronaviruses in peripheral blood monocytes feline infectious peritonitis virus-infected monocytes internalize viral membrane-bound proteins upon antibody addition virus complement evasion strategies live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis feline coronavirus type ii strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type i and canine coronavirus the complement system: its importance in the host response to viral infection a study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection in feline macrophages by monoclonal antibodies immunobiology, the immune system in health and disease measurement of virus antigens on the surface of hela cells persistently infected with wild type and vaccine strains of measles virus by radioimmune assay cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis morphologic features and development of granulomatous vasculitis in feline infectious peritonitis complement and immunity to viruses herpes simplex virus type 1 evades the effects of antibody and complement in vivo molecular mimicry between fc receptor and s peplomer protein of mouse hepatitis virus, bovine corona virus, and transmissible gastroenteritis virus a review of feline infectious peritonitis virus infection: 1963-2008 mechanism of injury of virus-infected cells by antiviral antibody and complement: participation of igg, f(ab )2, and the alternative complement pathway antibody-mediated destruction of virus-infected cells altered expression of hostencoded complement regulators on human cytomegalovirus-infected cells absence of viral antigens on the surface of equine herpesvirus-1-infected peripheral blood mononuclear cells: a strategy to avoid complement-mediated lysis antibodyinduced internalization of viral glycoproteins and ge-gi fc receptor activity protect pseudorabies virus-infected monocytes from efficient complementmediated lysis pathogenesis of feline infectious peritonitis, pathologic changes and immunofluorescence pathogenesis of feline infectious peritonitis, nature and development of viremia e. cornelissen and h.l. dewerchin were supported by the institute for the promotion of innovation through science and technology in flanders (iwt-vlaanderen). e. van hamme was supported by a doctoral grant from the special research fund of ghent university. key: cord-256343-dtfw8o4g authors: brandão, paulo eduardo title: the evolution of codon usage in structural and non-structural viral genes: the case of avian coronavirus and its natural host gallus gallus date: 2013-12-26 journal: virus res doi: 10.1016/j.virusres.2013.09.033 sha: doc_id: 256343 cord_uid: dtfw8o4g to assess the codon evolution in virus–host systems, avian coronavirus and its natural host gallus gallus were used as a model. codon usage (cu) was measured for the viral spike (s), nucleocapsid (n), nonstructural protein 2 (nsp2) and papain-like protease (pl(pro)) genes from a diverse set of a. coronavirus lineages and for g. gallus genes (lung surfactant protein a, intestinal cholecystokinin, oviduct ovomucin alpha subunit, kidney vitamin d receptor and the ubiquitary beta-actin) for different a. coronavirus replicating sites. relative synonymous codon usage (rscu) trees accommodating all virus and host genes in a single topology showed a higher proximity of a. coronavirus cu to the respiratory tract for all genes. the codon adaptation index (cai) showed a lower adaptation of s to g. gallus compared to nsp2, pl(pro) and n. the effective number of codons (nc) and gc(3%) revealed that natural selection and genetic drift are the evolutionary forces driving the codon usage evolution of both a. coronavirus and g. gallus regardless of the gene being considered. the spike gene showed only one 100% conserved amino acid position coded by an a. coronavirus preferred codon, a significantly low number when compared to the three other genes (p < 0.0001). virus cu evolves independently for each gene in a manner predicted by the protein function, with a balance between natural selection and mutation pressure, giving further molecular basis for the viruses’ ability to exploit the host's cellular environment in a concerted virus–host molecular evolution. codon usage (cu) refers to the frequency of the occurrence of each codon for at least two-fold degenerate codons (hershberg and petrov, 2008) , i.e., it is an indication of the 'preference' of a genome for one or more codons if more than one codon is possible for the same amino acid. natural selection for efficient protein synthesis speed and folding and genetic drift based on mutation pressure that leads to a homogeneous genome and the 3rd codon's gc%s are the most evident forces under codon usage evolution that could lead to detectable codon usage bias (cub) (yang and nielsen, 2008) , which has been increasingly used in studies on virus and host molecular evolution. avian coronavirus (nidovirales: coronaviridae: coronavirinae: gammacoronavirus), which originated approximately 4800 years ago (woo et al., 2012) and has a large number of serotypes and genotypes, primarily infects the respiratory tract of laying hens, broilers and breeders but can also infect the kidneys, intestines and reproductive tracts of both females and males (cook et al., e-mail address: paulo7926@usp.br 2012), depending on the pathotype. though affinity to different classes of cell membrane glycans could be one of the explanations for the existence of the different viral pathotypes (wickramasinghe et al., 2011) , the exact mechanism for this level of diversity is still unknown. the 27.6 kb single-stranded positive sense rna of a. coronavirus encodes 23 proteins, and the first two-thirds of the genome contains orf 1, which encodes 15 non-structural proteins involved in rna transcription and replication (masters, 2006; ziebuhr and snijder, 2007) . among these, the papain-like protease (pl pro ) is the proteolytic processor of the n-proximal domain of polyproteins pp1a and pp1ab (ziebuhr et al., 2000) . non-structural protein 2 (nsp2), the first in orf 1 because the a. coronavirus lacks nsp1, has a still undefined role, though a role on global rna synthesis has been suggested (graham et al., 2005) . of the structural proteins, the spike glycoprotein (s) has a strong interaction with the host immune system and is so highly polymorphic that mutations in only 10 amino acids on the amino terminal ectodomain (s1) could result in the loss of cross-reactivity (cavanagh, 2007) . while s1 allows the virus to attach to ␣2,3sia, which is widespread in chicken cells (winter et al., 2008) , the carboxy terminal s2 has the capacity to fuse virus-to-cell and cellto-cell membranes (masters, 2006) . the nucleocapsid (n) protein binds to the genomic rna due to its positively charged amino acid domains, and though under a more strict mutation constraint than s, positive selection plays a role in n evolution (kuo et al., 2013; masters, 2006) . the codon usage of a. coronavirus has been reported to be highly to moderately biased but closer to that found in the respiratory tract of gallus gallus when compared to other tissues (brandão, 2012) . however, that report was limited because codon usage was measured based on only the spike gene. the aim of this study was to assess the evolution of codon usage in viral structural and non-structural genes and their molecular relationship with host codon usage using a. coronavirus and its natural host g. gallus as a model. for a. coronavirus, sequences were chosen to promote diversity of geographic origin and serotype/genotypes, including the archetypical strains, with an effort to keep the same datasets if possible. because the number of complete genomes and genes for a. coronavirus available in genbank did not allow for the representation of such diversity, only partial genes were used in this study instead of complete ones to have the most diverse dataset possible. as the accuracy of codon usage measurements is lower for short sequences, sequences <100 codons in length (roth et al., 2012) were not included. sequence redundancy was avoided by keeping only one sequence if any 100% nucleotide identity was found. following these criteria, this study included 64 s protein sequences, codons 1-169 (14.6% of the 1162 s codons); 25 n protein sequences, codons 301-409 (26.7% of the 409 n codons); 18 nsp2 sequences, codons 1-245 (36.4% of the 673 nsp2 codons); and 15 papain-like protease sequences, codons 3-437 (99.5% of the 437 pl pro codons). the accession numbers are shown in fig. 1 . all indicated positions are relative to the complete genome of the avian infectious bronchitis virus strain m41 (dq834384.1). aiming to assess the codon usage of the different tissues in which a. coronavirus replicates in chicken, non-redundant complete codon sequences were retrieved from the genbank database and from the g. gallus genome project for cholecystokinin, expressed in the duodenum (nm 001001741.1 and gfc 000002315.3); lung surfactant pulmonary-associated protein a1 (sftpa1), expressed in the lungs (nm 204606.1 and gfc 000002315.3); vitamin d receptor, expressed in the kidneys (nm 205098.1 and gfc 000002315.3); and ovomucin alpha subunit, expressed in the oviduct (ab046524.1 and gfc 000002315.3). as a reference, the complete g. gallus beta-actin gene (l08165 and gfc 000002315.3) was included in the analyses as a ubiquitously expressed gene. all sequences used in this study can be found in supplementary material 1. rscu, the relationship between the observed and the expected frequency of a codon if the synonymous codon usage is random (roth et al., 2012) , was calculated for 59 codons, excluding the single codons of methionine and tryptophan and the three stop codons, using the equation rscu i = x i /( i x i /m) (nei and kumar, 2000) , where x i is the total count for a given codon, i x i is the sum of the count for all synonymous codons regarding the amino acid under consideration and m is the number of possible isoacceptors for that amino acid, implemented in mega 5.0 (tamura et al., 2011) . the continuous rscu values from a. coronavirus and g. gallus genes were converted to binary data using the value 1 for rscus >1, when a given codon was preferred for a specific amino acid, or 0 for rscus ≤ 1, when the codon was not preferred (rscu < 1) or was neutral (rscu = 1). finally, the combined dataset of the four viral and five host genes was used to build a binary 59 characters × 132 sequences matrix (supplementary material 2) for the presence or absence of a preferred codon, which was used to build a neighborjoining tree (1000 bootstrap replicates) using paup, version 4.1b (swofford, 2000) . the cai is a measure of codon usage derived from the geometric mean of the relative codon adaptiveness for each codon based on a set of translationally optimal codons used as a reference (roth et al., 2012) and can be calculated according to the equation here, w k is the relative adaptiveness of the kth codon (61 codons; the three stop codons were excluded), and x k,g is the fraction of the codon k relative to the total number of codons in the gene. values closer to 1 indicate a high fitness in terms of codon usage for a given codon sequence in relation to the reference system (sharp and li, 1987) , i.e., a high adaptation of viral genes to the host. the cai was calculated for sequences from both a. coronavirus and g. gallus using a reference set of highly expressed g. gallus genes available in the acua 1.0 software (vetrivel et al., 2007) . nc is a measure of the total number of different codons present in a sequence and shows the bias from equal use of all synonymous codons for a given amino acid, with each synonymous codon treated as an allele as in the calculation of the effective number of alleles in population genetics (roth et al., 2012) . nc values range from 20 to 61, with values closer to 61 indicating a lower bias (wright, 1990) . nc was calculated according to the equation nc = 2 + (9/f2) + (1/f3) + (5/f4) + (3/f6), where f is the average homozygosity for equal use of each synonymous codon for each class of degeneracy ranging from 2 to 6, using acua 1.0 (vetrivel et al., 2007) . the expected effective number of codons (enc), a measure of codon usage affected only by the gc 3% (the percentage of g or c at the third position of all codons in a sequence) as a result of mutation pressure and drift, was calculated using the equation enc expec = 2 + s + 29[s 2 + (1 − s) 2 ] −1 (wright, 1990) , where s is the gc 3% ranging from 0 to 100%. the enc and simulated gc 3% values were plotted as a curve together with the nc and observed gc 3% values; an nc × observed plot lying on the enc × simulated curve indicates genetic drift/mutational bias, while plots outside the curve indicate natural selection (wright, 1990 ). to assess the significance of each preferred codon on the molecular evolution of a. coronavirus, 100% conserved amino acid positions coded by the preferred codon(s), i.e., those with rscus >1, were counted for each gene, and the significance of the differences was assessed with fisher's exact test and the odds ratio (or). to understand the relationship between codon and protein selection, the occurrence of purifying or positive selection on a. coronavirus s, n, nsp2 and pl pro sequences was tested with fisher's exact test of neutrality for sequence pairs using the nei-gojobori method (nei and gojobori, 1986) for the difference between the synonymous and non-synonymous substitution distances (ds-dn) using mega 5 (tamura et al., 2011) . fig. 1 shows that g. gallus rscus segregate in a tissue-specific manner in a topology supported by bootstrap values of 100 for each gene analyzed. fig. 1 . neighbor-joining distance tree for the relative synonymous codon usage (rscu) for the avian coronavirus spike (s), nucleocapsid (n), non-structural protein 2 (nsp2) and papain-like protease (pl pro ) genes and the gallus gallus beta-actin, lung surfactant protein a (sftpa1, gray background), intestinal cholecystokinin (cck), oviduct ovomucin alpha subunit (osa) and kidney vitamin d receptor genes. the tree was based on binary data using the value 1 for rscus > 1 (codon is preferred) or 0 for rscus ≤ 1 when the codon is not preferred (rscu < 1) or is neutral (rscu = 1). enc (effective number of codons) values <40 and >45 are marked with an asterisk and a hash, respectively; sequences with enc values between 40 and 45 have no marks. the arrow indicates the separation between g. gallus and avian coronavirus clusters. numbers at each node are bootstrap values (1000 replicates, only values >50 are shown). the bar represents the codon usage preferences distance. for the a. coronavirus rscus, all genes segregated in genespecific clusters, except for the sequence eu526388.1 a2 pl pro , which segregated closer to the nsp2 cluster. all strains segregated in a cluster separated from g. gallus, with the internal nodes resulting in the genotype-specific sub-clusters for the s gene, including those for the archetypes connecticut, massachusetts and arkansas, with two sub clusters and the pl pro cluster between them. no pathotype-specific cluster was found. though the distinction between the a. coronavirus and g. gallus rscus clusters is also clear for the n, nsp2 and pl pro genes, a less resolved topology emerges because the distinction among the different genotypes is not sustained. for all four genes, a. coronavirus clusters show an increasing distance from the g. gallus clusters, with them being closer to sftpa1 (from the respiratory tract) and more distant from cholecystokinin (from the intestine) and with the ubiquitous beta-actin cluster being the most distant from both a. coronavirus and the other g. gallus clusters. mean cai values for the a. coronavirus s, n, nsp2 and pl pro genes were 0.66 (sd 0.01), 0.77 (sd 0.01), 0.69 (sd 0.01) and 0.7 (sd 0.01), respectively, while, for the g. gallus genes, the mean cai was 0.81 (sd 0.06), ranging from 0.71 for the pulmonary gene sftpa1 to 0.88 for the renal vitamin d receptor (mean values for two sequences). a boxplot representation of g. gallus and a. coronavirus cais (fig. 2) shows that, in relation to g. gallus, s has the lowest values (0.64-0.7) and n has the highest values (0.75-0.79), while nsp2 and pl pro have intermediate values (0.69-0.71), with non-overlapping medians. the mean nc values for a. coronavirus s, n, nsp2 and pl pro were 43 (sd 2.31), 44.9 (sd 3.64), 51.33 (sd 1.56) and 43.79 (sd 0.86), respectively, and for g. gallus, the mean nc values were 33.59 for vitamin d receptor, 40.03 for beta-actin, 46.48 for cholecystokinin, 50.21 for sftpa1 and 53.01 for ovomucin. the nc x gc 3% graphs (fig. 3) show that, regardless of the a. coronavirus gene under consideration (s, n, nsp2 or pl pro ), all plots fall either just below or in the vicinity of the enc × gc 3% expected curve. this same pattern was also found for the g. gallus genes, though with plots dislocated to the right side of the graph due to a higher gc 3% content. the number of 100% conserved amino acid positions coded by the preferred codons for genes s, n, nsp2 and pl pro was one, 20, 28 and 71, respectively. fisher's exact test showed that only the s gene presented a statistically significant lower number of occurrences (table 1 ) when compared to the other 3 genes (p < 0.0001), with ors conserved amino acid (aa) positions in the avian coronavirus spike (s), nucleocapsid (n), non-structural protein 2 (nsp2) and papain-like protease (pl pro ) genes coded by a preferred codon and the preferred codon for each aa in the gallus gallus beta-actin (b-act), lung surfactant protein a (sftpa1), intestinal cholecystokinin (cck), oviduct ovomucin alpha subunit (ovo) and kidney vitamin d receptor (vitd rec) genes. tryptophan and methionine, coded by a single codon, were excluded. codon preference was indicated by relative synonymous codon usage (rscu) >1. positions are provided only for avian coronavirus genes as g. gallus genes were used as the reference for comparison. nc: no 100% conserved amino acids positions coded by the preferred codon; nf: amino acid not found in the sequence. the mean number of amino acid residues in the sequences used for this study from the avian coronavirus spike (s), nucleocapsid (n), non-structural protein 2 (nsp2) and papain-like protease (pl pro ) genes coded by a preferred codon and the preferred codon for each aa in the gallus gallus beta-actin (b-act), lung surfactant protein a (sftpa1), intestinal cholecystokinin (cck), oviduct ovomucin alpha subunit (ovo) and kidney vitamin d receptor (vitd rec) genes. table 2 . the number of amino acids in the g. gallus proteins that presented the same codons used by at least one of the a. coronavirus genes in 100% conserved aa positions ranged from 1 (for vitamin d receptor) to 15 (for ovomucin alpha), and the most conserved preferred codon, found for all a. coronavirus genes, was uuu for f ( table 1 ). the positions of each of the conserved amino acids coded by preferred codons for a. coronavirus are also shown in table 1 . the sequences of n, nsp2 and pl pro from all the strains in this study were found to be under purifying selection as the p values from fisher's exact test were all above 0.05, with mean values of 0.99 for each gene and sd values of 0.06, 0.05 and 0.08, respectively. for s sequences, the mean p value was 0.97 (sd 0.13), but p values <0.05 were found between regardless of the gene being considered, all a. coronavirus sequences segregated in an exclusive cluster in the rscu tree, which, despite being consistently separate from the g. gallus cluster, was closer to the sftpa1 (a gene expressed in the respiratory tract of chicken) cluster. taking the codon usage for these genes as a reflection of the codon usage in the respiratory tract, both structural and non-structural genes show a codon usage closer to the chicken respiratory tissue translational environment than to the reproductive, renal and enteric ones. this similar codon usage could allow for an improved viral replication in the respiratory tract as a first site of viral replication, a feature common to all a. coronavirus strains in chickens, before the virus reaches other replication sites for each pathotype, as a result of the natural selection for codons and a more efficient translation of virus proteins, as already suggested for the s gene alone (brandão, 2012) . evidence of natural selection for codon usage as an evolutionary force acting upon a. coronavirus was found in the nc × gc 3% graphs (fig. 2 ) because for all four viral genes, observed gc 3% points fell outside the curve, indicating that codon usage for all the strains under analysis was not the sole result of the random accumulation of mutations. nonetheless, the nc × gc 3% plots show that a. coronavirus codon usage could also be a consequence of mutation pressure, as the points were in the vicinity of the curve, meaning that the gc% at the synonymous 3rd codon position follows the viral genomic gc% to some degree. it must be considered that both genetic drift derived from the mutation pressure and natural selection detected for a. coronavirus could also harbor some relationship with genomic rna secondary structure constraints and not only codon usage, as synonymous 3rd base mutations, though synonymous in terms of amino acid codification, could result in altered rna secondary structure (cardinale et al., 2013) and, consequently, impaired viral transcription, replication and assembly. as signals for rna replication and genome packaging in coronaviruses are rna secondary structuredependent (narayanan and makino, 2007; williams et al., 1999) , such structures must be under intense evolutionary constraints that balance with codon usage evolution. from the host side, mutation bias has also been shown to be the major driving force of g. gallus codon usage evolution, with minor participation of natural selection (rao et al., 2011) , in agreement with the results presented herein, suggesting a common evolutionary path for both virus and host. a marked difference was noticed regarding the degree of codon usage bias for each a. coronavirus gene studied: for s, n and pl pro , all mean values were just above 40, indicating a moderate bias (gu et al., 2004) , but for nsp2, the mean nc (53.01) indicated a lower codon usage bias. these results provide evidence that a. coronavirus genes have taken different codon evolution pathways depending on the function that each protein possesses. the function of nsp2 is still not clearly defined, but a role has been suggested as a co-factor for rna synthesis (graham et al., 2005) , possibly in the early stages of virus replication. despite the limited number of studies on nsp2 evolution, it can be speculated that a less biased codon usage for a protein involved in early stages of viral replication would allow for a less restricted trna preference and thus a more efficient start to the viral cycle. the finding that the most biased gene was s (mean nc = 43) might be linked to its relationship with the g. gallus immune system. the spike protein is the main target for neutralizing antibodies, and thus, theoretically, the more s protein that is expressed, the higher the generation of a humoral immune response against s and the lower cell infection by a. coronavirus. considering this stronger codon bias of s, the fact that s showed the lowest cai value when compared to the other three genes and the fact that genes with lower cais are expressed less efficiently (roth et al., 2012) , a deoptimization of s expression could have been selected for with the advantage of lower s expression, providing further evidence that viral proteins that participate in host recognition might have a codon usage less similar to that presented by the host (bahir et al., 2009) . regarding cai values for n, nsp2 and pl pro , fig. 3 suggests that the distributions were mostly above those for s, with the highest values for n (0.75-0.79). n protein plays a chief role in nucleocapsid assembly that is dependent on the association of positively charged amino acids with the genomic rna of coronaviruses (masters, 2006) and is thus under strong purifying selection, as shown herein by the fisher's exact test on ds-dn values. optimization of the codon usage in a manner closer to that of the host would endow a. coronavirus with a more efficient and accurate synthesis of the nucleocapsid protein. the distribution of cais for nsp2 and pl pro stayed between those for n and s (fig. 3) . considering that pl pro is a protease acting on the n-terminus domains of replicase polyproteins pp1a and pp1ab (ziebuhr et al., 2000) , an intermediate adaptation to the host's translational environment could have evolved as a balance between the conservation of structure of the enzymatic domain and the plasticity to follow amino acid mutations occurring on the pl pro cleavage sites of diverse a. coronavirus types as compensatory mutations, showing that epistasis could also be detected at the codon usage evolution level. it is noteworthy that none of the a. coronavirus strains showed no possible combinations of simultaneous occurrence maximum/minimum cai or nc (data not shown) for any of the four genes, meaning that cai and nc might be driven to different evolutionary pathways and that strains with a high cai, i.e., highly adapted to the host's transcription environment, are not necessarily the ones with the lower bias, i.e., with higher nc. the distribution of 100% conserved amino acid positions coded by the preferred codon is noteworthy when one compares the s gene with n, nsp2 or pl pro , as a single position was found in a region outside antigenic and hypervariable regions (cavanagh et al., 1988; kant et al., 1992) in the s gene, while for the other three genes, these positions (n = 20, 28 and 71, respectively) were scattered throughout the regions considered, with statistically significant differences when compared to s (p < 0.0001, or = 21.7-37.8). this low number of conserved amino acid positions coded by the preferred codon in s could be an additional molecular evolutionary mechanism for s antigenic diversity, as fine-tuning translation kinetics could result in high deoptimization of codon usage and a consequent increased fitness (aragonés et al., 2010) . on the other hand, possibly due to strong structural and functional constraints, n, nsp2 and pl pro have a higher number of amino acid positions coded by the preferred codon, which is the same codon preferred by the host (table 1) , which would allow higher fitness to the host transcription environment (zhou et al., 2012 ) in a concerted virus-host molecular evolution. thus, taking conserved amino acid positions coded by the preferred codons as a selection unit, it follows from the above mentioned differences that natural selection could either be positive for these positions, leading a protein under purifying selection (e.g., n, nsp2 and pl pro ) to show the same codons as the host for that amino acid, or negative if a protein is under positive selection (as shown for s). the most probable reason for the fact that non-100% conserved amino acid positions coded by a preferred codon for that amino acid (noted as nc in table 1 ) were only found in a. coronavirus genes and not in the g. gallus genes is that host genes are less susceptible to both the occurrence of putative amino acids and codon usage polymorphisms, contrary to what is observed and expected for virus genes. nc might be considered to be an accurate indicator of codon usage bias because the frequency of amino acids is normalized during the analysis and does not add bias; however, similarly to the cai, the outcome of the nc analysis is a single number, leading to a loss of deep evolutionary information similar to the loss of evolutionary information in nucleotide or amino acid distance-based phylogenetic analyses. taking into account informative sites during codon evolution studies, for instance, 100% conserved amino acid positions coded by the preferred codon for that amino acid, could unveil data that would otherwise be lost in the analysis and that could be used to gain a more comprehensive understanding of molecular evolution in association with the codon usage bias indicators and selection analysis. it would be interesting to use the analyses presented herein not only for a better understanding of virus evolution but also as supporting predictors of spill-over events, such as influenza (wahlgren, 2011) and the new human coronavirus (kindler et al., 2013) now named mers-cov, for which the role of codon usage evolution in virus adaptation to new hosts has been widely ignored. in conclusion, a. coronavirus codon usage evolves independently for each gene in a manner predictable by the protein function. proteins with high functional and structural constraints are more adapted to g. gallus, its natural host, with a balance between natural selection and mutation pressure, giving further molecular basis for the virus' ability to exploit the host's environment. 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proteolytic processing in the nidovirales analysis of base and codon usage by rubella virus key: cord-257487-xanqvdhn authors: carbajo-lozoya, javier; müller, marcel a.; kallies, stephan; thiel, volker; drosten, christian; von brunn, albrecht title: replication of human coronaviruses sars-cov, hcov-nl63 and hcov-229e is inhibited by the drug fk506 date: 2012-02-10 journal: virus res doi: 10.1016/j.virusres.2012.02.002 sha: doc_id: 257487 cord_uid: xanqvdhn recent research has shown that coronavirus (cov) replication depends on active immunophilin pathways. here we demonstrate that the drug fk506 (tacrolimus) inhibited strongly the growth of human coronaviruses sars-cov, hcov-nl63 and hcov-229e at low, non-cytotoxic concentrations in cell culture. as shown by plaque titration, qpcr, luciferaseand green fluorescent protein (gfp) reporter gene expression, replication was diminished by several orders of magnitude. knockdown of the cellular fk506-binding proteins fkbp1a and fkbp1b in caco2 cells prevented replication of hcov-nl63, suggesting the requirement of these members of the immunophilin family for virus growth. hcov-nl63 hcov-229e fk506 tacrolimus immunophilins fkbp1a (fkbp12) fkbp1b (fkbp12.6) inhibition of viral replication a b s t r a c t recent research has shown that coronavirus (cov) replication depends on active immunophilin pathways. here we demonstrate that the drug fk506 (tacrolimus) inhibited strongly the growth of human coronaviruses sars-cov, hcov-nl63 and hcov-229e at low, non-cytotoxic concentrations in cell culture. as shown by plaque titration, qpcr, luciferase-and green fluorescent protein (gfp) reporter gene expression, replication was diminished by several orders of magnitude. knockdown of the cellular fk506binding proteins fkbp1a and fkbp1b in caco2 cells prevented replication of hcov-nl63, suggesting the requirement of these members of the immunophilin family for virus growth. © 2012 elsevier b.v. all rights reserved. coronaviruses cause severe diseases of the respiratory and gastrointestinal tract and the central nervous system in animals (perlman and netland, 2009) . infection of humans with hcov-oc43 and hcov-229e are known since the 1960s to be associated with respiratory tract i.e. common cold diseases. severe acute respiratory syndrome-coronavirus (sars-cov) is the most aggressive human agent, causing the lung disease sars (drosten et al., 2003a) . hcov-nl63 and hcov-hku1 were discovered in , respectively (van der hoek et al., 2004 woo et al., 2005) . they cause upper and lower respiratory tract infections including bronchiolitis and pneumonia especially in young children, immunocompromised patients and the elderly (van der hoek, 2007) . until now effective treatment of coronavirus infection in humans is unavailable (stockman et al., 2006) , even though inhibitors of coronavirus enzymes (reviewed by (tong, 2009a,b) and compounds inhibiting in vitro replication have been described abbreviations: ec50, 50% effective inhibitory concentration; rlu, relative light units. * corresponding author. e-mail address: vonbrunn@mvp.uni-muenchen.de (a. von brunn). (kono et al., 2008; te velthuis et al., 2010; vincent et al., 2005) . coronaviruses represent the group of rna viruses with the largest rna genome known to date. compared to other rna viruses, the coronavirus polymerase has a rather low error frequency and may possess a rna proof reading activity (denison et al., 2011) . however, the development of resistant mutants in the presence of drugs targeting coronaviral proteins remains a serious concern. virus replication depends on a variety of host factors (de haan and rottier, 2006; vogels et al., 2011; zhang et al., 2010) , which in turn represent potential antiviral targets. these might be more preferable targets than viral proteins as development of resistance is much less likely. in a recent study we performed a genome-wide sars-cov yeast-two-hybrid interaction screen with human cdna libraries identifying cyclophilins and fk506-binding proteins as interaction partners of sars-cov non-structural protein 1 (nsp1). we identified cyclosporin a (csa) as a potent replication inhibitor of various human and animal covs (pfefferle et al., 2011) . inhibition of sars-cov, hcov-229e and, in addition, of mouse hepatitis virus was also confirmed by de wilde et al. (de wilde et al., 2011) . cyclophilins and fk506-binding proteins are collectively referred to as immunophilins because they bind the immunosuppressive drugs csa and fk506, respectively. furthermore, they share peptidyl-prolyl isomerase (ppiase) domains and chaperone functions facilitating protein folding (davis et al., 2010) . csa was recently shown to exert inhibitory effects on herpes simplex virus, vaccinia virus (damaso and keller, 1994) , bk polyoma virus (acott et al., 2008) , human immunodeficiency virus 1 (hiv-1) (briggs et al., 1999; wainberg et al., 1988) and hcv (fischer et al., 2010; nakagawa et al., 2004; watashi et al., 2003) . whereas cyclophilins were reported to be required for replication of hiv-1 (franke and luban, 1996) , vesicular stomatitis virus (bose et al., 2003) and hcv (nakagawa et al., 2005) , fk506-binding proteins were not found to play a role in the replication of hiv-1 (briggs et al., 1999) and hcv (nakagawa et al., 2004; watashi et al., 2003) . orthopoxviruses are inhibited by fk506 (reis et al., 2006) . fk506 was one of the first macrolides, discovered in 1984 in the bacterium streptomyces tsukubaensis, with immunosuppressive activity. although it is structurally unrelated to the cyclic undecapeptide csa both molecules display similar properties of binding to the catalytic pocket of the ppiase domains of their cellular binding partners resulting in inhibition of their ppiase activities (barik, 2006) . furthermore, the cyclophilin-csa and fkbp-fk506 complexes bind to the cellular phosphatase calcineurin (cna) which inhibits the dephosphorylation and activity of the nuclear factor of activated t-cells. the inhibition of this essential transcriptional regulator of important immune genes is the cause of immunosuppression. it is important to note that binding of the fkbp/fk506 complex to cna, which is required for immunosuppression, and inhibition of ppiase activitiy, which is responsible for antiviral activity, are two independent mechanisms. when studying the interaction of the sars-cov nsp1 protein with cyclophilins a and b we discovered that replication of various coronaviruses is sensitive to csa treatment including hcov-nl63 and hcov-229e (pfefferle et al., 2011) . since nsp1 also interacted with fk506-binding proteins fkbp1a and fkbp1b, we examined in this study whether fk506 inhibits replication of human coronaviruses. in a first experiment verofm cells were infected with sars-cov at a multiplicity of infection (moi) of 0.0001 in the presence of increasing concentrations of csa and fk506. virus rna concentrations were measured after 24 h in supernatant of infected cells by real-time rt-pcr (drosten et al., 2003b) . titers were reduced 128,740-fold by 33 m csa, and 11,112 fold by 49 m fk506, respectively. fig. 1a shows the percentage of sars-cov inhibition under the influence of fk506, with an ec 50 inhibitory concentration of about 6.9 m. in fig. 1b the corresponding log 10 titer reductions are represented. to examine whether fk506 exerts an inhibitory activity on other human coronaviruses, caco2 cells were infected with hcov-nl63 at moi = 0.004 (herzog et al., 2008) in the presence of increasing inhibitor concentrations. after two days rna from cell culture supernatant was harvested and viral rna was extracted using the high pure viral nucleic acid kit (roche) and quantified by real-time pcr using superscript tm iii one-step quantitative system with rox (invitrogen) allowing reverse transcription, cdna synthesis and pcr amplification in a single step. pcr primers used were nl-63rf2 for 5 -cttctggtgacgctagtacagcttat-3 (genome position nt 14459-14484) and nl-63rr2rev 5 -agacgtcgttgtagatccctaacat-3 (genome position nt 14573-14597) and nl-63 probe was 5 -fam-caggttgcttagtgtcccatcagattcat-tamra-3 (genome position nt 14532-14560). the ec 50 inhibitory concentration for hcov-nl63 was about 5.1 m and replication was completely inhibited at 36 m fk506 (fig. 1c) , reflecting a 732-fold reduction of virus replication (fig. 1d) . at this concentration cell viability (celltiterglo, promega) was still about 75%. inhibition of virus growth in caco2 cells was also confirmed by plaque titration assay (herzog et al., 2008) using serial virus dilutions in the presence of fk506 concentrations ranging from 0 m to 24 m. fig. 1e and f shows percentage and log reductions of inhibition of virus replication, respectively. under the conditions of the plaque assay involving virus growth for four days, the decrease of virus titers occurred in a more linear fashion with an ec 50 of about 13.4 m (e). complete inhibition was observed at 24 m with a reduction by more than five orders of magnitude (f). hcov-229e viruses expressing renilla luciferase (pfefferle et al., 2011) or gfp (cervantesbarragan et al., 2010) reporter genes were used to examine the inhibitory effect of fk506. huh7 cells were infected with moi = 0.1 and incubated for two days in the presence of increasing concentrations of fk506 in the culture medium. viral replication was determined by measuring renilla luciferase activity. fig. 2a and b shows percent and log reductions of virus growth, respectively. about 5.4 m fk506 were found to be effective as an ec 50 for hcov-229e-luc. cell viability was reduced to about 70% (at 24 m) as compared to untreated cells, indicating that virus reduction was due to the drug but not to cell death. fig. 2c and d shows a microscopic comparison of hcov-229e-gfp infection under increasing concentrations of csa and fk506. reduction of virus infection was quantified by determining fluorescence intensity from microscope images of infected huh7 cells. the pictures were analysed by using the software imagej 1.44 (abramoff et al., 2004; rasband, 1997 rasband, -2011 . the integrated density for three different regions of interest was measured in each picture and the obtained averages were used to calculate correlates of virus replication efficiency. the determined ec 50 was 12.2 m for fk506 under these conditions. gfp fluorescence was inversely correlated to inhibitor concentration, with dapi staining of cell nuclei indicating confluence of huh7 cells and equivalent cell densities at all concentration steps. virus growth was completely abolished between concentration of 8-12 m csa and 18-25 m fk506. the latter correlates well with the inhibitory concentration determined for hcov-229e-luc. for both hcov-229e and hcov-nl63, ec 50 values were higher in plaque and gfp titrations as compared to direct replication assays by either rt-pcr or luciferase measurement. however, deviations between both assay formats were only by factors of 3.2 (hcov-nl63) and 2.6 (hcov-229e). in order to examine whether the cellular fk506-binding proteins fkbp1a and fkbp1b are required for virus replication, caco2 knockdown cell lines were established using lentiviral expression of shrna (sirion gmbh, martinsried, germany). cells were transduced at moi 30 with missiontm lentiviral non-target control, cyclophilin b (ppib), fkbp1a, or fkbp1b shrna lentiviruses. sequences used for gene knockdown are listed in table 1a . stable shrna-expressing cells were generated through 3 weeks of bulk-selection in 10-15 g/ml puromycin-containing medium (dmem + 10% fcs + 2 mm l-glutamine + 1 mm na-pyruvate). the high puromycin concentration needed for selection of caco2 cells might result from an enhanced expression of mdr-1 (multi drug resistant protein 1 gene) depleting cells very efficiently from inhibitory drug molecules (takara et al., 2002) . from the bulk-selected knockdown and control cells mrna expression was quantified by real-time rt-pcr. for reverse transcription 1 g of total rna was used. amplification products were detected by sybr i, and amplicon integrity was verified by melting point analysis. cyclophilin a (ppia) served as a reference gene against ppib to determine the specificity of the knockdown. mrna expression levels of ppib, fkbp1a, and fkbp1b were then determined by real-time rt-pcr with primers listed in table 1b . fig. 3a shows ppib, fkbp1a and fkbp1b mrna expression in bulk-selected caco2 cells. in comparison to non-target controls a 98% and 96% knockdown was determined for ppib and fkbp1a/1b, respectively. plaque titration showed that hcov-nl63 grew to similar titers in caco2 wild type (not shown), caco2 shrna control and caco2-ppib knockdown cells indicating that expression of ppib was not necessary for viral growth. conversely, knockdown of fkbp1 a/b did not result in any plaque formation, identifying fkbp1a/b products as key proteins required for viral replication. similarly, real-time pcr amplification of rna genomes in the supernatant of the hcov-nl63-infected cell lines showed significantly reduced titers in the caco2-fkbp1a knockdown cells as compared to caco2sh and caco2-ppib knockdown cells (not shown), confirming the results of the plaque titrations. in summary, fk506 inhibits the replication of sars-cov, hcov-nl63 and hcov-229e at non-toxic, low-micromolar concentrations with a reduction of virus titers by several orders of magnitude to undetectable levels. in a comparative study of the effect of csa and fk506 on hiv-1, the former inhibited virus replication completely (ic 50 : 1-2 g/ml) whereas latter had an inhibitory effect on chronically but not on newly infected cells [up to 10 g/ml fk506 (briggs et al., 1999) ]. also in the case of hcv, fk506 had no effect on the replication of replicon rna up to concentrations of 3 g/ml (watashi et al., 2003) . considering the higher drug concentrations of csa and fk506 needed for inhibition of coronavirus replication it can be speculated that also hiv-1 and hcv might be inhibited by higher fk506 concentrations. it also remains to be clarified why the low nanomolar affinities of fkbp1a/b to fk506 do not reflect the ec50 inhibitory concentrations of coronaviruses which reside in the low micromolar range. this might be explained by a stronger affinity of coronavirus proteins to the cellular proteins as compared to fk506. lack of growth of hcov-nl63 in stable caco2-fkbp1a and -fkbp1b, but not -ppib knockdown mutants indicates the requirement of fkbp1a and fkbp1b for hcov-nl63 replication. as the two proteins share about 83% homology on the amino acid level (not shown) they do not substitute for each other upon individual knockdown. a common structural feature of fkbp proteins is the ppiase domain, also called fk506-binding domain. its peptidylprolyl cis/trans isomerase activity influences cellular pathways by binding to cellular proteins (kang et al., 2008) . these activities are common to many fkbp family members containing a ppiase domain and separate chaperone functions can be discriminated from them (barik, 2006) . the two less pathogenic coronaviruses hcov-nl63 and hcov-229e and the highly pathogenic sars-cov are sensitive to csa and fk506, indicating the involvement of cyclophilins and fk506binding proteins in viral replication. however, it is not clear what functions of these cellular proteins are required for virus replication. for sars-cov we have shown that nsp1 binds to cyclophilins and to fkbp1a and fkbp1b by y2h and lumier assay (pfefferle et al., 2011) . also, nsp1 proteins of hcov-nl63 and -229e interact with fkbp1a and fkbp1b in y2h (not shown). the mechanism and function of these interactions have to be analysed in more detail. furthermore it has to be clarified whether other viral proteins are involved in the relation of coronaviruses and immunophilins. in vitro effect of cyclosporin a on primary and chronic bk polyoma virus infection in vero e6 cells immunophilins: for the love of proteins requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype comparison of the effect of fk506 and cyclosporin a on virus production in h9 cells chronically and newly infected by hiv-1 dendritic cell-specific antigen delivery by coronavirus vaccine vectors induces long-lasting protective antiviral and antitumor immunity cyclosporin a inhibits vaccinia virus replication in vitro structural and biochemical characterization of the human cyclophilin family of peptidyl-prolyl isomerases hosting the severe acute respiratory syndrome coronavirus: specific cell factors required for infection cyclosporin a inhibits the replication of diverse coronaviruses coronaviruses: an rna proofreading machine regulates replication fidelity and diversity identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome: identification of the etiological agent cyclophilin inhibitors for the treatment of hcv infection inhibition of hiv-1 replication by cyclosporine a or related compounds correlates with the ability to disrupt the gag-cyclophilin a interaction plaque assay for human coronavirus nl63 using human colon carcinoma cells fkbp family proteins: immunophilins with versatile biological functions inhibition of human coronavirus 229e infection in human epithelial lung cells (l132) by chloroquine: involvement of p38 mapk and erk specific inhibition of hepatitis c virus replication by cyclosporin a suppression of hepatitis c virus replication by cyclosporin a is mediated by blockade of cyclophilins coronaviruses post-sars: update on replication and pathogenesis the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors fk506, a secondary metabolite produced by streptomyces, presents a novel antiviral activity against orthopoxvirus infection in cell culture sars: systematic review of treatment effects digoxin up-regulates mdr1 in human colon carcinoma caco-2 cells zn(2+) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture therapies for coronaviruses. part 2. inhibitors of intracellular life cycle therapies for coronaviruses. part i of ii. viral entry inhibitors human coronaviruses: what do they cause identification of a new human coronavirus chloroquine is a potent inhibitor of sars coronavirus infection and spread identification of host factors involved in coronavirus replication by quantitative proteomics analysis the effect of cyclosporine a on infection of susceptible cells by human immunodeficiency virus type 1 cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia quantitative proteomics analysis reveals bag3 as a potential target to suppress severe acute respiratory syndrome coronavirus replication this work was supported by the "bundesministerium fuer bildung und forschung" of the german government (zoonosis network, consortium on ecology and pathogenesis of sars, project code 01ki1005a,f; http://www.gesundheitsforschungbmbf.de/de/1721.php#sars). we are grateful to julia schöpf and nicole senninger for technical help. we also thank drs. c. thirion and m. salomon (sirion, martinsried, germany) for production of the caco2 knockdown cell lines via lentiviral technology. we greatly appreciate the provision of huh7 lunet cells by ralf bartenschlager (heidelberg, germany). key: cord-327682-i3uim0zi authors: santti, juhana; vainionpää, raija; hyypiä, timo title: molecular detection and typing of human picornaviruses date: 1999-08-25 journal: virus res doi: 10.1016/s0168-1702(99)00036-2 sha: doc_id: 327682 cord_uid: i3uim0zi picornaviruses include several important clinical pathogens which cause diseases varying from common cold to poliomyelitis and hepatitis. introduction of rt-pcr methods for the detection of these viruses has significantly facilitated the diagnosis of picornavirus infections and elucidated their etiological role in clinical illnesses. partial sequence analysis of the genomes has been used for typing of the viruses and in studies of molecular epidemiology of picornaviruses. these molecular approaches are likely to become the most predominant techniques for the diagnosis and epidemiological analysis, particularly in the enterovirus infections. in the picornaviridae family, enteroviruses, hepatoviruses, parechoviruses, and rhinoviruses include human pathogens while aphtho-and cardioviruses cause disease in animals. based on pathogenicity in experimental animals, human enteroviruses have been subgrouped into polioviruses (pv; 3 serotypes), coxsackie a viruses (cav; 23 serotypes), coxsackie b viruses (cbv; 6 serotypes), echoviruses (ev; 28 serotypes), and enteroviruses 68-71. the outcome of enterovirus infections in man varies from minor respiratory illnesses to paralysis, myocarditis, meningoencephalitis and generalized infections with multiorgan failure in newborns (grist et al., 1978) . most of the enterovirus infections, however, are assumed to be asymptomatic. although several enterovirus serotypes can cause many of the illnesses mentioned, there are disease entities that are typically caused by one or only a few serotypes. these include epidemic poliomyelitis (pvs), hand-footand-mouth disease (mainly cav16), acute hemorrhagic conjunctivitis (cav24 and enterovirus 70), herpangina (certain cavs) and pleurodynia (cbvs) (grist et al., 1978) . over 100 human rhinovirus (hrv) serotypes have been identified. they are considered to be the most frequent cause of the common cold. moreover, hrv infections may result in sinusitis, otitis media, or exacerbation of asthma (pitkäranta and hayden, 1998) . due to its unique pathogenicity and genetic distance when compared to other picornaviruses, hepatitis a virus (hav), previously designated as enterovirus 72, was reclassified in hepatovirus genus (minor, 1991) . more recently, two echoviruses (serotypes 22 and 23) with exceptional molecular properties have been assigned to a new genus, parechovirus stanway et al., 1994; mayo and pringle, 1998) . examples of clinical diseases associated with picornavirus infections are shown in table 1 . conventional diagnostic methods for human picornavirus infections include culturing of the virus in susceptible cell lines, followed by neutralization typing (enteroviruses, parechoviruses) or acid lability testing (hrvs). serological detection of enterovirus-specific antibodies provides indirect evidence of the etiology of the disease. diagnosis of hav infections is based on demonstration of specific igm-class antibodies. in general, the isolation methods currently used for picornavirus identification are cumbersome, laborious and insensitive. even at its best, several days are required for specific diagnosis. hence, the introduction of newly developed molecular techniques into picornavirus diagnosis that provide means for faster and more sensitive virus detection has been highly appreciated. these methods improve not only laboratory diagnosis and clinical management of picornavirus infections, but increase knowledge of the clinical importance of human picornaviruses. the authors summarize here recent advances in rt-pcr and sequence analysis of human picornaviruses which, together with in situ hybridization techniques, have significantly improved the virological laboratory diagnosis of these infections. sequence analysis and the use of cdna clones for the detection of several enterovirus serotypes by one single probe had suggested that some genome regions are highly similar among members of the genus. moreover, hrvs were shown to be closely related to human enteroviruses. based on these observations, attempts were made to select primers and probes which could be used for the development of rt-pcr assays for these virus groups. the picornavirus genome is an : 7000-9000 nucleotides (nt) long, single-stranded rna molecule. the long open reading frame, coding for the capsid and nonstructural proteins, is preceded by a 5% noncoding region (5%ncr; 750 nt in enteroviruses). this region contains several conserved motifs which participate in the formation of secondary structures required for translation and replication of the virus genome. most of the rt-pcr assays described have taken advantage of these homologous regions which are shared by entero-and rhinoviruses ( fig. 1 ). because the amplification products are usually of a similar length, a specific oligonucleotide probe is needed for discrimination between the two picornavirus groups (hyypiä et al., 1989; santti et al., 1997) . alternatively, a primer may be targetted to a conserved region in the vp2 capsid protein gene and used together with another primer from the 5%ncr (olive et al., 1990 ). an advantage with this primer pair is that the entero-and rhinovirus amplicons differ in length, due to an apparent deletion in the hrv genome in the region preceding the initiation codon. it has been shown in a number of studies that virtually all the enterovirus serotypes and most of the hrv isolates can be detected using these primer sequences (hyypiä et , 1989; horsnell et al., 1995; pulli et al., 1995; arola et al., 1996; huttunen et al., 1996; pitkäranta et al., 1997; hyypiä et al., 1998; oberste et al., 1998) the authors became convinced of the usefulness of this technique during a recent outbreak of aseptic meningitis in finland. the first cases of the illness, subsequently shown to be caused by echovirus 30, were diagnosed in the late summer 1996 and the peak of the epidemic occurred in september and october. specimens including csf, throat swabs, stool samples and sera were collected from the first eight patients with typical symptoms. the etiological agent was identified as an enterovirus from csf by rt-pcr (arola et al., 1996) , after which this technique was mainly used for virological diagnosis. rt-pcr was positive from seven of the eight csf samples, while virus isolation was positive from three csf, throat swabs and stool specimens, all from the same three patients (table 2) . rt-pcr from serum samples gave positive diagnosis in five patients including one patient whose csf remained negative in the rt-pcr test. enterovirus igm was positive in four sera by enzyme immunoassay. microbial etiology of the common cold in young adults was recently examined using conventional diagnostic methods and hrv rt-pcr by mäkelä et al. (1998) . hrvs were demonstrated in 53%, coronavirus infections in 9% and other viruses in 13% of the patients. bacterial infections were rare and found in b5% of these 200 patients with signs and symptoms of the disease. when virus isolation was compared with rt-pcr, the sensitivities in the detection of hrvs were 66 and 98%, respectively . hrv rna has also been detected in maxillary aspirates and nasal swabs of patients with sinusitis (pitkäranta et al., 1997) . during one epidemic year (july 1997-june 1998) 51 enteroviruses and 28 hrvs were detected from a total of 435 samples by using an rt-pcr assay at the department of virology, university of turku. enteroviruses were identified in 24 (18%) of the 135 csf specimens analyzed while most of the other positive findings were from nasopharyngeal samples. the assay utilizes common 5%ncr primers and specific probes for entero-and rhinoviruses (halonen et al., 1995) . thirteen samples, positive by rt-pcr based on the detection of an amplicon of the expected size in agarose gel electrophoresis, were negative in the hybridization assay, indicating need for further improvements in the reactivity of the probes. analysis of enterovirus genomes has shown that members of the genus form four genetic clusters when the coding region is used in comparisons (fig. 2) : cluster a includes some cav serotypes and enterovirus 71; cluster b contains cav9, cbvs and evs; polioviruses (cluster c1) and certain cavs (cluster c2) are closely related; while enterovirus 70 is a representative of cluster d [for review, see hyypiä et al. (1997) ]. partial sequence analysis of virtually all enterovirus serotypes has shown that they belong to these four clusters (pulli et al., 1995; huttunen et al., 1996) and that the vp4/vp2 region sequence can be used for molecular typing of clinical isolates (arola et al., 1996) . it is notable, however, that, for instance, polioviruses cannot be discriminated from cavs in the c cluster by this means and other genomic regions are required for the purpose. in the 5%ncr the clustering of enterovirus serotypes is different and does not provide a useful tool for typing, but can be used in some epidemiological studies. although genetic clustering can also be detected among hrvs (horsnell et al., 1995) , the correlation of this grouping with pathogenesis of infection is not currently clear. partial sequence analysis of clinical isolates has been extensively used in the epidemiologic studies of polioviruses and resulted in a large sequence database (rico-hesse et al., 1987; zheng et al., 1993; huovilainen et al., 1995; kew et al., 1995; mulders et al., 1995; chezzi et al., 1997; morvan et al., 1997; fiore et al., 1998) . the global eradication program has significantly benefitted from these studies. a region of 150 nt in the junction of the structural gene vp1 and the viral protease gene 2a has been universally adopted for this purpose. the term genotype, which is defined as a cluster of genetically related viruses with b 15% nucleotide sequence divergence, has been used for poliovirus strains (rico-hesse et al., 1987) . within each serotype, the independent genotypes usually have distinct geographic distribution, consistent with the existence of separate foci of endemicity. these studies have provided an invaluable insight into the pathways of virus transmission and epidemiologic links. in addition to polioviruses, several other enteroviruses have also been subjected to detailed epidemiologic analysis. these include the two serotypes responsible for large outbreaks of acute hemorrhagic conjunctivitis, cav24 variant (cav24v; ishiko et al., 1992) and enterovirus 70 (takeda et al., 1994) . regression analysis of the genetic distances between isolates has enabled estimation of the time of emergence of these viruses. the viruses were predicted to originate from single focal places; cav24v in november 19639 21 months, about 7 years before the first isolation of the virus in singapore, and enterovirus 70 in august 19679 15 months, 2 years before recognition of the virus in a pandemic of acute hemorrhagic conjunctivitis. the molecular epidemiology of cav9, cbv1 and cbv5 as well as echovirus 30 which is characteristically involved in epidemics of meningitis has also been studied (chang et al., 1992; zoll et al., 1994; kopecka et al., 1995; gjøen et al., 1996) . in hepatitis a virus infections, the molecular epidemiology has been investigated by many reseach groups and an extensive analysis of partial sequences from different geographic locations has been used to establish a useful database for further studies (robertson et al., 1992) . recently, an rt-pcr assay was also developed for the detection of parechovirus 1 in clinical samples and the amplicons, representing the 5%ncr, were used in epidemiological analysis (joki-korpela and hyypiä, 1998) . increased knowledge of the molecular biology of picornaviruses and advances in laboratory techniques have allowed the development of rt-pcr assays which are more sensitive and rapid than the previously used isolation methods in the detection of entero-and rhinoviruses in clinical samples. moreover, rt-pcr products can be used for typing and molecular epidemiological analysis of the viruses. the availability of rt-pcr assays has also enabled analysis of the presence of enteroviruses in various acute and chronic disease conditions, such as myocarditis and cardiomyopathies, central nervous system infections, fig. 2 . dendrogram based on partial sequences from vp2 capsid proteins shows genetic relationships between human enterovirus serotypes. molecular clusters (a, b, c and d; hyypiä et al., 1997) are indicated. the dendrogram was generated by using the pileup program from the genetics computer group software (devereux et al., 1984) . generalized neonatal infections, insulin-dependent diabetes mellitus and chronic fatigue syndrome. information on different picornavirus geno-types will rapidly increase and, together with advances in mapping of pathogenic and antigenic determinants, this may allow more precise molec-ular grouping of picornaviruses. it is possible that deduction of phenotypic characteristics from the genomic sequence may become possible in the near future. identification of enteroviruses in clinical specimens by competitive pcr followed by genetic typing using sequence analysis the nucleotide sequences of wild-type coxsackievirus a9 strains imply that an rgd motif in vp1 protein is functionally significant molecular epidemiology of type 1 polioviruses from a comprehensive set of sequence analysis programs for the vax antigenic and molecular characterization of wild type 1 poliovirus causing outbreaks of poliomyelitis in albania and neighboring countries in 1996 intratypic genome variability of echovirus type 30 in part of the vp4/vp2 coding region enteroviruses in human disease detection of enteroviruses and rhinoviruses in clinical specimens by pcr and liquid-phase hybridization molecular relationships between 21 human rhinovirus serotypes genetic divergence of poliovirus strains isolated in the karachi region of pakistan the major echovirus group is genetically coherent and related to coxsackie b viruses polymerase chain reaction for human picornaviruses a distinct picornavirus group identified by sequence analysis classification of enteroviruses based on molecular and biological properties molecular diagnosis of human rhinovirus infections: comparison with virus isolation phylogenetic analysis of a coxsackievirus a24 variant: the most recent worldwide pandemic was caused by progenies of a virus prevalent around 1981 diagnosis and epidemiology of echovirus 22 infections molecular epidemiology of polioviruses genotypic variation in coxsackievirus b5 isolates from three different outbreaks in the united states viruses and bacteria in the etiology of the common cold virus taxonomy -1997 picornaviridae the molecular epidemiology of type 1 poliovirus in central african republic molecular epidemiology of wild poliovirus type 1 in europe, the middle east, and the indian subcontinent molecular phylogeny of all human enterovirus serotypes based on comparison of sequences at the 5% end of the region encoding vp2 detection and differentiation of picornaviruses in clinical samples following genomic amplification detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr what's new with common colds? complications and management molecular comparison of coxsackie a virus serotypes geographic distribution of wild poliovirus type 1 genotypes genetic relatedness of hepatitis a virus strains recovered from different geographical regions comparison of pcr primer pairs in the detection of human rhinoviruses in nasopharyngeal aspirates molecular and biological charasteristics of echovirus 22, a representative of a new picornavirus group molecular evolution of the major capsid protein vp1 of enterovirus 70 distribution of wild type 1 poliovirus genotypes in china intratypic genome variability of the coxsackievirus b1 2a protease region the authors thank tapani hovi and glyn stanway for helpful comments during the preparation of the manuscript. the original studies were supported by grants from the academy of finland and the sigrid juselius foundation. key: cord-020087-gs0pc6ee authors: nan title: cumulative contents for 2010 date: 2010-11-18 journal: virus res doi: 10.1016/s0168-1702(10)00397-7 sha: doc_id: 20087 cord_uid: gs0pc6ee nan myristoylation of the small envelope protein of porcine reproductive and respiratory syndrome virus is non-essential for virus infectivity but promotes its growth 294 issues 1 japan) inhibitory effect on the tobacco mosaic virus infection by a plant ring finger protein 50 usa) influenza virus budding does not require a functional aaaϩ atpase pr china) molecular anatomy of subcellular localization of hsv-1 tegument protein us11 in living cells 71 ) mapping of amino acid residues responsible for adhesion of cell culture-adapted foot-and-mouth disease sat type viruses 82 renault (france) detection and description of a particular ostreid herpesvirus 1 genotype associated with massive mortality outbreaks of pacific oysters, crassostrea gigas kotait (brazil, ecuador) human rabies transmitted by vampire bats: antigenic and genetic characterization of rabies virus isolates from the amazon region china) classical swine fever virus ns3 is an ires-binding protein and increases ires-dependent translation 106 ) immunogenicity of novel dengue virus epitopes identified by bioinformatic analysis usa) evaluation of innate immunity and vector toxicity following inoculation of bovine occult hepatitis b virus infection with low viremia induces dna damage, apoptosis and oxidative stress in peripheral blood lymphocytes 143 porcine reproductive and respiratory syndrome virus (prrsv) could be sensed by professional beta interferon-producing cao (china) genetic analysis of a hantavirus strain carried by niviventer confucianus in yunnan province mansoor (pakistan) cotton leaf curl disease in sindh province of pakistan is associated with recombinant begomovirus components 161 a negative element in the downstream region of the rice tungro bacilliform virus promoter is orientation-and positionindependent and is active with heterologous promoters 166 hayashi (japan) lifelong persistent ebv infection of rabbits with eber1-positive lymphocyte infiltration and mild sublethal hemophagocytosis 172 hikichi (japan) the 126-and/or 183-kda replicases or their coding regions are responsible both for inefficient local and for systemic movements of paprika mild mottle virus japanese strain in tomato plants the capsid protein of grapevine rupestris stem pitting-associated virus contains a typical nuclear localization signal and targets to the nucleus 212 distinct propagation efficiencies of h5n1 influenza virus thai isolates in newly established murine respiratory region-derived cell clones 218 usa) genetic control of host resistance to porcine reproductive and respiratory syndrome virus (prrsv) infection porcine reproductive and respiratory syndrome virus (prrsv) in serum and oral fluid samples from individual boars: will oral fluid replace serum for prrsv surveillance usa) use of a production region model to assess the efficacy of various air filtration systems for preventing airborne transmission of porcine reproductive and respiratory syndrome virus and mycoplasma hyopneumoniae: results from a 2-year study 177 morrison (usa) control and elimination of porcine reproductive and respiratory syndrome virus 185 cumulative author index for cumulative subject index for cumulative contents for key: cord-312848-vbadg8ki authors: jeong, jae-ho; kim, gye-yeop; yoon, soon-seek; park, su-jin; kim, you-jung; sung, chang-min; shin, sung-shik; lee, bong-joo; kang, mun-il; park, nam-yong; koh, hong-bum; cho, kyoung-oh title: molecular analysis of s gene of spike glycoprotein of winter dysentery bovine coronavirus circulated in korea during 2002–2003 date: 2004-08-26 journal: virus res doi: 10.1016/j.virusres.2004.07.003 sha: doc_id: 312848 cord_uid: vbadg8ki since the molecular analysis of spike (s) glycoprotein gene of bovine coronavirus (bcov) has been conducted and compared mainly among american and canadian isolates and/or strains, it is unclear whether bcov circulated in the other countries are distinctive in genetic characteristics. in the present study, we analyzed the s glycoprotein gene to characterize 10 winter dysentery (wd) coronavirus strains circulated in korea during 2002–2003 and compared the nucleotide (nt) and deduced amino acid (aa) sequences with the other known bcov. the phylogenetic analysis of the entire s glycoprotein gene revealed that the aa sequences of all korean wd strains were more homologous to each other and were very closely related to respiratory bovine coronavirus (rbcv) strain ok and enteric bovine coronavirus (ebcv) strain ly-138, but were distinct from the other known bcovs. based on the phylogenetic analysis of the hypervariable region of the s1 subunit, all korean wd strains clustered with the respiratory strain ok, bcq3994 and the enteric strain ly-138, while the canadian bcq calf diarrhea and wd strains, and the american rbcv lsu, french ebcv f15 and avirulent vacc, l9, and mebus strains clustered on a separate major branch. these data suggest that the wd strains circulated in korea had a genetic property of both rbcv and ebcv and were significantly distinct from the ancestral enteric strain. 1997). the virion contains five major structural proteins: the nucleocapsid (n) protein, the transmembrane (m) protein, the hemagglutinin/esterase (he) protein, the spike (s) protein, and the small membrane (e) protein (lai and cavanagh, 1997) . the variation in host range and tissue tropism of coronaviruses is largely attributable to variations in the s glycoprotein (gallagher and buchmeier, 2001) . the bcov had point mutations or deletions especially in the s1 subunit of s glycoprotein (chouljenko et al., 1998; hasoksuz et al., 2002; gelinas et al., 2001; rekik and dea, 1994) . moreover, 0168-1702/$ -see front matter © 2004 elsevier b.v. all rights reserved. doi:10.1016/j.virusres.2004.07.003 molecular analysis of s gene of bcov has been conducted and compared mainly between american and canadian isolates and/or strains (chouljenko et al., 1998; hasoksuz et al., 2002; gelinas et al., 2001; rekik and dea, 1994) . in this study, we performed molecular analysis on the s glycoprotein gene to characterize wd coronaviruses circulated in korea during 2002 korea during -2003 , compared the nucleotide, and predicted aa sequences with the other known bcov. ten wd strains, kwd1-kwd10, were originally isolated in the g clone of human rectal tumor cells (hrt-18g) from fecal samples of adult dairy or beef cows, with wd in south korea, and identified as a bcov by immunoelectron microscopy, elisa with bcov-specific antisera and monoclonal antibodies, and rt-pcr specific for the part of bcov n gene (jeong et al., 2004) . rna was extracted from the supernatant fluids of hrt-18g cells infected with 10 kwd strains, based on the acid quanidinium-phenol-chloroform rna extraction method as described by cho et al. (2001a) . as a negative control, rna was extracted from mock-infected hrt-18g cells. the oligonucleotide primers used in the rt-pcr were designed from the published sequence of the s gene of the mebus strain (genbank accession no. u00735). the sequence of primers (positions calculated from the start codon of the s gene) are as follows: 1st upstream primer, 5 -atgtttttgatacttttaatttcc-3 (1-24); 1st downstream primer, 5 -acaccagtagatggtgctat-3 (901-920); 2nd upstream primer, 5 -gggttacacctct-cacttct-3 (782-801); 2nd downstream primer, 5 -gcaggacaagtgcctatacc-3 (1531-1550); 3rd upstream primer, 5 -ctgtccgtgtaaattggatg-3 (1459-1478); 3rd downstream primer, 5 -tgtagagtaatc-cacacgt-3 (2268-2286); 4th upstream primer, 5 -ttc acgacagctgcaaccta-3 (2151-2170); 4th downstream primer, 5 -ccatggtaacaccaatcc ca-3 (3003-3022); 5th upstream primer, 5 -ccctgt-attaggttgtttag-3 (2691-2710); 5th downstream primer, 5 -accactaccagtgaacatcc-3 (3587-3606); 6th upstream primer, 5 -gtgcagaatgct-ccatatggt-3 (3439-3459); 6th downstream primer, 5 -ttagtcgtcatgtgatgttt-3 (4073-4092). a one-step rt-pcr assay was performed as described by cho et al. (2001a,b) . the rt-pcr products were purified using a genclean ii kit (bio 101, inc., lajolla, ca) according to the manufacturer's instructions. the dna sequencing was done using an automated dna sequencer (abi system 3700, applied biosystem inc., foster city, ca). using the dna basic module (dnasis max, alameda, ca), nucleotide sequences of our bcov strains were first compared for the s sequence of bcov as follows: the cd strains including mebus (genbank accession no. u00735), bcq2070 (genbank accession no. u06090), bcq571 (genbank accession no. u06093), bcq20 (gen-bank accession no. u06092), bcq9 (genbank accession no. u06091), and bcq1523 (genbank accession no. aag40601); the wd strains including bcq7373 (genbank accession no. aag40595) and bcq2590 (genbank accession no. ak01071); the rbcv strains including bcq3994 (genbank accession no. aak14398), lsu (genbank accession no. aaf25509), and ok (genbank accession no. aaf25519), and ebcv strains including ly-138 (genbank accession no. aaf25499) and f15 (genbank accession no. d00731). the deduced aa sequences were then assembled and analyzed on the amino acid basic module (dnasis max, alameda, ca). a sequence similarity search was performed for the bcov s glycoprotein using the lalign query program of the genestream network server at institut de génétique humaine. phylogenetic analyses were conducted using phylodraw program at the graphics application laboratory, pusan national university. all of the s genes of the 10 kwd strains contained an orf of 4092 nucleotides, encoding a predicted protein of 1363 aa residues and having a molecular weight of approximately 150 kda. spike protein further divided it into s1 and s2 segments of approximately 86 and 65 kda, respectively, at the cleavage site aa 768. among all strains analyzed, a total of 77 and 52 polymorphic nucleotides were identified in the s1 and s2 subunit of the spike glycoprotein of bcov, respectively, compared with the mebus strain (data not shown). these polymorphisms led to 46 and 24 aa changes at 41 and 20 distinct sites, respectively. we have shown recently that the respiratory and enteric isolates collected concurrently from two different calves in usa were significantly distinct from the ancestral enteric strain, mebus (hasoksuz et al., 2002) . by drawing comparisons of deduced aa between our strains and other known bcov, the two most similar sequences were those of kwd10 strain and rbcv lsu strain (96.6%) in paired comparisons, and the most distant were those of the kwd5 strain and the mebus strain (93.4%). all the virulent-kwd strains tended to be distant from the ancestral enteric strain, mebus, suggesting that allelic variation resulted in genetic drift over time (hasoksuz et al., 2002) . based on the total number of aa substitutions, a phylogenetic tree of all s gene sequences was analyzed with prototype strain mebus, rbcv strains lsu and ok, ebcv strains f15 and ly-138, and avirulent ebcv l9 (fig. 1) . the aa sequences of 10 kwd strains were more homologous to each other and were very closely related to the virulent rbcv ok and ebcv ly-138 strains, among the other known bcov studied in paired comparisons (fig. 1) . these data showed that the wd bcovs circulated in korea were genetically similar to both rbcv and ebcv. in addition, the phylogenetic comparison of korean wd strains with other known bcovs, on the sequences of the hypervariable region, revealed that all korean wd strains were also clustered in groups with the virulent rbcv ok and bcq3994 strains, and the virulent ebcv ly-139 strain (fig. 2) . the canadian bcq cd and wd strains, the american rbcv lsu strain, french ebcv f15 strain and avirulent vacc, l9 and mebus strains were clustered on a separate major branch (fig. 2) . therefore, these results indicated that the bcov strains may be diverging from an enteric tropism to a dual (respiratory and rekik and dea, 1994) of respiratory (rbcv), enteric (ebcv), calf diarrhea (cd), winter dysentery (wd), and avirulent strains. the cd strains including mebus, bcq2070, bcq571, bcq20, bcq9, and bcq1523; the wd strains including bcq7373, bcq2590, and kwd1-kwd10; the rbcv strains including bcq3994, lsu, and ok; ebcv strains including ly-138 and f15. phylogenetic analyses were conducted using phylodraw at the graphics application laboratory, pusan national university. enteric) tropism over time via intermediates (hasoksuz et al., 2002) . this speculation could be supported by our previous paper (cho et al., 2001a) in which gnotobiotic and colostrumdeprived calves were inoculated with respiratory isolates of bcov shed viruses, both nasally and rectally. this subsequently induced diarrhea. moreover, feedlot cattle, which had shown respiratory disease signs at arrival time, developed diarrhea 4 days after arrival, which peaked at 4-7 days post arrival (cho et al., 2001b) . further study will be needed on the differences in pathogenicity, antigenicity, or any other physicochemical characteristics between the kwd strains. in the s glycoprotein, the hypervariable region spanning aa 452-593 of the s1 subunit contains the s1b immunoreactive epitope which is the target for virus neutralizing monoclonal antibodies (mabs) (cavanagh, 1995) . the hypervariable region showed varied antigenicity when reacting with mabs directed against the s glycoprotein. therefore, chouljenko et al. (1998) reported that four rbcv-specific aa substitutions fig. 3 . comparison of the predicted amino acid sequences of the bcov s glycoprotein specified by different strains. light-gray boxes contain aa sites known for respiratory bovine coronavirus-specific (chouljenko et al., 1998) , dark boxes contain virulent-specific (chouljenko et al., 1998) , and clear boxes contain korean strains-specific aa changes. at aa 510, 531, 543, and 578 can be predictive and mabsspecific strains for this region may also be distinguished among respiratory, enteric, and vaccine bcov strains. in the present study, three aa sites including aa 510, 543, and 578 of rbcv ok and lsu strains were identical in all korean wd strains (fig. 3) . in our previous paper (hasoksuz et al., 2002) , enteric and respiratory bcov pairs collected concurrently from two feedlot calves in usa also had identical aa substitution sites at the same sites. furthermore, aa 531 (n → g) substitution of rbcv ok and lsu strains compared with the mebus strain was also detected in the cd strain bcq 1523 (data not shown). since these changes were observed in wd, cd, and rbcv strains, our data does not support the speculation of chouljenko et al. (1998) . in addition, aa 13 and 744 of the s1 subunit have been reported for ebcv-specific sites, which were observed in ebcv strains f15 and ly (chouljenko et al., 1998) . however, aa 13 and 744 of the s1 subunit of all korean wd strains were identical with those of lsu and ok strains of rbcv, bcq7373, and bcq2590 of wd isolates, mebus, and l9 avirulent strains (fig. 3) . in particular, aa 744, reporting its sequence in many isolates and/or strains, are the same as other known bcov including rbcv, cd, wd, and ebcv. these results imply that these aa sites are ebcv f15 and ly strain-specific, or that there are no ebcv-specific sites in the s gene. the n-terminal region of the s1 subunit of 10 kwd strains (aa 1-330; laude et al., 1995) , being shown to function as a receptor-binding domain in mouse hepatitis virus, had a total of 15 aa changes compared with the mebus strain (data not shown). amino acid substitutions in this region could alter the tropism of the coronavirus. for example, the first 200 aa of the s1 subunit of the prcv were associated with respiratory tropism and reduced enteropathogenicity (wesley et al., 1991) . therefore, these 15 aa substitutions in the korean wd strains compared with that of the mebus strain may induce either an alteration of the receptor-binding ability during viral invasion or pathogenicity to cattle. the s protein of bcov has a cleavage site located at aa 763-768 (krrsrr) and is divided into the peripheral s1 glycoprotein from the transmembrane domain of the s2 glycoprotein (abraham et al., 1990) . the sequence krrsrr at the predicted proteolytic cleavage site was identified in all kwd strains. interestingly, all kwd strains had an ala to ser change at aa 769, immediately upstream of the cleavage site. since this change was observed exclusively in the respiratory isolates lsu and ok, it was speculated that the change in aa 769 was a potential marker of respiratory tropism (chouljenko et al., 1998) . although changes in aa surrounding the cleavage site of the ha molecule of the influenza a virus and sendai virus have been demonstrated to modulate its cleavability (rott et al., 1984; hsu et al., 1987) , rekik and dea (1994) reported that this change (769; a → s) did not appear to modulate cleavability, the rate of viral replication or the type of cytopathic changes induced in hrt-18 cells for their enteric bcov isolates. in our previous paper (hasoksuz et al., 2002) , this change (769; a → s) was observed in both respiratory and enteric strains. taken all together, the aa substitution (a → s) at aa 769 does not appear to be a potential marker of respiratory tropism. of the two cleavage products of s, the s2 subunit is highly conserved among coronaviruses (gallagher and buchmeier, 2001) . in the present study, the aa sequences of the s2 subunit of korean wd strains were comparatively well conserved compared with the s1 subunit of the mebus strain. the notable aa changes were observed only in the first hydrophobic region of all kwd strains. in this region, there were four aa substitutions (aa 959, 965, 984, and 988), resulting in a marked increase of hydrophilicity in comparison with that of the mebus strain (data not shown). interestingly, the other known virulent bcov, including rbcv lsu and ok strains, ebcv ly and f15 strains revealed an increase in hydrophilicity in the first hydrophobic region compared with avirulent strains vacc, l9, and mebus (data not shown). the membrane fusion activity is most likely conferred by the internal hydrophobic sequences within the s2 subunit (luo and weiss, 1998; yoo et al., 1991) . therefore, aa substitutions inducing an increase in hydrophilicity observed in our strains and other virulent bcov may alter the fusion activity or pathogenicity of the viruses. putative virulent-specific sites of bcov were reported to exist at 10 aa sites (six within the s1 subunit and four within the s2 subunit) in the s gene through comparison with avirulent and virulent strains (zhang et al., 1991) . in the present study, the s glycoprotein of all kwd strains contained seven aa substitutions (aa 33, 40, 248, 470, 965, 1241, and 1341) which were common for all virulent strains (fig. 3) . this is in agreement with chouljenko et al. (1998) , who identified seven virulent-specific aa changes in the s gene by comparison with the rbcv, ebcv, and vaccine strains. since these virulent-specific sites were observed in all virulent bcov, but not in avirulent bcov, these aa changes may affect the structure and function of the s glycoprotein and alter the pathogenetic potential of these viruses (chouljenko et al., 1998; zhang et al., 1991) . deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site the coronavirus surface glycoprotein detection and isolation of coronavirus from feces of three herds of feedlot cattle during outbreaks of winter dysentery-like disease cross-protection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and rt-pcr and nested pcr for their detection evaluation of concurrent shedding of bovine coronavirus via the respiratory tract and enteric route in feedlot cattle nucleotide and predicted amino acid sequences of all genes encoded by the 3 genomic portion (9.5 kb) of respiratory bovine coronaviruses and comparisons among respiratory and enteric coronaviruses bovine coronavirus the genome organization of the nidovirales: similarities and differences between arteri coronavirus spike proteins in viral entry and pathogenesis bovine coronaviruses associated with enteric and respiratory diseases in canadian dairy cattle display different reactivities to anti-he monoclonal antibodies and distinct amino acid changes in their he, s and ns4. 9 protein molecular analysis of the s1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates protease activation mutants of sendai viruses: sequence analysis of the mrna of the fusion protein (f) gene and direct identification of the cleavage-activation site detection and isolation of winter dysentery bovine coronavirus circulated in korea during the molecular biology of coronaviruses antibody titers against bovine coronavirus and shedding of the virus via the respiratory tract in feedlot cattle corona and related virus: funtional domains in the spike protein of transmissible gastroenteritis virus roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein comparative sequence analysis of a polymorphic region of the spike glycoprotein s1 subunit of enteric bovine coronavirus isolates studies on the adaptation of influenza viruses to mdck cells winter dysentery in dairy herds: electron microscopic and serological evidence for an association with coronavirus infection isolation of respiratory bovine coronavirus, other cytocidal viruses, and pasteurella spp. from cattle involved in two natural outbreaks of shipping fever genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus the s2 subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells comparison of the nucleotide and deduced amino acid sequences of the s genes specified by virulent strains of bovine coronaviruses this study was supported by the technology development program for agriculture and forestry, ministry of agriculture and forestry, republic of korea. key: cord-279827-921kvrrz authors: murata, takayuki; takashima, yasuhiro; xuan, xuen; otsuka, haruki title: growth behavior of bovine herpesvirus-1 in permissive and semi-permissive cells date: 1999-06-11 journal: virus res doi: 10.1016/s0168-1702(99)00023-4 sha: doc_id: 279827 cord_uid: 921kvrrz bovine herpesvirus-1 (bhv-1) can replicate well in bovine-derived cell lines such as madin darby bovine kidney (mdbk) but grows poorly in hamster lung (hmlu-1). virus replication, dna synthesis, and immediate-early gene expression are severely restricted in hmlu-1. we compared adsorption and penetration of bhv-1 in permissive mdbk and semi-permissive hmlu-1 cells. at a low multiplicity of infection, bhv-1 attached to permissive mdbk cells twice as much as to hmlu-1. the presence of heparin inhibited the attachment of bhv-1 to mdbk cells by about 60%, but over 90% of the attachment was inhibited in hmlu-1. to investigate the penetration of bhv-1, we performed the quantitative measurement of viral dna by quantitative competitive (qc)pcr in infected cells. in mdbk cells, virions attached to the cell surface, penetrated into the cells and were transported to the nucleus. however in hmlu-1, only a small fraction of the virions attached to the cell surface were allowed to penetrate. our results indicated that the replication of bhv-1 in semi-permissive hmlu-1 was not dramatically restricted at one certain point but at some various stages including adsorption and penetration. some a-herpesviruses such as herpes simplex virus (hsv) and pseudorabies virus (prv) have a broad host cell range in vitro. many cell lines derived from a wide variety of animals, such as human, monkeys and rodents, are permissive to hsv and prv, and most of the research on these viruses were carried out using permissive cell systems. however, it is also important to study the growth behavior of herpesviruses in various nonpermissive or semi-permissive cell systems to understand and dissect the fine details of the stages of their viral growth cycles. there have been some examples of such research. swine testis (st) cells were abortive to herpes simplex virus (hsv) because the virus was unable to enter the cells through the plasma membrane (subramanian et al., 1994 (subramanian et al., , 1995 . for st cells, virus entry was critically reduced, dna from the input virus could not be detected, and virus proteins were not synthesized. murine resident peritoneal macrophages (respmo) produced viral immediate early (ie) protein in the course of hsv-1 infection, but early promoter activities were markedly reduced probably under the influence of infected cell-specific protein0 (icp0) and therefore were not permissive to hsv (leary et al., 1985; tufaro et al., 1987; sit et al., 1988; mittel and field, 1989; morahan et al., 1989; wu et al., 1993) . tufaro et al. (1987) isolated a mutant line of mouse l cells, in which the growth of hsv was abortive. hsv entered the mutant cells normally, negotiated the early stages of infection, yet were impaired at a late stage of virus maturation. it was concluded that the export of newly made glycoproteins was defective in the mutant cells. these studies indicated that the growth of herpesvirus could be arrested at various stages in different systems. bovine herpesvirus-1 (bhv-1), which causes economically important diseases in the respiratory tract of cattle, belongs to the a-herpesvirus group (armstrong et al., 1961; roizman et al., 1981) but unlike other members of the group, such as hsv-1 or prv, it has a relatively narrow host range in vitro. bhv-1 can replicate well in bovine-derived cell lines such as madin darby bovine kidney (mdbk) cells but grows poorly in rodent cell lines such as hmlu-1 cells derived from hamster lung. but little knowledge has been obtained about the host specificity. in this study, we investigated at which step bhv-1 replication was restricted in semi-permissive hmlu-1 cells. the virus strain used in this study was the bhv-1 los angeles (la) strain re-cloned in our laboratory by limiting dilution. a recombinant bhv-1/rsv/p32 was obtained by inserting a 852 bp bamhi fragment. this fragment contained the whole open reading frame of the major surface antigen p32 of theileria sergenti (matsuba et al., 1993) in the thymidine kinase gene of bhv-1 (la) (otsuka and xuan, 1996) . the p32 gene was flanked with the rous sarcoma virus long terminal repeat (ltr) at the 5% side and with an sv40 poly a signal at the 3% side (sakai et al., 1990) . mdbk and hmlu-1 (kurogi et al., 1976 ) (hamster lung fibroblast, re-cloned in our laboratory) cells were cultured in eagle's mem (autoclavable, nissui, japan) containing heat inactivated 7.5% fetal calf serum (fcs) and 60 mg/ml kanamycin. the fetal calf serum was negative for bhv-1 antibody. mdbk or hmlu-1 cells in 35 mm dishes were incubated with 1.0× 10 5 , 1.0 ×10 4 or 1.0 × 10 3 plaque forming unit (pfu) of bhv-1 at a multiplicity of infection (moi) of 0.1, 0.01 or 0.001, respectively, at 4°c for 2 h to allow the virus to be adsorbed into the cells. after the virus inoculum was removed, the monolayers were rinsed three times with 1 ml pbs, added to 1 ml medium and incubated at 37°c. at 0, 12, 24, 48, and 72 h post infection (p.i.) the cells were washed with pbs, treated with trypsin at 37°c for 10 min and collected by centrifugation at 3000 rpm for 5min. the cell pellets were washed three times with pbs, diluted with the medium and added to mdbk monolayers formed in 24 well plates. the cultures were incubated for 48 h, fixed and stained and the number of plaques was counted. confluent monolayers of mdbk or hmlu-1 cells in 60 mm dishes were infected with bhv-1(la) at a moi of 5. at various times p.i., total dna (cellular and viral dna) was extracted by the method described previously (xuan et al., 1990) . briefly, infected cells were washed with pbs, lysed in an extraction buffer (0.1 m tris -hcl (ph 9.0), 1% sds, 0.1 m nacl and 1m m edta), and then treated overnight with 1 mg/ml of pronase e. the lysates were extracted with phenol-chloroform, and the dna was precipitated by ethanol. dna samples (0.5 mg) were digested with ecori and subjected to 0.7% agarose gel electrophoresis in tris -borate buffered solution (90 mm tris -borate (ph 8), 2 mm edta, and 1 mg/ml ethidium bromide). after photographing on a uv light trans-illuminator, the agarose gel was soaked in 0.2 m hcl for 5 min, in 0.5 m naoh containing 0.6 m nacl for 45 min, and then neutralized in 1.0 m tris -hcl (ph 7.0) buffer containing 0.6 m nacl. the dna fragments were transferred to a biodyne nylon membrane b (pall) by capillary action with 20×ssc (3 m nacl, 0.3 m trisodium citrate dihydrate). after air drying and baking at 80°c, the nylon membrane was hybridized with nick-translated probes in a buffer containing 50% formamide, 0.6 m nacl, 0.2 m tris -hcl (ph 8.0), 20 mm edta and 0.5% sds at 42°c overnight. the membrane was then washed three times in the same buffer, dried and exposed to fuji medical x-ray film. a bhv-1 specific probe was prepared from the xhoi-bglii 1.7 kbp fragment in the open reading frame (orf) of the bhv-1 gc gene (fitzpack et al., 1989) . this probe was used to detect bhv-1 dna. confluent monolayers of mdbk or hmlu-1 cells in 75-cm 2 flasks were infected with bhv-1 at moi 5, and incubated at 37°c. at various times total rna was extracted with guanidium thiocyanate (chomczynski and sacchi, 1987) . briefly, the cells were rinsed three times with ice-cold pbs and lysed with 2 ml denaturing solution d (50% guanidium thiocyanate, 10 mm tris -hcl (ph 7.5), 0.5% sarcosyl, 0.1 m 2-mercaptoethanol). the viscous lysate was forced to pass through a 23g needle several times to reduce viscosity and mixed with 0.2 ml 2 m sodium acetate, 2 ml water-saturated phenol, and 0.4 ml chloroform containing 2% isoamyl alcohol. the mixture was centrifuged at 10 000× g for 20 min, and the rna was precipitated from the supernatant with 50% isopropanol. the rna was re-dissolved in 1 ml of the denaturing solution, re-precipitated with isopropanol and finally dissolved in a 10% mops (3-(n-morpholino)propanesulfonic acid) buffer containing 50% formamide and 16% formaline. pol a (+ ) mrna was purified by oligotex-dt30 (takara; 9020), and 30% of each rna sample was heated at 65°c for 15 min and separated in 1.0% agarose gel (1% agarose, 10% mops buffer) by electrophoresis in a running buffer (10% mops, 16.6% formalin) according to the method described earlier (chomczynski and sacchi, 1987) . rna was transferred to a biodyne nylon membrane b by capillary action with 20× ssc. after air drying and baking at 80°c, the membrane was hybridized with a dna probe in a hybridization buffer (50% formamide, 0.6 m nacl, 0.2 m tris-hcl (ph 8.0), 20 mm edta, 0.5% sds) at 42°c overnight. the membrane was then washed three times with the same buffer, dried and exposed to fuji medical x-ray film. the nick-translated 11 kbp linear dna digested with ecori and hindiii (wirth et al., 1992; schwyzer et al., 1993) was used as a probe to detect transcripts of bicp4 and bicp0. the 7.6 kbp xhoi fragment of the bicp4 gene (wirth et al., 1991; schwyzer et al., 1993) was inserted at the sali site of the pgemex2 plasmid (promega) so that the coding region of the bicp4 gene was in frame. escherichia coli jm109 (de3), a host strain which contained an iptg-inducible gene for t7 rna polymerase, was transformed with the recombinant plasmid, and the bicp4 antigen was expressed as a t7 gene 10 fusion protein by incubating in l-broth containing 0.5 mm iptg. the bicp4 antigen was extracted and precipitated according to the instruction in the promega technical bulletin. mice (ddy) were inoculated i.p. with the bicp4 antigen, once in freund's complete adjuvant, and twice in freund's incomplete adjuvant at 7 day intervals. blood was collected from the mice 7 days after the last inoculation, and the prepared sera were used for western blot analysis. confluent monolayers of mdbk or hmlu-1 cells in 60mm dishes were infected with bhv-1 (la) at a moi of 5. after incubating over various intervals, the cells were washed with pbs, scraped in pbs, centrifuged and dissolved in 200 ml of a sample buffer (62.5 mm tris -hcl (p h6.8), 2% sds, 0.7 m mercapto-ethanol, 10% glycerol and 0.01% bromophenol blue). the samples were sonicated, heated in a boiling-water bath for 5 min and subjected to sds-polyacrylamide gel electrophoresis (sds-page). protein bands were electroblotted to a polyvinylidene difluoride paper (millipore) as described previously (towbin et al., 1979) . the blotted paper was incubated at 37°c for 1 h with antibodies against bicp4. after washing with pbs, it was incubated with horseradish peroxidase labeled goat anti-mouse antibodies for 1 h, washed again with pbs and developed with pbs containing 0.5 mg/ml diaminobenzidine and 0.01% hydrogen peroxide until visible bands appeared. the reaction was stopped by rinsing the paper with pbs. monolayers of mdbk cells grown in 100 mm dishes were infected with bhv-1 (la) at moi 3. the infected cells were incubated in a methioninefree medium containing 100 mci [ 35 s]methionine between 5 and 24 h p.i. virions were collected from the medium at 24 h by centrifugation at 100 000× g for 2 h in a hitachi ultracentrifuge (type 55p-72) with a rps40t rotor. the pellet was suspended in 1 ml pbs, layered onto a linear potassium tartrate gradient (10 -40%) and centrifuged with the same rotor at 100 000 × g for 2 h. the virion band was collected by fractionation and stored at −80°c. labeled bhv-1 (1 ml) was subjected to an attachment assay after diluting 1000-fold with eagle's mem. cells in 12 hole plates were infected with labeled bhv-1 at 4°c. at indicated times, cells were washed with pbs three times, lysed with 100 ml extraction buffer (0.1 m tris-hcl (ph 9.0), 1% sds, 0.1 m nacl and 1 mm edta), put onto a glass filter and air dried completely. each filter was then soaked in econofluor2 (nen) in a vial, and the radioactivity was assayed using a solvent partition method and presented as the net dpm of products formed per hour. the fractionation of cells was performed as follows. mdbk or hmlu-1 cells in 60 mm dishes were infected with bhv-1/rsv/p32 at 4°c for 1 h, washed three times with ice-cold pbs and incubated at 37°c in the medium containing 400 mg/ml phosphonoacetic acid (paa). at various times, the cells were washed with pbs, trypsin solution added (0.8% trypsin (w/v), 0.05% edta (w/v) in pbs) and incubated at 37°c for 15 min. after the trypsin treatment, the cells were washed three times with pbs. dna was extracted by the method described in section 2.3. the isolation procedure for nuclei was described previously (preston, 1979) . cells in 60 mm dishes were infected with bhv-1/rsv/p32 at moi 5 at 4°c for 1 h, washed three times with ice-cold pbs and incubated at 37°c with a medium containing paa. at various times, the cells were washed with pbs, scraped into pbs and centrifuged at 5000 rpm for 3 min by a minifuge at 4°c. the cell pellets were suspended in 200 ml lysis buffer (10 mm tris-hcl (ph 7.5), 2 mmmgcl 2 , 10 mm nacl, 5 mm 2-mercaptoethanol, 0.5% (v/v) nonidet p-40) and homogenized with a dounce homogenizer until almost all the cells were broken and the nuclei released under microscope examination. homogenates were centrifuged at 2000× g for 2 min, and the pellets were washed three times with lysis buffer and once with sucrose buffer (10 mm tris-hcl (ph 7.5), 2 mm mgcl 2 , 10 mm nacl, 0.32 m sucrose) at 2000×g for 4 min. dna was extracted from the nuclear preparation by the method described above. all work was carried out on ice or at 4°c except where described otherwise. after ethanol precipitation, each dna sample was dissolved in 20 ml water and a 1 ml aliquot was subjected to quantitative competitive (qc)pcr reaction. the qcpcr reactions to measure the amount of bhv-1/rsv/ p32 in infected cells or nuclei were designed based on the method described by gariglio et al. (1997) . the primers used to detect t. sergenti p32 gene were as follows; 5%-aagctcgacctttcccat-3% and 5%-caggcgatgagaagagc-3%. using the p32 gene in plasmid or bhv-1/rsv/p32 as a template, these primers yielded a 517 bp fragment by pcr amplification. a mutant template plasmid pucp32cdel1, which contained a deletion in the p32 gene, was constructed by cleaving the parental plasmid pucp32 at the tthiii1 site and deleting some nucleotides with exonuclease iii (takara) and mung bean nuclease (takara) according to the manufacturer's instructions. using pucp32cdel1 as a template, a 350 bp fragment was amplified by pcr using the same pair of the primers. known amounts of mutant templates and samples (1 ml) were added to each reaction tube with taq dna polymerase recombinant (gibco brl) and amplified according to the instructions using a geneamp pcr system 2400 (perkin-elmer). the conditions were as follows; incubation at 94°c for 2 min, 35 or 40 cycles at 94°c for 1 min, 55°c for 1 min and 72°c for 2 min, followed by a single extension at 72°c for 5 min. amplified dna fragments (517 and 350 bp) were electrophoretically separated in 1.5% agarose gel. the dna bands were compared using luminous imager ver. 1 for macintosh (ai-c). the equivalent points, where equal amounts of 517 and 350 bp dnas were synthesized, were determined according to the standard linear regression curves. replication of bhv-1 in permissive mdbk and semi-permissive hmlu-1 was compared. while we could observe distinct cytopathic effects (cpe) in bhv-1 infected the mdbk cells within 6 h p.i., and the cells were completely rounded by 12 h p.i., little cpe were observed in the hmlu-1 cells at 24 h p.i. however at 48 or 72 h p.i., most of the hmlu-1 cells in the infected culture were rounded (data not shown). fig. 1a shows one step and multi step growth curves for bhv-1 in the mdbk and hmlu-1 cells. when infected at a moi 5, the titer of bhv-1 in the mdbk cells started growing exponentially after 3 h, reached a plateau between 12 and 24 h and declined afterwards. in contrast, the titer in hmlu-1 increased very little up to 12 h and increased very slowly afterwards until it reached a plateau at around 48 h. the yield of infectious viruses in the hmlu-1 cells was two orders of magnitude lower than that in the mdbk cells. in the case of infection at a low moi, the difference in the growth in permissive and semi-permissive cells appeared to be greater. in the mdbk cells, bhv-1 replicated exponentially between 16-24 h p.i. reaching up to 10 7 pfu or more at around 48 h p.i. regardless of the initial input of the virus. however, in the hmlu-1 cells, the onset of the virus replication was delayed, starting around 24 h p.i., and the yield of infectious virus was three orders of magnitude lower than in the mdbk cells. when the input virus was less, the yield was much lower. additionally, we performed infectious center assays. cell monolayers were infected with bhv-1 at a low moi at 4°c and then incubated at 37°c. after the incubation period, the cells were trypsinized, and the infectious centers were scored on permissive mdbk cells (fig. 1b) . most of the infected mdbk cells (about 10 6 ) became infectious centers at 24 h p.i. in contrast only a small population of bhv-1 infected hmlu-1 cells were infectious centers at 24 h p.i. 2.1 × 10 4 , 6.0 ×10 2 and 2.0×10 1 infectious centers were formed when infected with 10 5 , 10 4 and10 3 pfu bhv-1, respectively. in order to investigate whether replication of viral dna was restricted in the hmlu-1 cells, viral and cellular dna were extracted together hmlu-1 cells grown in 35 mm dishes were infected with 5.0× 10 6 pfu (at an moi of 5, indicated as mdbk, hmlu), 1.0×10 5 pfu (moi = 0.1, m5 and h5), 1.0 × 10 4 pfu (moi = 0.01, m4 and h4), and 1.0 ×10 3 pfu (moi =0.001, m3 and h3) bhv-1. after 2 h adsorption, the media were removed, the cells were washed three times with pbs, and 1 ml fresh medium was added. at various times, both the cells and media were harvested and stored at −80°c. the samples were diluted in eagle's mem and seeded onto mdbk cells. the monolayers were overlaid with eagle's mem containing 0.5% methylcellurose. after 2 days incubation, the monolayers were fixed with ethanol, stained with 0.15% crystal violet solution, and plaque numbers were scored (a). the numbers of infectious centers were counted and shown in (b). the cells in the 35 mm dishes were attached with 1.0× 10 5 (moi = 0.1, m5 and h5), 1.0×10 4 (moi=0.01, m4 and h4), 1.0× 10 3 (moi = 0.001, m3 and h3) pfu of bhv-1 at 4°c for 2 h. after the adsorption period, the virus inocula were removed, the cells were washed extensively and shifted to 37°c. at 0, 12, 24, 48, and 72 h p.i., the cells were trypsinized, washed three times with pbs and layered onto mdbk cells. the numbers of infectious centers were determined after crystal violet staining. from bhv-1 infected cells and analyzed by southern blot hybridization using the virus specific probe. equal amounts of ecori-digested dna samples were electrophoresed in an agarose gel and visualized with ethidium bromide (fig. 2a) . dna bands in this gel were blotted, and hybridized with a nick translated probe containing the coding region of the bhv-1 gc gene. as shown in fig. 2b , strong signals of bhv-1 dna were observed in permissive mdbk cells starting at 6 h p.i., while no viral dna was detected in semi-permissive hmlu-1 cells up to 12 h p.i., and very weak signals were seen at 24 and 48 h p.i. it would appear that only a very low level of replication of viral dna took place in the hmlu-1 cells. total cellular rnas were isolated from bhv-1 infected mdbk and hmlu-1 cells. equal amounts of rna samples were separated by agarose gel electrophoresis, transferred to a membrane and hybridized to a nick translated probe. schwyzer et al. (1993) and wirth et al. (1991) reported the ie gene structure of bhv-1. we prepared the nick translated probe to detect both of the transcripts of the bicp4 and bicp0 genes according to their reports. as shown in fig. 3 , abundant transcripts of the bicp4 and bicp0 genes were detected in the mdbk cells at 3 and 6 h p.i., but only very low levels of these transcripts were found in the hmlu-1 cells until 12 h p.i. when rna extracted from the mdbk cells at 6 h p.i. was diluted and compared with rna from the hmlu-1 cells, it was found that the level of mrna of bicp4 and bicp0 in the hmlu-1 cells was 0.1-0.01% of that in the mdbk cells (data not shown). western blot hybridization analysis of bicp4 using mouse anti-serum revealed that the level of expression of bie (200, 190 , and 160 kda) in the hmlu-1 cells was very low (fig. 4) , indicating that the rate limiting step of bhv-1 growth in the hmlu-1 cells was before the transcription of the ie genes. we compared the attachment and penetration of bhv-1 in permissive and semi-permissive cells fig. 2 . detection of bhv-1 dna in permissive mdbk and semi-permissive hmlu-1 cells. confluent monolayer cultures of mdbk cells or hmlu-1 cells were infected with bhv-1 at an moi of 5 and at 3, 6, 12, 24, and 48 h p.i., dna was extracted as described in section 2. equal amounts were digested with ecori and electrophoresed in agarose gel. bhv-1 specific dna fragments are depicted with a large arrow on the right side. small fragments (small arrow indicated on the left side) should be mdbk cells specific dna, not viral dna (a). the dna was then blotted and hybridized to a [ 32 p]-labeled nick-translated dna probe which reacted to the bhv-1 specific gene. the arrow indicates bhv-1 specific bands (b). attached to both mdbk and hmlu-1 cells, but the amount of bhv-1 attached to hmlu-1 was about half of that attached to mdbk. bhv-1, as well as other herpesviruses, is known to bind to heparan sulfate on the cell surface, and this binding is blocked by heparin (okazaki et al., 1991) . when labeled bhv-1 was incubated at 4°c for 1 h in a medium containing 10 u/ml heparin and then added to the mdbk cells, the attachment of bhv-1 to the mdbk cells was reduced by about 60%, suggesting that about 40% of the labeled bhv-1 attached to the mdbk cells not via heparan sulfate but by other mechanisms. on the other hand, 90% of the adsorption of labeled bhv-1 to hmlu-1 was blocked by heparin treatment. this indicates that bhv-1 attached to the hmlu-1 cells mostly through heparan sulfate and only a small fraction of virus attached in a heparan sulfate independent manner. using isotope labeled virions. the mdbk or hmlu-1 cells grown in 12 well plates were infected with [ 35 s]methionine labeled bhv-1 at 4°c. at 1, 2, and 4 h p.i., the cells were washed extensively and harvested, and the amounts of radioactivity associated with the cells were determined by a liquid scintillation counter. the results shown in fig. 5a indicate that labeled bhv-1 was fig. 4 . immunoblot hybridization analysis of bicp4, the major transcription regulatory protein, from bhv-1 infected hmlu-1 and mdbk cells. the cell proteins were harvested at 3, 6, 12, and 24 h p.i., and equal quantities were sds-polyacrylamide gel electrophoresed and immunoblotted. molecular weight markers are indicated on the left side. ments we used a bhv-1 recombinant, bhv-1/ rsv/p32, which carries the p32 gene of t. sergenti in the bhv-1 genome, in place of wild type bhv-1. the reasons are as follows: (1) bhv-1 dna is difficult to amplify by pcr due to a high gc content, (2) we established that the p32 gene can be easily amplified by pcr even it was integrated into bhv-1 dna, and (3) the recombinant bhv-1/rsv/p32 behaved in the same way fig. 5 . adosorption of radioisotope labeled bhv-1. mdbk and hmlu-1 cells were infected with [ 35 s]methionine labeled bhv-1 (mdbk, hmlu) or infected with labeled bhv-1 previously treated with heparin (mdbk heparin treated, hmlu heparin treated) at 4°c. at 0, 1, 2, and 4 h p.i., the cells were washed with pbs three times and collected, and the radioactivities associated with the cells were counted (a). the cells were infected with [ 35 s]methionine labeled bhv-1 at 4°c. at 0, 1, and 2, h p.i., the cells were washed with pbs (mdbk pbs wash, hmlu pbs wash) three times or inoculated with heparin (500 mg/ml in pbs) for 2 min followed by washing with pbs twice (mdbk heparin wash, hmlu heparin wash). the cells were collected and radioactivities associated with the cells were counted (b). the effect of heparin wash on bhv-1 which were already attached to the cells was studied. monolayers were infected with [ 35 s]methionine labeled bhv-1 at 4°c. at 0, 1, and 2 h p.i., the cells were washed three times with pbs or incubated with heparin solution followed by washing twice with pbs. the cells were harvested, and the radioactivities associated with the cells were determined. as shown in fig. 5b , once bhv-1 was attached onto either the mdbk or hmlu-1 cells, only 10% of the attached virions were released by washing with heparin. these results seemed to imply that immediately after the attachment via heparan sulfate molecules, bhv-1 virions became bound to the surface of the mdbk and hmlu-1 cells via non-heparan sulfate molecules. after attaching to the cell membrane, herpesvirus enters into the cells by ph-independent fusion between the viral envelope and the cell plasma membrane (wittels and spear, 1990) . in order to measure the viral dna that fused to the cells, qcpcr was used. in this series of experi-as the wild type bhv-1 in the mdbk or hmlu-1 cells. to quantitate viral dna in the sample, a series of known amounts of a competitor template plasmid pucp32cdel1 was mixed with the sample dna, and the series of mixtures of the sample and the competitor were subjected to pcr amplification using the pair of primers described above. the pcr products were separated by 1.5% agarose gel electrophoresis as shown in fig. 6a . bhv-1/rsv/p32 contains the wild type p32 gene which yields a 517 bp pcr product, and the competitor plasmid pucp32cdel1 contains the p32 gene with a deletion which yields a 350 bp pcr product. when an equi-molar amount of bhv-1/rsv/p32 and the competitor plasmid were present in the sample mixture, the same amounts of the 517 bp product and the 350 bp product would be present in the pcr product. the amount of viral dna in the samples was determined by estimating such a point by analyzing the data in fig. 6a as described in section 2. the mdbk or hmlu-1 cells were infected with bhv-1/rsv/p32 at 4°c for 1 h, washed extensively with cold pbs and incubated with medium at 37°c for 0, 1, 2, and 5 h. the virions attached onto the surface of cells during incubation at 4°c and penetrated into the cells when the temperature was shifted to 37°c (huang and wagner, 1964) . to prevent the replication of viral dna, 400 mg/ml phosphonoacetic acid (paa) was included in the medium. total dna (cellular and viral dna associated with cells) was extracted before and after trypsin treatment, and the amount of viral dna was quantitated as above. the trypsin treatment removes attached virions from the cell surface but does not remove fused (penetrated) virions. the results are shown in fig. 6b . after incubating at 4°c for 1 h, 1.02 pg equivalent (mean of three independent samples) bhv-1/rsv/p32 dna was attached to the mdbk cells. however, trypsin treatment removed over 90% of the attached viral dna, and only 0.095 pg equivalent viral dna was found to be associated with the cells. the attached virions became more resistant to trypsin treatment when infected mdbk cells were incubated at 37°c; 0.37, 0.38, and 0.50 pg equivalent viral dna were detected in the cells after 1, 2, and 5 h incubation periods, respectively. in the mdbk cells, therefore, about 49% of the attached virions became trypsin resistant after 5 h incubation at 37°c. in semi-permissive hmlu-1 cells, qcpcr detected 0.42 pg equivalent of viral dna that was attached to the cells after incubating at 4°c for 1 h. this indicated that bhv-1 attached hmlu-1 was about 40% of that attached to the mdbk cells, and this ratio was comparable to the isotope-labeled virus attachment experiments. after 1, 2, and 5 h incubation at 37°c, however, only 0.050, 0.093, and 0.01 pg equivalent dna were detectable in trypsin treated hmlu-1 cells, respectively. the results indicated that after 2 h incubation at 37°c (3 h p.i.), the amount of virions which penetrated into the hmlu-1 cells was 24% of that which penetrated into the mdbk cells. it was also noted that the amount of trypsin resistant, cell associated virions in the hmlu-1 cells decreased after 5 h incubation while in the mdbk cells it increased. it can be concluded that bhv-1 can attach to hmlu-1 cells, but the fusion and penetration process may be at a low level. 3.6. nuclear transport of 6iral dna additionally, we analyzed the nuclear transport of bhv-1 in permissive mdbk and semi-permissive hmlu-1 cells by qcpcr. after adsorption of bhv-1/rsv/p32 onto the cells for 1 h at 4°c, the cells were washed extensively with ice cold pbs and incubated with medium at 37°c. at 1, 3, 6, and 12 h p.i., the cells were washed with pbs, and the nuclei were isolated as described in section 2. the dna was extracted from the nuclear fraction, and the amount of viral dna was quantitated by qcpcr. as shown in fig. 6b , in permissive mdbk cells, 0.0100, 0.173, 0.364, and 0.116 pg equivalent dna were transported to the nuclei at 1, 3, 6, and 12 h p.i., respectively. the amount of bhv-1 dna in the nuclei of mdbk decreased at 12 h p.i. probably because the viral dna was degraded when the viral dna replication was inhibited by paa. in the nuclei of hmlu-1, the amount of viral dna was below the detection level of the method employed here (b 0.001 pg equivalent). after the attachment period, the cells were washed and temperature shifted to 37°c. at 1, 2, 3, and 6 h p.i. (0, 1, 2, and 5 h incubation at 37°c), the cells were washed, trypsinized, and the virus dna associated with the cells were measured. the right graph is bhv-1 transported to the nucleus. after the attachment period, the cells were washed and temperature shifted to 37°c. dna was extracted from the nucleus at 1, 3, 6, and 12 h p.i. (that was 0, 2, 5, and 11 h after adsorption), and the virus dna were measured. four experiments of penetration (3 h p.i.) and three experiments of attached were performed independently, and their s.d. were depicted. 'o' on the bars indicates that the quantities were less than 0.001 pg equivalent. replication of the a-herpesvirus group, to which bhv-1 belongs, is well studied in permissive cell systems (roizman and sears (1990) and others). when permissive cells are infected with a virus, the following sequential events take place: (1) the virus particles attach onto the cell surface, (2) the viral envelope fuses to the cell plasma membrane and the virus capsid is released into the cytoplasm, (3) the released capsid is transported to the nucleus, (4) the ie class of the viral genes are expressed, (5) the ie products induce other classes (early, late) of viral genes, (6) the virus dna replicats, (7) the newly synthesized virus dna and proteins assemble automatically, (8) particles surrounded by envelopes bud from the cells. in semi-or non-permissive systems, the growth of the virus is arrested at a point or points of the above mentioned steps. to study the growth behavior of the virus in a semi-or non-permissive system is very important because it allows for the analysis of the fine details of the particular step where the growth is arrested. in this study we investigated and elucidated the growth behavior of bhv-1 in semi-permissive hmlu-1 cells compared with that in permissive mdbk cells. the hmlu-1 cells have been used to propagate akabane virus (kurogi et al., 1976; liao et al., 1996) , bovine corona virus (fukai et al., 1998) , bluetongue virus (ianconescu et al., 1996) and prv (shibata et al., 1991; matsuda tsuchida et al., 1992) . however, bhv-1 grows poorly in hmlu-1 cells. when the hmlu-1 cells were infected with bhv-1 at low moi, very little cpe was observed, and the virus titer hardly increased after prolonged incubation periods. infection at high moi caused distinct cpe, and the virus titer increased slightly (less than 1% of that in the mdbk cells). the timing of the virus replication was also delayed in the hmlu-1 cells. the transcripts of the bicp4 and bicp0 genes were synthesized in the mdbk cells immediately after infection by bhv-1, but only a very small quantity of both transcripts was found in the hmlu-1 cells at 12 h p.i. or later. it would appear that the cause of the poor growth in the hmlu-1 cells exist in the step or steps before the induction of the ie gene. it has been reported earlier that a-herpesvirus adsorbed onto the cell surface in two distinct ways . one adsorption mechanism is through the interaction between viral glycoproteins (gc and gb) and heparan sulfate on the cell surface. this adsorption mechanism is inhibited by heparin (wudunn and spear, 1989) . the other adsorption mechanism, which is not inhibited by heparin, is not well understood (lee and fuller, 1993) . radioisotope labeled bhv-1 was adsorbed onto non-permissive hmlu-1 cells although the amount of adsorbed virus was about 50% of that adsorbed onto permissive mdbk cells. when the virus was treated with heparin before infection, the adsorption onto hmlu-1 cells was reduced by 90%. this suggests that 90% of bhv-1 was adsorbed onto semi-permissive hmlu-1 cells through heparan sulfate, and only 10% was adsorbed through the other mechanism. on the other hand, 60% of bhv-1 was adsorbed onto permissive mdbk cells through heparan sulfate and 40% through the heparan sulfate independent mechanism. therefore, it would appear that the hmlu-1 cells are deficient in their ability to adsorb bhv-1 through the heparan sulfate independent mechanism. because the heparin wash did not remove the virus which was already attached to either permissive mdbk or semi-permissive hmlu-1 cells, we concluded that after heparan sulfate binding, bhv-1 quickly bound to a cellular factor other than heparan sulfate and became resistant to the heparin wash. virus particles fused with the plasma membrane are resistant to trypsin treatment while particles which are just adsorbed on the cell surface are susceptible to trypsin. quantitative analysis by qcpcr revealed that bhv-1 adsorbed to permissive mdbk cells penetrated into the cells, became trypsin resistant, and gradually were transported to the nuclei. on the other hand, the greater part of virus adsorbed to hmlu-1 remained trypsin sensitive after prolonged incubation and virus dna was not detectable by the method employed in this study. however, a small number of bhv-1 virions must have reached the nuclei and replicated because a small fraction of bhv-1 infected hmlu-1 became infectious centers as shown in fig. 1b. our results suggest that the major bottleneck for the growth of bhv-1 in the hmlu-1 cells may exist at the level of the penetration of the virion. many viral and cellular factors are involved in the herpesvirus penetration which is probably a multistep event. montgomery et al. (1996) cloned herpesvirus entry mediator (hvem/hvea) and demonstrated that hvem mediated the entry of several wild type hsv strains into non-permissive cho-k1 and st cells. mutations in the hsv envelope glycoprotein d (gd) significantly reduced hvem-mediated entry, suggesting that hvem functions via hsv gd (montgomery et al., 1996) . other evidence existed that gd was important for fusion. abortive cells could be made resistant to hsv entry while retaining the ability to bind a virus by transfection with plasmid expressing hsv gd (campadelli-fiume et al., 1988; johnson and spear, 1989) . soluble forms of hsv gd inhibited virus entry into cells (johnson et al., 1990) . and recently, two other cell surface proteins named poliovirus receptor-related protein 1 (prr1)/hveb and prr2/hvec, were found to mediate hsv entry also by interaction with gd warner et al., 1998) . for bhv-1, gd expressing cells were reported to resist bhv-1 penetration into the cells (chase and letchworth, 1994) . therefore it might be possible that hmlu-1 cells might lacked the hvem like factor for bhv-1 gd while mdbk cells express such a factor allowing the penetration and the nuclear transport of bhv-1. prv grows well in hmlu-1 cells as well as in mdbk cells. we have constructed bhv-1 recombinants which express prv glycoproteins (otsuka and xuan, 1996) . in our preliminary experiments it was observed that the penetration into the hmlu-1 cells was greatly improved by bhv-1 recombinants which express prv gb. further analyses of these recombinants will shed some light on the question of virus -cell tropism. observations on the virus of infectious bovine rhinotracheitis virus, and its affinity with the herpesvirus group entry of herpes simplex virus 1 in bj cells that constitutively express viral glycoprotein d is by endocytosis and results in degradation of the virus bovine herpesvirus 1 giv-expressing cells resist virus penetration single-step method of rna isolation by acid guanidium thiocyanate -phenolchloroform extraction nucleotide sequence of bovine herpesvirus type 1 glycoprotein giii, a structure model for giii as a new member of the immunoglobulin superfamily, and implications for the homologous glycoproteins of other herpes viruses evaluation of serum neutralizing antibodies to bovine coronavirus in cows and their calves using hmlu-1 cells suppression of high mobility group protein t160 expression impairs mouse cytomegarovirus replication entry of alphaherpesviruses mediated by poliovirus receptor related protein 1 and poliovirus receptor penetration of herpes simplex virus into human epidermoid cells comparative susceptibility of a canine cell line and bluetongue virus susceptible cell lines to a bluetongue virus isolate pathogenic for dogs soluble forms of herpes simplex virus glycoprotein d bind to a limited number of cell surface receptors and inhibit virus entry into cells herpes simplex virus glycoprotein d mediated interference with herpes simplex virus infection epizootic congenital arthrogryposis-hydranencephaly syndrome in cattle: isolation of akabane virus from affected fetuses comparison of herpes simplex virus type 1 dna replication and virus production in murine bone marrow-derived and resident peritoneal macrophages herpes simplex virus type 1 and pseudorabies virus bind to a common saturable receptor on vero cells that is not heparan sulfate the isolation of akabane virus (iriki strain) from calves in taiwan analysis of mixed parasite populations of theileria sergenti using cdna probes encoding a major piroplasm surface protein protection from pseudorabies virus challenge in mice by a combination of purified gii, giii and gvi antigens cell-specific kinetics and efficiency of herpes simplex virus type 1 entry are determined by two distinct phases of attachment analysis of bovine herpesvirus type 1 thimidine kinase (tk) gene from wild-type virus and tk-deficient mutant herpes simplex virus-1 entry into cells mediated by a novel member of the tnf/ngf receptor family molecular localization of abortive infection of resident peritoneal macrophages by herpes simplex virus type 1 bhv-1 adsorption is mediated by the interaction of glycoprotein giii with heparin-like moiety on the cell surface construction of bovine herpesvirus-1 (bhv-1) recombinants which express pseudorabies virus (prv) glycoproteins gb, gc, gd and ge abnormal properties of an immediateearly polypeptide in cells infected with herpes simplex virus type 1 mutant tsk herpesviridae definition, provisional nomenclature, and taxonomy functional comparison of transactivation by human retrovirus rev nd rex genes promoter, spliced leader, and coding sequence for bicp4, the largest of the immediate-early proteins of bovine herpesvirus 1 avirulent ts and thymidine kinase-deficient mutant of aujeszky's disease virus effect of macrophage activation on resistance of mouse peritoneal macrophages to infection with heroes simplex virus type 1 and 2 swine testis cells contain functional heparan sulfate but are defective in entry of herpes simplex virus defective entry of herpes simplex virus type 1 and 2 into porcine cells and lack of infection in infant pigs indicate species tropism electro-phoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications identification and characterization of a mouse cell mutant defective in the intracelluler transport of glycoproteins a cell surface protein with herpesvirus entry activity (hveb) confers susceptibility to infection by herpes simplex virus type 2, mutants of herpes simplex virus type 1 and pseudorabies virus the three major immediate-early transcripts of bovine herpesvirus 1 arise from two divergent and spliced transcription units immediate-early rna 2.9 and early 2.6 of bovine herpesvirus 1 are 3% coterminal and encode a putative zinc finger transactivator protein penetration of cells by herpes simplex virus does not require a low ph-dependent endocytic pathway regulation of herpes simplex virus type 1 gene expression in nonpermissive murine resident peritoneal macrophages initial interaction of herpes simplexvirus with cells is binding to heparan sulfate restriction endonuclease analysis of canine herpesviruses isolated in japan t. sergenti p32 gene was provided by dr m. onuma (hokkaido university, hokkaido, japan). this work was supported by grants from the ministry of education, science, sports and culture of japan. key: cord-260835-ck9z5xsd authors: kamau, anthony ndirangu; park, jung-eun; park, eui-soon; yu, jung-eun; rho, jaerang; shin, hyun-jin title: porcine amino peptidase n domain vii has critical role in binding and entry of porcine epidemic diarrhea virus date: 2017-01-02 journal: virus res doi: 10.1016/j.virusres.2016.10.004 sha: doc_id: 260835 cord_uid: ck9z5xsd porcine epidemic diarrhea virus (pedv) infects swine intestinal cells causing enteric disease. research has shown that the entry into these cells is through porcine aminopeptidase n (papn) receptor. to gain insights into mechanisms of pedv-papn interactions, the present study aimed at identifying the domain that is critical for pedv binding. to this end, nih3t3 cell lines constitutively expressing papn or papn mutants were generated. the mutants were; domain vii deletion mutant and domains iv–vi deletion mutant. in the latter, domain vii was linked to the transmembrane segment through domain iii. results showed pedv infection was restricted to papn and papn domain vii expressing nih3t3 cells. further, reducing pedv titre 10 fold resulted in 37.8% decrease in foci indicating positive correlation. a time course test at 12, 24, 36, 48 and 60 h showed that foci increased 6 fold in the overall time range. also, pedv harvested from papn or domain vii expressing nih3t3 cells was induced indirect plaques in vero cells confirming successful entry and replication. collectively, our results demonstrate that pedv recognizes papn and that the main interactive point is lodged within domain vii of the papn. these findings are important for therapeutic development as well as creating a platform for future studies on pedv. coronaviruses (covs) are enveloped, positive-stranded rna viruses potentially pathogenic in respiratory and intestinal tracts of humans, animals and birds (david and kathryn, 2007; susan and navas-martin, 2005) . analyses by serological cross-reactivity and genome phylogeny distinguish these viruses into four genera which include alpha, beta, gamma and delta-cov (de groot et al., 2011) . studies have speculated alpha and beta-covs to have originated from bats whereas gamma and delta-covs gene source is the birds (woo et al., 2012) . pedv belongs to the genus alpha-cov (de groot et al., 2011) . host specificity, tissue tropism and virulence of covs are dictated by interactions between the spike glycoproteins of the viruses and cellular glycoproteins on surface receptors. receptor binding and cell entry is essential steps for virus entry into host cell, which leads to internalization of the viruses (park et al., 2014; liu et al., 2015) . an envelope-anchored spike protein mediates coronavirus entry into cells (liu et al., 2015) . the spike ectodomain consists of a receptor-binding subunit, s1, and a membrane fusion subunit, s2. two domains in s1, calls s1-ntd and s1-ctd, can potentially function as receptor-binding domains (li, 2012 (li, , 2015 in some covs, entry mechanisms progress via sialic acid interactions with spike and/or hemagglutinin esterase glycoproteins (haijema et al., 2003; kuo et al., 2000; phillips et al., 1999; sanchez et al., 1999) . the spike protein of murine hepatitis virus (mhv) binds carcinoembryonic cell adhesion molecule 1a for cell penetration while human (hcov-nl63) and severe acute respiratory syndrome (sars)-cov recognize human angiotensin-converting enzyme 2 (hace-2) on host cells (dveksler et al., 1991 (dveksler et al., , 1993 . aminopeptidase n (apn) serves as receptor for feline (fcov), canine (ccov), transmissible gastroenteritis virus (tgev), pedv as well as human (hcov -229e) (delmas et al., 1992; tresnan et al., 1996; yeager et al., 1992; kolb et al., 1998; li, 2015; liu et al., 2015) . apn/cd13 is a 150 kda, zinc dependent metalloprotease consisting of 967 amino acids (rawlings and barrett, 1995) . it is ubiquitously present in diverse organs, tissues and cell types (luan and xu, 2007) . it is expressed as a glycosylated homodimer on the surface of epithelial cells of liver, intestine, kidney and respiratory tract fibroblasts and leukocytes (kenny and maroux, 1982; lendeckel et al., 2000; nam and lee, 2010) . additionally, apn is found on endothelial cells such as cerebral astrocytes as well as pericytes and synaptic membrane in the central nervous system (kay et al., 1994; soichiro et al., 1983; mina-osorio et al., 2008) . apn cleaves the n-terminal amino acids from peptides. in addition, apn plays role as a receptor and signaling molecule (mina-osorio et al., 2008) . a seven domain organization has been described for apn structure. the first domain constitutes a short cytoplasmic tail on the n-terminal end (amino acids [aa] 1-7). domain ii (aa 8-39) consists of the transmembrane segment and links to a stalk forming domain iii (aa 40-70). domain iv spans from aa 71 to 252. the peptidase activity is housed by domains v and vi (aa 253-580). located on the c terminus is domain vii consisting of aa 581-967 (sjostrom et al., 2000) . previous investigations by virus overlay protein binding assay showed that pedv bound papn in permissive cells and it was also shown that anti-papn blocked this binding (oh et al., 2003) . further, expression of papn in non-permissive madin-darby canine kidney cells resulted in susceptibity of these cells to pedv (li et al., 2009) . however, pedv fails to infect native porcine apn expressing swine testis cells which are highly susceptible to tgev (hofmann and wyler, 1988 ). in the current study, by constitutive expression of papn in non-susceptible nih3t3 cells, we have sought to annotate the previous findings stipulating papn receptor function for pedv. in addition, this study aimed at identification of using papn protein domain(s) that are critical for its binding and entry. vero cells were cultured in alpha minimum essential medium ([␣-mem] gibco). mouse embryonic fibroblasts (nih3t3) and human embryonic kidney (hek 293t) cells were cultured in dulbecco's modified eagle medium ([dmem] gibco). all growth media were supplemented with 10% fetal bovine serum ([fbs] gibco). the pedv strain used in this study was korean isolate. the clones used in this study are illustrated (fig. 1b) . three clones were designed and denoted as follows; (1) wild type papn (papn wt ), (2) domain vii deletion mutant of papn (papn di-vi ) and (3) domains iv-vi deletion papn mutant (papn dvii ). these were amplified by pcr (horton, 1993) using pfu polymerase (solgent). primers were designed using genbank sequences (accession number hq824547) and all antisense primer sequences were fused in frame to flag epitope sequence. the wild type papn was amplified using sense 5 -ccc aag ctt acc atg gcc aag gga ttc tac-3 and anti-sense 5 -ccc ctc gag tca ctt gtc gtc atc gtc ttt gta gtc gct gtg ctc tat gaa cca-3 primers. mutants papn di-vi pcr primers were sense 5 -ccc aag ctt acc atg gcc aag gga ttc tac-3 and anti-sense 5 -ccc ctc gag tca ctt gtc gtc atc gtc ttt gta gtc gct gtg ctc tat gaa cca-3 while papn dvii was amplified using 5 -cgg tac cgc gcc tcg gtt atc agg atg ctc-3 and 5 -ccc ctc gag tca ctt gtc gtc atc gtc ttt gta gtc gct gtg ctc tat gaa cca-3 sense and anti-sense primers respectively. the cdnas encoding papn wt and mutants were cloned into the hind iii and xho i sites of pcdna 3.0 (invitrogen). hek 293t cells were transfected using plasmid constructs containing papn clone or mutants cdna using fugene hd (roche) according to the manufacturer. at 24 h after transfection, cells were assayed for papn expression by immunoblotting using mouse anti-papn polysera and anti-flag antibody (santa-cruz). extracellular expression of papn was determined by detection of flag epitope. cells were harvested using cells dissociation solution (sigma). these were washed two times in 2 ml of 1 x phosphate buffered saline (pbs), centrifuged at 300 × g for 5 min and supernatants were discarded. cells were resuspended in 100 l of staining buffer (1 x pbs containing 2% fbs) and surface antigens were stained with anti-flag antibodies. after 2 times wash with 2 ml of 1 x pbs and re-suspension in 100 l staining buffer, cells were incubated with phycoerythrin (pe) conjugated secondary antibody (bd biosciences) and analyzed by flow cytometry (facs calibur, becton-dickinson). nih3t3 cells were transfected with pcdna3.0 with or without papn wt or mutants using fugene hd (roche) as suggested by the manufacturer and 24 h after transfection, cells were subjected to 400 g/ml g418 selection pressure. the cell lines generated were designated as follows; nih3t3 stably harboring only vector (nih3t3 pcdna3.0 ), expressing papn wt (nih3t3-papn wt ), expressing papn di-vi (nih3t3-papn di-vi ) and papn dvii mutant (nih3t3-papn dvii ), respectively . after antibiotic selection, the cells were analyzed for expression by western blot. the kpedv-9 was propagated in vero cells using serum free ␣-mem in the presence of 10 g/ml trypsin (sigma) as described (oh et al., 2003) . for the quantitation of virus, plaque assay was performed as described (dulbecco, 1952) . briefly, vero cells (2.5 × 10 5 cells/ml) were seeded in a 6-well plate 24 h prior to inoculation. cells were washed with 1 ml of 1x pbs and 200 l of inoculum was added. after 1 h adsorption, inoculum was removed and cells were overlaid with serum free ␣-mem containing 0.9% methylcellulose and 10 g/ml trypsin. these were incubated at 37 • c with 5% co 2 until plaques formed. the overlay medium was discarded and cells washed with 1 ml 1x pbs then fixed with 2 ml of fixation solution (5% methanol and 10% acetic acid) for 30 min at room temperature. cells were washed with 1 ml of 1x pbs and incubated with 1 ml of 1% crystal violet for 20 min at room temperature. the dye was then discarded and cells washed were gently with distilled water then air-dried. a plaque count was conducted to deduce the number of plaque forming units (pfu) per ml of virus suspension. this virus culture technique was adapted for inoculation of nih3t3 cells with pedv at a multiplicity of infection (m.o.i.) of 0.06-0.6. the inocula were removed after 1 h adsorption and replaced with fresh medium. after various time periods after inoculation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% triton x-100. cells were blocked with 4% skim milk for 30 min at room temperature and detection of viral antigens was performed using mouse anti-pedv polysera for 1 h at 37 • c. after 3 times wash with 200 l of 1 x pbs, cells were incubated with horseradish peroxidase (hrp) -conjugated anti-mouse igg (santa-cruz) for 1 h, 37 • c. the cells were washed 3 times with 1 x pbs. reaction was visualized by addition of 3,3 -diaminobenzidine chromogenic substrate (vector lab). pedv titer attained in nih3t3 cell types was analyzed indirectly in vero cells as previously described (yu et al., 2010) . nih3t3 cell lines expressing or not expressing surface papn wt or mutants were seeded at a density of 2.5 × 10 5 per ml. at 24 h post inoculation (p.i.) with pedv at m.o.i. 0.6, virus was harvested after by 3 cycles of freezing-thawing. the supernatants were re-inoculated into vero cells along with pedv control inoculation. since dead or defective virus particle are incapable of forming plaques, virus viability in nih3t3 cell lines was verified by plaque assay. western blot assays using anti-flag antibody and mouse anti-papn polysera revealed papn wt was expressed at approximately 150 kda in both 293t and nih3t3 cells ( fig. 2a and b) . additionally, surface expression was confirmed by flow cytometry using anti-flag and p.e. labeling. the results confirmed 47.88% (293t) and 62.23% (nih3t3) surface expression ( fig. 2c and d) . to investigate susceptibility of nih3t3-papn wt , pedv was inoculated into nih3t3-papn wt , wild type nih3t3 and nih3t3 pcdna3.0 . although the typical cytopathic effects (cpe) such as cell rounding and syncytia formation were not observed in nih3t3-papn wt cells, detection of pedv antigens in fixed cells showed stained foci, but not in wild type nih3t3 cells nor in nih3t3 pcdna3.0 (fig. 3a) . this result confirmed that papn wt is important for pedv infection. further, pedv was serially diluted by a factor of 10 and inoculated to nih3t3-papn wt . comparison of pedv at m.o.i. 0.6 and 0.06 showed that number of foci was positively correlated to concentration (fig. 3b ). the number of foci formed in nih3t3-papn wt at an m.o.i of 0.6 reduced by 37.8% at an m.o.i. of 0.06. although nih3t3-papn wt was susceptible to pedv, the number of progeny virions produced was much lower than those in vero cells. to identify papn wt protein domain(s) important for pedv binding, deletion mutants were generated and their stable expression in nih3t3 was verified by western blotting. as shown in fig. 4 , papn dvii and papn di-vi expression was detected at 61 kda and 55 kda, respectively. susceptibility assays showed infection in papn domain-vii expressing cells but virus entry was abrogated in absence of seventh domain (papn di-vi ) as shown in (fig. 5a) . also, there was no observable infection of both nih3t3 wild type and nih3t3 pcdna3.0 controls. analysis of infection between 12 and 60 h p.i. showed that the average number of foci increased more than 6 times in nih3t3-papn dvii . similarly, pedv induced foci increased more than 6 fold over time (12 h-60 h) in papn wt expressing cells. also notable was the increase in foci spread through time especially in nih3t3-papn wt (fig. 5b) . lysates of pedv or mock-infected nih3t3 cell lines were re-inoculated in vero cells for the indirect plaque assay. nih3t3-papn wt and nih3t3-papn dvii lysates induced plaques but nih3t3, nih3t3 pcdna3.0 and nih3t3-papn di-vi lysates did not (fig. 6a) . on quantification, nih3t3-papn wt had more plaque forming particles (992 pfu/ml) on average while nih3t3-papn dvii lysate had 725 pfu/ml (fig. 6b) . the hallmark of coronaviruses is the spike glycoprotein which is their functional attachment molecule to the surface of host cells. as of fact and characteristic of all viruses, the attachment involves definite interactions with precise receptors which determine host specificity and tissue tropism (sandrine et al., 2012) . the extensively studied coronavirus prototype, mhv is able to penetrate the hepatocytes through the biliary glycoprotein on the surface of the liver and cause hepatitis (godfraind and coutelier, 1998) . in alphacoronaviruses, papn on the microvillar membrane of the small intestine is the known gateway for tgev infections in swine (delmas et al., 1992 (delmas et al., , 1994 while human apn (cd13) is the attachment receptor for hcov-229e which targets infections to lungs where it has been associated with common cold or severe pneumonia in immunocompromised persons (lachance et al., 1998; pene et al., 2003) . apn/cd13 is a monomeric or homodimeric type ii membrane-bound zinc-dependent metalloproteinase, which cleaves neutral or basic amino acids from the n-terminus of oligopeptides and can release neutral and basic amino acid from the n-terminal of peptides (mou et al., 2009) . it is widely expressed on various cells such as epithelial cells of the intestine and kidney, hepatocytes, osteoclasts, endometrial cells, fibroblasts, endothelial cells, bone marrow stromal cells and on neuronal synaptic membranes. apn is involved in many physiology and pathology processes such as hydrolysis of nutrients, inactivation of bioactive peptides, inflammatory bowel diseases, rheumatoid arthritis and cancer (bank et al., 2008; yamashita et al., 2007; shimizu et al., 2002) . in 2006, the 3d structure of apn has been studied according to the co-crystal complex of apn and bestatin by ito (ito et al., 2006) . based on this report on overall structure and subunits of apn, it is composed of consists of 26 ␣helices and 26 ␤-strands and can be divided into four domains, an n-terminal ␤-domain (met 1 -asp 193 ), a catalytic domain (phe 194 -gly 444 ), a middle ␤-domain (thr 445 -trp 546 ), and a c-terminal ␣-domain (ser 547 -ala 870 ). they also reported the c-terminal ␣domain might be important as the active site. more recently, shan et al. reported the protein structure on papn by swiss-model web server (shan et al., 2015) . they reported papn contains small intra-cellular domain and large extra cellular domain divided into 3 subdomains named spa, spb and spc. in virology, it has been reported that apn is involved in binding of corona viruses and mediating cytomegalovirus infection and antigen presentation (shahwan et al., 2013; regan et al., 2012; li et al., 2007; kasman, 2005) . it has been established that tgev binds the papn receptor on the surface of swine enterocytes (delmas et al., 1992 (delmas et al., , 1994 . additionally, tgev along with ccov, tgev and hcov-229e exhibit cross-binding activities to feline apn (fapn), which is the major receptor for feline infectious peritonitis virus and it has been demonstrated that these viruses can utilize this receptor to infect fapn-expressing bhk21 and nih3t3 cells (tresnan et al., 1996; tusell and holmes, 2006; tusell et al., 2007) . pedv was also reported to interact with papn in porcine enterocytes (li et al., 2009) , and is capable of infecting mdck and st cells expressing the papn receptor (oh et al., 2003; nam and lee, 2010) . more recently, li et al. reported papn is important receptor for pedv infection in swine small intestine (iecs). they found higher expression increased the susceptibility by pedv, and this was confirmed by sirna inhibition results. they also found pedv enters via apical plasma membrane and released from there in polarized epithelial cells (cong et al., 2015) . other group reported that porcine and human apn serve as efficient receptor for pedv infection (li, 2015) . they found although tgev spike protein bound porcine apn much more tightly than human apn, pedv spike protein efficiently bound both porcine and human apn. they also reported that neu5ac as the pedv coreceptor. very recently, shirato et al. reported papn is not functional receptor for pedv but promotes the its infection through protease activity (shirato et al., 2016) . taken together it is acceptable that papn as the pedv receptor still little bit controversial. because pedv infects porcine enterocytes causing intestinal lesions that are indistinguishable from those caused by tgev (ducatelle et al., 1982; hooper and haelterman, 1969) , it is conceivable that the two viruses may recognize the same receptor. curiously, tgev readily infects porcine primary cells whereas attempts to isolate pedv in both primary and secondary fetal porcine cells have not been successful (hofmann and wyler, 1988; kusanagi et al., 1992) . this ambiguity is further compounded by the fact that vero cells, with no surface papn, are susceptible to pedv (hofmann and wyler, 1988) . to clarify these doubts, the current study employed the nih3t3 cells which are non-susceptible to pedv infection. by constitutive expression of papn wt on these cells, the receptor function would be established by the conversion of the cells into a susceptible cell line (nih3t3-papn wt ). our results demonstrated that there was extracellular expression of papn wt with a surface presence of 62.23% by flow cytometry. whether this surface receptor would allow pedv entry was investigated by infection of the cells along with experimental controls, nih3t3 wt and empty vector harboring cells (nih3t3 pcdna3.0 ). results of our analyses showed that although cytopathic effects such as cell rounding and or syncytia formation were not vividly evident, pedv antigens were detectable immunocytochemistry. in addition, the number of foci formation was positively correlated with the virus titer and the time allowed for virus replication. this development could only be attributed to the papn wt because the wild type nih3t3 and nih3t3 pcdna3.0 cells were not permissive to pedv infection. since nih3t3 cells being fibroblasts, have a morphology that may not allow close association between cells, spaces in between cells may have resulted in the lack of syncytia or plaque formation in nih3t3. in addition, pedv has already been adapted to vero cells and it is assumable that nih3t3 may be an unfamiliar environment for pedv replication. indeed, even new isolates of pedv are able to show distinct signs of cpe in vero cells only after several passages (pan et al., 2010) , which could probably be an adaptation period. as the functional domain study on papn, shan et al. reported that spc region on papn might be putative functional domain for pedv infection (shan et al., 2015) . they found mdck cells expressing only spc region successfully infected by pedv and even shown cytopathic effects (cpe). they also reported that pedv titer in mdck cell expressing only spc region generated same number of virus in papn expressing mdck and vero cells. in our study, to determine the specific interactive point of papn wt with pedv, domain mutants were expressed stably in nih3t3 cells. the cloning strategy of the mutants followed cues that coronaviruses identify certain intermittent motifs on fapn which are amino acids (aa) 288-290, aa 732-746 (called r1), and aa 764-788 (called r2) which determine their tropism (tusell et al., 2007) . thus, two mutants; (david and kathryn, 2007) expressing domains i-vi of papn wt and (susan and navas-martin, 2005) expressing domain vii of papn wt were constructed and designated papn d1-vi and papn dvii, respectively. nih3t3 stable cell lines expressing these mutants were assayed for their susceptibility to pedv and similar to tgev (delmas et al., 1994) , pedv successfully infected papn dvii expressing cells but that was abrogated in the absence of the seventh domain (papn di-vi ). also noteworthy, pedv harvested from nih3t3-papn wt and nih3t3-papn dvii induced plaques on vero cell monolayers indicating virus entry and propagation in those cells. taken together, all our data confirmed that pedv replicated in both nih3t3-papn wt and nih3t3-papn dvii suggesting successful entry into the cell lines. we therefore concluded that domain vii of papn is critical for pedv entry. although structural domain analysis on papn used in this study is little bit different from those from shan et al. study, but functional binding domain from both studies are identical. especially, our results promising because we studied stable expression of wild type and deletion mutants from stable cell lines expressing those proteins. even if our studies confirmed with stable cell lines but still don't know how those mutations on papn affected on its stability, 3-d structure and especially function. still more studies required to answer on those questions. inflammatory bowel diseases: multiple benefits from therapy with dipeptidyl-and alanyl-aminopeptidase inhibitors porcine aminopeptidase n mediated polarized infection by porcine epidemic diarrhea virus in target cells coronavirus binding and entry virus taxonomy: ninth report of the international committee on taxonomy of viruses aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site pathology of experimental cv777 coronavirus enteritis in piglets. ii. electron microscopic study production 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of a recombinant canine coronavirus with a distinct receptor-binding (s1) domain targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence mechanisms of coronavirus cell entry mediated by the viral spike protein sialic acid binding properties of soluble coronavirus spike (s1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus identification of the functional domain of the porcine epidemic diarrhoea virus receptor cd13/aminopeptidase n-induced lymphocyte involvement in inflamed joints of patients with rheumatoid arthritis porcine aminopeptidase n is not a cellular receptor of porcine epidemic diarrhoea virus, but promotes its infectivity via aminopeptidase activity structure and function of aminopeptidase n distribution and biosynthesis of aminopeptidase n and dipeptidyl aminopeptidase iv in rat small intestine coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i molecular interactions of group 1 coronaviruses with feline apn mutational analysis of aminopeptidase n, a receptor for several group 1 coronaviruses, identifies key determinants of viral host range discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus involvement of aminopeptidase n in enhanced chemosensitivity to paclitaxel in ovarian carcinoma in vitro and in vivo human aminopeptidase n is a receptor for human coronavirus 229e protein arginine methyltransferase 1 regulates herpes simplex virus replication through icp27 rgg-box methylation this research was supported by the basic science research program through the national research foundation of korea (nrf) funded by the ministry of science, ict and future planning (grant number 2015r1a2a2a01003785). key: cord-258294-ny3xrjzc authors: gao, xiang; zhao, donghong; zhou, peng; zhang, liping; li, mingxia; li, weiyan; zhang, yongguang; wang, yonglu; liu, xinsheng title: characterization, pathogenicity and protective efficacy of a cell culture-derived porcine deltacoronavirus date: 2020-04-02 journal: virus res doi: 10.1016/j.virusres.2020.197955 sha: doc_id: 258294 cord_uid: ny3xrjzc porcine deltacoronavirus (pdcov) is a novel swine enteropathogenic coronavirus that causes acute diarrhea, vomiting, dehydration and mortality in neonatal piglets, resulting in significant economic losses to the pig industry. however, there is currently little information on vaccine studies and commercially available vaccines for pdcov. hence, herein, a pdcov strain, ch/xjyn/2016, was successfully isolated and serially propagated in vitro, and its biological characteristics were determined. compared to that of previously reported and recently isolated pdcov strains from china and the united states, the s gene of the ch/xjyn/2016 strain contains novel mutations. infection studies revealed that ch/xjyn/2016 is pathogenic to suckling piglets and conventional weaned pigs. in addition, the median pig diarrhea dose (pdd(50)) of pdcov in conventional weaned pigs was determined (2.0 log(10)pdd(50)/3 ml). furthermore, an inactivated cell-adapted ch/xjyn/2016-based vaccine candidate was developed with different adjuvants. compared with nonvaccinated pigs, conventional weaned pigs given the inactivated vaccine developed a potent humoral immune response and showed no clinical signs or viral shedding after challenge, indicating a potent protective effect of the vaccine against pdcov infection. therefore, the pdcov vaccine developed in this study is a promising vaccine candidate that can be used for the control of pdcov infection in pigs. porcine deltacoronavirus (pdcov) is an enveloped, single-stranded, positive-sense rna virus belonging to the deltacoronavirus genus of the coronaviridae family (hu et al., 2015) . pdcov was first reported in pigs in hong kong in 2012 (woo et al., 2017) and was subsequently detected in swine herds in the united states and isolated from clinical cases of diarrhea in young pigs in 2014 (hu et al., 2015; marthaler et al., 2014; wang et al., 2014) . to date, pdcov has been successively detected in canada (marthaler et al., 2014) , south korea (lee and lee, 2014) , mainland china , mexico (lee, 2015) , thailand (janetanakit et al., 2016; madapong et al., 2016) , vietnam and lao pdr (saeng-chuto et al., 2017) . hence, pdcov has recently become prevalent in pigs worldwide and has caused serious economic losses for the pig industry. the complete genome of pdcov is approximately 25 kb long and contains the 5'-untranslated region, open reading frame (orfs) organized in the order orf1a/1b, spike (s) glycoprotein gene, envelope (e) gene, membrane (m) gene, non-structural protein 6 (nsp6) gene, nucleoprotein (n) gene, nsp7 gene , and the 3'-utr (lee and lee, 2014; zhang, 2016) . orf1a/b encodes two overlapping viral replicase polyproteins that are processed into mature nonstructural proteins. although the general characteristics of the structural and nonstructural proteins of pdcov are similar to those of other swine coronaviruses, the detailed functions and roles of the structural and nonstructural pdcov proteins in host cells are largely unknown (jung et al., 2016) . among these structural proteins, the n protein is known to be the most abundant and multifunctional viral component . typical clinical symptoms of pdcov infection include diarrhea, dehydration, variable vomiting and mortality in nursing piglets. however, compared with those of other swine coronaviruses, such as porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev), the clinical symptoms of pdcov infection are milder and the mortality rates are lower in affected nursing pigs jung et al., 2016; zhang, 2016) . previous reports have shown that pdcov mortality rates range from 40% to more than 80% among neonatal pigs song et al., 2015) . similar to that of pedv and tgev, the strongest tissue tropism of pdcov is in villous enterocytes of the small and large intestines, leading to marked villous atrophy in the small intestine but not in the large intestine hu et al., 2015; jung et al., 2016) . pdcov has been successfully isolated and serially propagated in llc porcine kidney (llc-pk, atcc no: cl-101) and swine testicular (st, atcc no: crl1746) cells supplemented with trypsin or pancreatin (dong et al., 2016; hu et al., 2015; jang et al., 2018; zhang et al., 2019b) . in addition, the porcine enterocyte cell line, ipec-j2, is also susceptible to pdcov infection but apoptosis may not be induced in the infected cells (jung et al., 2018) . as is the case for pedv, trypsin contributes to a significant increase in pdcov growth after several passages in llc-pk cells, but not in st cells (hu et al., 2015) . in addition, pdcov can also be propagated in llc-pk cells without supplemental trypsin; however, it does not produce cytopathic effects (cpes) and the viral titer is reduced (hu et al., 2015; jung et al., 2016) . therefore, in general, llc-pk and st cells are suitable for the in vitro isolation and propagation of pdcov field strains under optimal cell culture conditions. previous phylogenetic analysis showed that the chinese pdcov strain is more closely related to other pdcov strains in china than to the strains from southeast asia, usa, japan, and south korea, suggesting the diversity of genetic relationships and regional and epidemic characteristics among these strains . additionally, the recent study shows that the chinese pdcov strains isolated from china had the same discontinuous amino acid deletions in nsp2 and nsp3 regions as thailand, vietnam and laos, indicating that pdcov may have undergone a high degree of variation since pdcov was first detected in china (sun et al., 2020) . all of these studies show that the persistent occurrences of pdcov infection in asia and north america implies a potential risk of pandemic outbreaks. therefore, effective vaccines are required to control and limit the spread of pdcov. however, there are currently no vaccines available for pdcov. therefore, the main purpose of the present study was to isolate and serially propagate a pdcov strain and to develop a vaccine candidate that can elicit a potent humoral immune response against pdcov and effectively protect pigs against pdcov infection. in this study, the pdcov strain, ch/xjyn/2016 (genbank no: mn064712), was successfully isolated and serially propagated using llc-pk, and its biological characteristics and pathogenicity were determined. notably, the infectious titer (the median pig diarrhea dose, pdd50) of pdcov in conventional weaned pigs was determined for the first time. in addition, the immunoprotective effect of the cell-adapted strain ch/xjyn/2016-based inactivated vaccine candidate was evaluated. the results presented in this study indicated that the inactivated vaccine is a promising vaccine candidate for the prevention and control of pdcov in china. all pigs used in this study were well taken care of during the experiment and euthanized according laboratory procedure at the end of the experiment. all animal experiments were performed in biosecurity laboratory. animal care and use protocols were reviewed and approved by the institutional animal use and care committee of lanzhou veterinary research institute. in february 2016, twelve clinical samples, including porcine intestinal contents and feces, were collected from a small-scale pig farm (100 sows) with diarrhea outbreaks (more than half of the sucking piglets developed severe diarrhea and about one-third of piglets died) in yining, xinjiang uygur autonomous region, china. the samples were stored at a low temperature with dry ice after collection. subsequently, the samples were diluted 1:10 with serum-free dulbecco's modified eagle's medium (dmem, invitrogen, usa) containing 1% penicillin-streptomycin (10,000 units/ml penicillin and 10,000 µg/ml streptomycin; gibco, usa) and then centrifuged at 1500×g at 4°c for 30 min. the supernatant was filtered using a 0.22-μm pore-size filter (merck millipore, germany) to remove bacteria, and then the viral rna was extracted using an rneasy mini kit (qiagen, germany) according to the operating instructions. the virus isolated from these samples was identified as pdcov by n gene-based reverse transcription pcr (rt-pcr; developed by our lab). llc-pk cells (atcc no: cl-101) were purchased from atcc and were cultured in minimum essential medium (mem, gibco, usa) supplemented with 5% heat-inactivated fetal bovine serum (fbs, gibco, australia), 1% mem nonessential amino acids (neaa, gibco, usa), 1% antibiotic-antimycotic (gibco, usa) and 1% n-2-hydroxyethylpiperazine-n-2-ethane sulfonic acid (hepes, gibco, usa). the cells were maintained at 37°c in a humidified 5% co2 incubator. mouse anti-pdcov n protein monoclonal antibodies (mcabs) were prepared and stored in our laboratory. pdcov isolation was performed using llc-pk cells as described previously (hu et al., 2015) , with some modifications. in brief, 100% confluent llc-pk cell monolayers were prepared in a t-25 flask (corning, usa) and washed three times with sterile phosphate-buffered saline (pbs; ph 7.2, gibco, usa)to completely remove fbs. then, the prepared supernatant of the pdcov-positive samples was added as an inoculum for incubation. after incubation at 37℃ in 5% co2 for one hour, the inoculums were removed, and 2 ml of virus growth medium (mem supplemented with 1% neaa, 1% antibiotic-antimycotic and 1% hepes) was added. after that, the flasks were maintained at 37°c in a humidified 5% co2 incubator and monitored daily until cpes were observed in > 90% of the cells. then, the flask was subjected to three rounds of freezing and thawing, and the cell culture-harvested virions were collected as seed stock for the next serial passage. the tissue culture infectious dose (tcid50) assay was performed using a procedure described previously with some modification (liu et al., 2019) . the diluted virus supernatant of twenty passages (p20) of the cell culture-adapted ch/xjyn/2016 strain (200 tcid50/0.1 ml) was used as virus stock for incubation. cpes in llc-pk cells were monitored for 72 to 96 h, and the viral titers were calculated according to the reed and muench method. after forming a 100% confluent cell monolayer in 6-well plates, llc-pk cells were infected with the p10 of cell culture-adapted pdcov strain ch/xjyn/2016 at a multiplicity of infection (moi) of 0.01. the infected cells were subsequently cultured and were fixed using 4% paraformaldehyde (solarbio, china) at 4°c for 30 min at 0, 12, 24 and 36 h post-infection, respectively, and then permeabilized with 0.25% triton x-100 (solarbio, china) in pbs at room temperature (rt) for 10 min. after washing with pbs (gibco™, usa), 5% bovine serum albumin (bsa; solarbio, china) was added for blocking at rt for 1 h. then, the cells were incubated with pdcov n protein mcabs #51 (prepared by our laboratory) and goat anti-mouse igg conjugated to alexa fluor 488 (abcam, uk) as primary and secondary antibodies, respectively. after washing, the cell nuclei were stained with 4',6-diamidino-2-phenylindole (dapi; sigma) for 5 min at rt, followed by observation with a fluorescence microscope (olympus, japan). to image the virion particles of pdcov, electron microscopy was used, and samples were prepared according to methods described in a previous study (liu et al., 2019) . briefly, llc-pk cells infected with pdcov were harvested when cpes were observed in more than 90% of the cells. the harvested cell cultures were frozen and thawed three times and then centrifuged at 8,500×g at 4°c for 30 min. the cell debris in the supernatant was removed by filtration through a 0.22-mm filter, and then polyethylene glycol 8000 (peg-8000; solarbio, china) was incubated overnight at a 10% final concentration. after that, the suspensions were ultracentrifuged at 12,000×g at 4°c for 2 h to pellet the viral particles. the purified viral particles were resuspended using tris buffered saline solution (tbs, solarbio, china) and then negatively stained with 2% phosphotungstic acid. the image was observed with a transmission electron microscope (jem-1200ex, japan). viral rna was extracted from the original fecal samples using the rneasy mini kit (qiagen, germany) according to the manufacturer's instructions. reverse transcription and pcr were performed with specific primers, which were designed and stored in our laboratory. primer sequences: pdcov-sf1: 5'-attatctatggtgatgattcca-3', pdcov-sr1:5'-acaattcagcatgagtggtt-3'; pdcov-sf2: pdcov-sr3:5'-aaccaagacgcgtcagtagta-3'. the pcr-amplified target fragments were purified and recycled, and then were cloned into topo vector (invitrogen, usa). the positive plasmids identified by restriction analysis were sent to sangon biotech (shanghai, china) for sequencing. the full-length s gene were assembled and analyzed with dnastar 7.0 software. phylogenetic trees were constructed by the neighbor-joining method with bootstrap values calculated for each node from 1000 replicates. all phylogenetic tree figures were produced using mega 4.0 software (tamura et al., 2013) . an indirect elisa based on pdcov nucleocapsid (n) antigen, which was expressed as a recombinant protein in 293 cells in our laboratory, was developed to assess igg antibody in pigs. in brief, a concentration of 0.25 ng/ml purified pdcov n protein in 0.05 m nahco3 was used to coat elisa plates (corning, usa), which were then incubated with serum samples (1:400 dilution) at 37°c for 1 h. after the plate was washed with pbst 3 times, an hrp-conjugated goat anti-pig igg mcab (1:8000 dilution, abcam, usa) was added and the plate was incubated at 37°c for 30 min. then, the plate was washed with pbst 3 times, 3, 3', 5, 5'-tetramethylbenzidine (tmb) substrate was added and the plate was incubated at 37°c for 10 min. then, 50 μl of stop solution was added, and the absorbance was determined at 450 nm. to determine the pathogenicity of the isolated virus in suckling piglets, infection experiments were performed using 4-day-old conventional suckling piglets, as described in our previous study (zhang et al., 2019a) . briefly, pregnant pdcov-naïve sows that tested seronegative for pdcov in the indirect elisa were selected from a commercial pig farm with no previous herd history of pdcov infection. after delivery, six piglets were randomly allotted into two groups: the infection group (n=4) and the mock control group (n=2). each piglet was housed in an individual steel cage, and each group of pigs was housed in different rooms. all piglets were artificially fed bovine milk and had free access to water. piglets in the infection group and mock control group were separately inoculated with 3 ml of mem containing 1.0×10 4 tcid50 of the cell culture-adapted pdcov strain ch/xjyn/2016-p6. all piglets were observed daily for clinical signs after inoculation. rectal swabs were collected daily from all piglets, tested by real-time pcr and scored for fecal consistency as follows: 0 = normal; 1 = pasty; 2 = semiliquid; and 3 = liquid (zhang et al., 2019a) . the piglets were euthanized when obvious clinical symptoms were observed and fecal viral rna shedding was detected in the rectal swab samples. in addition, necropsy examinations of small intestinal tissues were also performed, as described previously (zhang et al., 2019a) . intestinal samples were collected for hematoxylin and eosin (he) staining and immunohistochemistry (ihc) examination. histopathology and ihc examinations were performed using a procedure described previously (liu et al., 2019) . after euthanization, the intestinal tissue specimens of each piglet were removed, fixed in 4% paraformaldehyde solution at rt for 48 h, and then processed and embedded in paraffin. the paraffin-embedded tissues were sectioned by microtome (leica, germany) and then deparaffinized and washed in ethanol. subsequently, the intestinal tissue specimens were routinely stained with he (baso, china) for histopathology or subjected to ihc using pdcov n-specific mcabs (prepared in our laboratory). to determine a standardized and validated dose for the subsequent pig challenge experiments, the median pig diarrhea dose (pdd50) of p6 of the cell culture-adapted pdcov strain, ch/xjyn/2016, was determined by using conventional weaned pigs as described in our previous study (zhang et al., 2019a) . in detail, a total of twenty-five 45-day-old conventional weaned pigs were randomly divided into five experimental groups (g1-g5; table 1 ). each group of pigs was housed in a different room, and each piglet was housed in an individual steel cage. to ensure that all pigs were pdcov-naïve, fecal and blood samples were collected before inoculation and then tested by real-time pcr (developed by our laboratory) and indirect elisa. the primer and probe sequences used in real-time pcr were as follows: sense, 5'-acgtcgtaagacccagcatc-3'; antisense, 5'-cccacctgaaagttgctctc-3'; probe, j o u r n a l p r e -p r o o f 5'-fam-gtatggctgatcctcgcatcatggc-bhq1-3'. the pcr conditions were as follows: 42 °c for 5 min; 95 °c for 10 s, and followed by 40 cycles of 95 °c for 10 s and 57 °c for 20 s. in groups 1 to 4, pigs were separately inoculated orally with 3 ml of 10-fold serially diluted ch/xjyn/2016-p6 (diluted from 10 0 to 10 -3 ; the original titer of virus stock: 4 log10 tcid50/ml) (table 1) . pigs in group 5 were inoculated orally with 3 ml of pbs (gibco, usa) as a mock control (table 1) . after inoculation, typical clinical signs were observed daily and scored for fecal consistency until 7 days post-infection (dpi). in addition, the intestinal faeces were collected daily using rectal swabs to detect viral rna shedding by real-time pcr (above method). the pdd50 was defined as the reciprocal of the virus dilution at which 50% of pigs developed serious diarrhea during 7 dpi using the reed and muench method (liu et al., 2015) . the experimental inactivated vaccines were prepared according to our previous study (liu et al., 2019) , with some modifications. in brief, the p30 of the cell culture-adapted ch/xjyn/2016 (10 6 tcid50/ml) strain was chemically inactivated using binary ethyleneimine (bei). after three rounds of freezing and thawing, the supernatant of the viral cultures was collected by centrifugation and then inactivated by the addition of 0.2 m bei to a final concentration of 2 mm at 30°c for 24 h. then, 20% sodium thiosulfate was added to neutralize the remaining bei. the effect of inactivation was assessed by the absence of viral growth in llc-pk cell cultures. subsequently, the inactivated vaccines were prepared by mixing bei-inactivated cell culture-adapted ch/xjyn/2016 p30 with 206 adjuvant (china agricultural vet. bio. science) and imject tm alum adjuvant (thermo, usa) and were then stored at 4°c until use. thirteen two-week-old pdcov-naïve piglets that tested seronegative for pdcov-specific antibodies by indirect elisa were obtained from a commercial pig farm with no previous herd history of pdcov outbreaks. all of the piglets were raised in the laboratory animal facility at the lanzhou veterinary research institute. each group was housed in separate rooms for the duration of the experiment. the animal use protocols were approved by the institute. all piglets were randomly allocated to two experimental groups (n=5 per group) and one mock control group (n=3). the two experimental groups were immunized with different formulations of the inactivated vaccine as follows: group 1, inactivated virus with imject tm alum adjuvant (n=5); group 2, inactivated virus with 206 adjuvant (n=5) ( table 2) . piglets in the two experimental groups were immunized intramuscularly at 0 days postvaccination (dpv) with 2 ml of the prepared inactivated vaccines and were given two booster immunizations at 14 and 28 dpv. the mock control group received only pbs (n=3). blood samples were collected at 0, 7, 14, 21, 28, 35 and 42 dpv for antibody detection. all of pigs in three groups were challenged orally with 3 ml of 1000 pdd50 cell cultured ch/xjyn/2016-p6 at 42 dpv. after challenge, typical clinical symptoms of pdcov infection were observed daily until 7 dpi, and scores of fecal consistency were rated according to our previous study (liu et al., 2019) . samples of the intestinal faeces were collected daily to monitor viral shedding. pigs will be identified as pdcov infection-positive when the scores of fecal consistency were ≥ 2 and virus shedding could be detected in fecal samples at the same time. neutralizing antibody responses induced by the experimental inactivated vaccines were assessed by vn test, as described previously (liu et al., 2019) . neutralizing antibody titers were calculated as the reciprocal of the highest serum dilution that inhibited cpes. statistical analyses of the data in this study were performed using spss 16.0 software. one-way anova with tukey's multiple comparisons test was used to determine statistical significance among the different groups. differences between groups were considered significant when the p value was less than 0.05. after centrifugation and filtration, the supernatant of the diluted clinical fecal samples containing one pdcov strain, named ch/xjyn/2016, was used to inoculate llc-pk cells for a series of passages. obvious cpes were first observed at the fifth passage at 36 h postinoculation (hpi), and cpes were observed in more j o u r n a l p r e -p r o o f than 90% of cells at 96 hpi. compared with the uninoculated cells, the pdcov-inoculated cells were characterized as enlarged, rounded, and clustered at the early stage, and then the infected cells showed signs of lysis and detachment from the cell monolayer at the late stage (data not shown). the propagation of the 10th passage of strain ch/xjyn/2016 was confirmed by detecting pdcov antigens with ifa using a pdcov n-specific mcabs and dapi. the results showed that distinct green signals were observed in the pdcov strain ch/xjyn/2016-infected llc-pk cells starting at 12 hpi, and the signal intensity tended to increase significantly from 12 to 36 hpi (figure 1a ). viral particles of pdcov strain ch/xjyn/2016 purified from infected llc-pk cell cultures were imaged with transmission electron microscopy (tem). tem observations revealed that the viral particles were the typical crown-shaped coronavirus particles, approximately 80 to 160 nm in diameter and with spiky surface projections, which were morphologically indistinguishable from other coronaviruses (figure 1b) . the viral titers of the serially passaged pdcov ch/xjyn/2016 strain were determined as the tcid50 at 10-passage intervals. the infectious titers of the cell-adapted virus from p0 to p70 ranged from 10 3.2 to 10 7.8 tcid50/ml (figure 1c) . the results of the sequence analysis indicated that compared with that of other reported pdcovs, the ch/xjyn/2016 strain s gene contained some new mutations. details, thirty-seven single nucleotide substitutions and a 3-nt insertion were observed in s gene of the ch/xjyn/2016 strain. the complete s genes of strain ch/xjyn/2016 shares 98.74% -97.70% nucleotide identity with the other 100 pdcov reference strains in genbank. in addition, phylogenetic analyses based on the full-length s gene demonstrated that strain ch/xjyn/2016 clusters with other pdcov strains isolated from china, the united states, japan and south korea, whereas the strains isolated from thailand, vietnam and lao clustered into a large clade ( figure 2 ). however, compared with that of other reported chinese strains, the genetic distance of the ch/xjyn/2016 strain is closer to the original hong kong strain hku15-44 (jq065042) (woo et al., 2012) , sharing 98.74% nucleotide identity. these results suggest that the us, japan and south korea clade and the chinese clade might share a common parent. to determine the pathogenicity of the ch/xjyn/2016 strain, 4-day-old conventional suckling piglets were orally inoculated with p6 of the cell culture-adapted virus. before inoculation, all piglets were lively, showed no clinical symptoms, had normal fecal consistency, and no viral rna was detected in their fecal samples by real-time pcr. all the pigs from the infection group (n=4) developed severe diarrhea with high levels of fecal viral rna shedding from 1-2 dpi, and the symptoms persisted until the piglets were euthanized (figure 3a ). all the pigs from the infection group with severe diarrhea were underweight, depressed and vomiting. the pigs (n=2) from the mock control group were active and fleshy during the experiment and negative for fecal pdcov rna shedding. at necropsy, typical macroscopic intestinal lesions were found, especially in the small intestine (figure 3b ). in detail, the intestinal tract was distended, transparent, filled with yellow fluid, and mesenteric congestion was present (figure 3b ). further histopathological examination of all the pigs from the infection group showed viral enteritis, such as vacuolar degeneration in the enterocytes and shortening, fusion and sloughing of the villi (figure 3c and d) . in addition, immunohistochemical examinations revealed that pdcov antigens were present mainly in the villous enterocytes of the small intestine (figure 3f and g). the piglets in mock control group exhibited normal intestinal histopathology and no pedv antigens were present in the small intestines ( fig. 3e and h) . a total of twenty-five 45-day-old conventional weaned pigs were used to determine the pdd50 of the ch/xjyn/2016 strain. before inoculation, all pigs were healthy and had no viral rna shedding detected in fecal samples by real-time pcr. during the 7 dpi, obvious clinical symptoms, including watery diarrhea, vomiting, weight loss and depression, were observed in pigs from g1 and g2 (original and 10 -1 diluted p6 virus), whereas none of the pigs in g3-g5 developed diarrhea, and all of their rectal swab samples were negative for pdcov (table 1) . by 7 dpi, viral rna fecal shedding in rectal swab samples became positive in g1 and g2 pigs, with cycle threshold (ct) values ranging from 13.43 to 28.78 (table 1) . hence, based on the clinical observation and viral rna fecal shedding results, 100% (5/5) of pigs in g1 and g2 and no pigs (0/5) in g3, g4 and g5 (table 1) were infected. the cutoff time point was set as 7 dpi for determination of the pdd50, which was 2.0 log10pdd50/3 ml, equivalent to 100 tcid50. this result indicates that the ch/xjyn/2016 strain is pathogenic in conventional weaned pigs, which is very useful for further pig challenge studies. virus inactivation was verified by the absence of viral growth in llc-pk cell cultures, and no obvious cpes were observed in cell cultures within 96 h after inoculation (data not shown). these findings indicate that the virus was completely inactivated, and no live virus was present in the prepared vaccine. in addition, pig fecal rectal swabs collected 5 days postvaccination were negative for pdcov, further confirming that no infectious pdcov was present in the vaccine (data not shown). all pigs in the study were confirmed to be seronegative for pdcov antibodies at day 0 by an indirect elisa (data not shown). as shown in figure 4a , compared with the mock control, two experimental inactivated vaccines induced significant pdcov-specific serum igg responses in pigs (p < 0.05). the levels of igg antibodies in the two vaccine groups increased continuously after two booster immunizations at 14 and 28 dpv (figure 4a) , indicating that the two experimental inactivated vaccines could induce a strong pdcov -specific igg response in pigs. the vn antibody titers also increased continuously in the two vaccine groups from 7 dpv and were significantly higher than those in the mock control group (p < 0.05, figure 4b ). these results suggest that both of the experimental inactivated vaccines could induce a strong humoral immune response in pigs. all of the pigs in the three groups were apparently healthy and had no clinical symptoms before challenge. at 42 dpv, all pigs were challenged orally with 3 ml of 1000 pdd50 cell culture-adapted ch/xjyn/2016 p6. upon challenge, the clinical signs and viral shedding were monitored closely. as shown in table 2 , in the mock control group (n=3), serious diarrhea was observed, and viral shedding was detected in all 3 pigs after 2 dpi. in the two experimental inactivated vaccine groups, all of the pigs developed no obvious diarrhea, and no virus shedding was detected during the 7 dpi timeframe (table 2), indicating that the experimental vaccines provide potent protection against pdcov infection in pigs. in general, vaccination plays a significant role in the prevention and control of various emerging infectious diseases. recent outbreaks of pdcov in pig herds in multiple countries have caused significant economic losses for the pig industry (dong et al., 2016; janetanakit et al., 2016; jang et al., 2018; saeng-chuto et al., 2017; wang et al., 2014) . like those of pedv and tgev, pdcov infections cause similar severe clinical disease and lesions, but the lack of an effective preventive vaccine for pdcov has impeded the prevention and control of pdcov in pig farms. notably, reported data suggest that pdcov has existed in mainland china for at least 11 years (dong et al., 2015) and results in an increased mortality rate (more than 80%) among suckling piglets (song et al., 2015) . in addition, because pdcov is an emerging virus, the lack of a comprehensive understanding of the pathogenic characteristics of have pdcov also deterred the development of prevention and control methods (jung et al., 2016) . therefore, the infectious titer (pdd50) of pdcov in two-month-old conventional pigs and the protection efficacity of an inactivated vaccine based on the cell-adapted strain ch/xjyn/2016 were determined for the first time in the present study. this information will be helpful for future pig infection experiments and vaccine studies. most of the reported chinese strains were isolated from the eastern and central regions of china (dong et al., 2016; dong et al., 2015; wang et al., 2015; zhang et al., 2019c) . besides, pdcov isolate, chn-sc2015, was isolated from sichuan province in southwest china . the pdcov strain ch/xjyn/2016 in this study is a field strain that was isolated in early 2016 in northwest china. phylogenetic analyses based on the full-length s gene demonstrated that the ch/xjyn/2016 strain clusters with other pdcov strains isolated from china, the united states, japan and south korea. however, compared with those of the other reported chinese strains, the genetic distances of the ch/xjyn/2016 strain are closer to the original hong kong strain hku15-44 (jq065042) (woo et al., 2012) , sharing 98.74% nucleotide identity. the sequence information of pdcov strain ch/xjyn/2016 will contribute to the genetic and evolutionary characterization of pdcov in china. for the following several reasons, in this study, the ch/xjyn/2016 strain-p6 were used as challenge virus stocks. first, as we mentioned in results, the obvious cpes of the ch/xjyn/2016 strain were first observed at the fifth passage at 36 h postinoculation (hpi). therefore, in order to obtain enough virus stocks for next challenge study, the passage p6 was chosen. second, in general, virus stocks for challenge study needs the passage as low as possible. because the pathogenicity of the viruses will decrease with the passage numbers increases. besides, the cell culture-adapted ch/xjyn/2016-p30 was used for activated vaccine is because it keeps the greatest of the antigen concentrations and the lowest of passage at same time. consistent with previous reports, all pigs from the infection group (n=4) developed severe diarrhea with high levels of fecal viral rna shedding from 1-2 dpi, and the symptoms persisted until the piglets were euthanized (figure 3a) . notably, no pdcov-associated deaths were observed in this study. these results demonstrate that ch/xjyn/2016 is pathogenic to newborn piglets. compared to that in 4-day-old neonatal piglets, the virus incubation period, time to shedding of fecal viral rna and clinical disease onset (3-4 dpi) was increased in two-month-old pigs. these results confirmed that nursing pigs have increased susceptibility to pdcov infection, which is similar to the results of a previous study of pedv infection . inactivated vaccines do not contain any live components; therefore, they are safe, with a reduced risk of inducing disease and are widely used for the prevention and control of various diseases, especially viral disease. for example, pedv and tgev, for which many inactivated vaccines have been developed and have been widely used in many countries, such as china, korea, japan and the united states (gerdts and zakhartchouk, 2017; lee, 2015) . in china, inactivated vaccines have been routinely used for ped. pdcov infections, which have similar clinical symptoms as those of pedv and tgev, can also be prevented and controlled using inactivated vaccines. to provide a vaccine that could be used to control pdcov transmission, we developed an inactivated vaccine based on the cell-adapted strain ch/xjyn/2016. the inactivated pdcov experimental vaccines showed good safety and immunogenicity in pigs and significantly protected pigs against live virus challenge. in the immune protection experiment, compared with the mock control, both inactivated vaccines induced significant pdcov-specific humoral immune responses in pigs compared (p < 0.05). in addition, the levels of specific antibodies in the two vaccine groups increased continuously after two booster immunizations, indicating that a three immunization protocol is an effective immune procedure for pdcov inactivated vaccines. further animal challenge studies showed that the two inactivated vaccines both fully protected the pigs against 1000 pdd50 pdcov infection; the pigs showed no obvious diarrhea, and no viral shedding was detected during the 7 dpi follow-up period. these results suggest that the inactivated pdcov vaccine developed in this study could be used as a very promising vaccine candidate in the future. in conclusion, we developed a pdcov vaccine candidate that induces a potent humoral immune response in pigs and provides good protection against pdcov infection. this vaccine candidate can potentially be used for the prevention and control of pdcov in the future. in addition, this is the first 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respiratory tract of pigs and first discovery of coronavirus quasispecies in 5'-untranslated region discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, prevalence and genetic evolution biological characterization and pathogenicity of a newly isolated chinese highly virulent genotype giia porcine epidemic diarrhea virus strain genomic characterization and pathogenicity of porcine deltacoronavirus strain chn-hg-2017 from china detection and spike gene characterization in porcine deltacoronavirus in china during characterization and pathogenicity of the porcine deltacoronavirus isolated in southwest china key: cord-301301-ilsenpus authors: mihalov-kovács, eszter; martella, vito; lanave, gianvito; bodnar, livia; fehér, enikő; marton, szilvia; kemenesi, gábor; jakab, ferenc; bányai, krisztián title: genome analysis of canine astroviruses reveals genetic heterogeneity and suggests possible inter-species transmission date: 2017-03-15 journal: virus res doi: 10.1016/j.virusres.2016.12.005 sha: doc_id: 301301 cord_uid: ilsenpus canine astrovirus rna was detected in the stools of 17/63 (26.9%) samples, using either a broadly reactive consensus rt-pcr for astroviruses or random rt-pcr coupled with massive deep sequencing. the complete or nearly complete genome sequence of five canine astroviruses was reconstructed that allowed mapping the genome organization and to investigate the genetic diversity of these viruses. the genome was about 6.6 kb in length and contained three open reading frames (orfs) flanked by a 5′ utr, and a 3′ utr plus a poly-a tail. orf1a and orf1b overlapped by 43 nucleotides while the orf2 overlapped by 8 nucleotides with the 3′ end of orf1b. upon genome comparison, four strains (hun/2012/2, hun/2012/6, hun/2012/115, and hun/2012/135) were more related genetically to each other and to uk canine astroviruses (88–96% nt identity), whilst strain hun/2012/126 was more divergent (75–76% nt identity). in the orf1b and orf2, strains hun/2012/2, hun/2012/6, and hun/2012/135 were related genetically to other canine astroviruses identified formerly in europe and china, whereas strain hun/2012/126 was related genetically to a divergent canine astrovirus strain, ita/2010/zoid. for one canine astrovirus, hun/2012/8, only a 3.2 kb portion of the genome, at the 3′ end, could be determined. interestingly, this strain possessed unique genetic signatures (including a longer orf1b/orf2 overlap and a longer 3′utr) and it was divergent in both orf1b and orf2 from all other canine astroviruses, with the highest nucleotide sequence identity (68% and 63%, respectively) to a mink astrovirus, thus suggesting a possible event of interspecies transmission. the genetic heterogeneity of canine astroviruses may pose a challenge for the diagnostics and for future prophylaxis strategies. astroviruses (astv), family astroviridae, are non-enveloped viruses with a diameter of 28-30 nm and with a typical star-like shape. the genome is a single strand of positive sense, rna of 6.4-7.3 kb in size, containing three overlapping orfs (orf1a, orf1b and orf2) with a 3 poly(a) tail (mendez and arias, 2007) . orf1a encodes a serine 3c type of viral protease. orf1b is separated from orf1a by a heptameric frame shift signal (aaaaaac) and encodes the viral rna polymerase. orf2 encodes an 87-kda polypeptide, which functions as the capsid precursor. astv infection is associated with gastroenteritis in many animal species and humans, and they are also associated with extra-intestinal diseases, such as nephritis in chickens, hepatitis in ducks and shaking syndrome in minks (imada et al., 2000; fu et al., 2009; blomström et al., 2010) . the evolution of astvs is driven by mechanisms of genetic drift, recombination and, possibly, inter-species transmission (finkbeiner et al., 2008; de benedictis et al., 2011; martella et al., 2014) . so far, astvs are classified into two distinct genera, mamastrovirus (mastv) and avastrovirus (avastv) with 19 mastv (mammalian) species and 3 avastv (avian) species listed officially by the international committee on taxonomy of viruses (ictv) (bosch et al., 2010) . however, taking advantage of broad-range pcr primers for astvs and metagenomic protocols, several novel astvs have been identified in a number of mammalian and avian species (chu et al., 2008; finkbeiner et al., 2008; bosch et al., 2010; de benedictis et al., 2011) . using the classification criteria adopted http://dx.doi.org/10.1016/j.virusres.2016.12.005 0168-1702/© 2016 elsevier b.v. all rights reserved. in the 9th ictv report that is based on genetic analysis of the full-length orf2, an additional 14 mastv and 4 avastv candidate species have been defined recently (schultz-cherry, 2013) . astv-like particles have been detected only occasionally in dogs by em, either alone or in co-infection with other enteric viruses (williams, 1980; marshall et al., 1984; vieler and herbst, 1995; toffan et al., 2009 ). more recently, astvs have been identified in dogs with enteric signs and characterized molecularly, suggesting that the detected viruses may represent a distinct astv species (toffan et al., 2009) . also, a canine astv, strain ita/08/bari, was successfully adapted to replicate in vitro on mdck cells, and astv-specific antibodies were detected in convalescent canine sera (martella et al., 2011a (martella et al., ,b, 2012 . the prevalence of astv infection seems higher in dogs with enteric disease than in asymptomatic animals (martella et al., 2011a,b; zhu et al., 2011; caddy and goodfellow, 2015; takano et al., 2015) . also, monitoring of natural infection by astv in dogs has revealed that the acute phase of gastroenteritis overlaps with peaks of viral shedding (martella et al., 2012) . at present, limited and partial information on canine astv genomes is available in genbank. this limited amount of information seems to suggest that canine astvs are genetically heterogeneous (martella et al., 2011a,b; caddy and goodfellow, 2015) , thus posing a challenge for the diagnostic and for the understanding of the genetic and biological properties of these viruses in dogs. in this study, the complete or nearly complete genome sequence of five canine astvs and the partial genome sequence of one canine astv were determined and analysed, providing information on the genome organization and genetic diversity of these viruses. during 2012 samples were collected from diarrheic and nondiarrheic dogs from a hungarian shelter. there was no age restriction. a total of 63 samples obtained from 50 animals were tested for astv by using a pan-astrovirus specific primer set (chu et al., 2008) as described elsewhere (mihalov-kovács et al., 2014) and 37 (from 33 dogs) were randomly selected for viral metagenomics. fecal samples were diluted 1: 10 in pbs (phosphate buffered saline) and homogenized. the homogenates were centrifuged at 10000g for 5 min and the supernatant was collected for extraction of viral rna (zymo directzol viral rna extraction kit, zymo research). templates for deep sequencing were prepared as described previously (mihalov-kovács et al., 2015) . in brief, viral rna samples were denatured at 97 • c for 5 min in the presence of 10 m random hexamer tailed by a common pcr primer sequence (djikeng et al., 2008) . reverse transcription was performed with 1 u amv reverse transcriptase (promega, madison, wi, usa), 400 m dntp mixture, and 1× amv rt buffer at 42 • c for 45 min following a 5 min incubation at room temperature. then, 5 l cdna was added to 45 l pcr mixture to obtain a final volume of 50 l and a concentration of 500 m for the pcr primer, 200 m for dntp mixture, 1.5 mm for mgcl2, 1 × taq dna polymerase buffer, and 0.5 u for taq dna polymerase (thermo scientific, vilnius, lithuania). the reaction conditions consisted of an initial denaturation step at 95 • c for 3 min, followed by 40 cycles of amplification (95 • c for 30 s, 48 • c for 30 s, 72 • c for 2 min) and terminated at 72 • c for 8 min. we subjected 0.1 g of random pcr product to enzymatic fragmentation and adaptor ligation (nebnext fast dna fragmentation & library prep set for ion torrent kit, new england biolabs, ipswich, ma, usa). the barcoded adaptors were retrieved from the ion xpress barcode adapters (life technologies, carlsbad, ca, usa). the resulting cdna libraries were measured on an qubit 2.0 device using the qubit dsdna br assay kit (invitrogen, eugene, or, usa). the emulsion pcr that produced clonally amplified libraries was carried out according to the manufacturer's protocol using the ion pgm template kit on an onetouch v2 instrument. enrichment of the templated beads (on an ion one touch es machine) and further steps of presequencing setup were performed according to the page 3 of 11 200-bp protocol of the manufacturer. the sequencing protocol recommended for ion pgm sequencing kit on an 316 chip was strictly followed. to determine the actual 5 end genomic sequence of two astv strains, a 5 race protocol was used, following the manufacturer's instructions (invitrogen ltd). the amplicons were visualized on 1.5% agarose gel, and cleaned up with qiagen qiaquick gel extraction kit, according to the manufacturer's instructions. the amplicons were cloned using topo xl pcr cloning (invitrogen ltd) and the clones were subjected to sequencing in both directions using big dye v3.1 chemistry on a 3730xl instrument from applied biosystems (foster, ca). for sequencing accuracy, a minimum of three independent clones for each fragment type were selected for sequencing in both directions using the universal m13f/r primers. the first strand iii kit (invitrogen ltd) was used to generate cdna from the poly-a tailed rna target, as described previously (martella et al., 2011a,b) . forward primers were designed about 1 kb upstream of the 3 terminus of the genome of the various astv strains and used with a reverse poly-t oligonucleotide with a 5anchored tail. the 3 race products were sequenced on ion torrent pgm. sequence data generated by the ion torrent pgm were trimmed and analysed by the clc bio software (www.clcbio.com). the same software package was utilized to map sequence reads to reference eucaryotic viral sequences retrieved from genbank. sequence editing, annotation and analysis were carried out using geneious software v9.1.4 (biomatters ltd, new zealand). open reading frames (orf) were predicted with the orf finder. multiple alignments were prepared and manually adjusted, whereas phylogenetic analysis was performed by using the mega6 software (tamura et al., 2013) with a neighbor-joining method, jukes cantor genetic distance model and bootstrapping over 1000 replicates. mean amino acid genetic distances were computed using the p-distance method of mega6. the protein sequence analysis and classification were prepared with the aid of embl-ebi website (http://www.ebi.ac.uk/interpro/). transmembrane domains were determined using phobius web server (käll et al., 2007) . nuclear localization signals (nls) were predicted by http://www.moseslab. csb.utoronto.ca/nlstradamus/. astv genomic sequences were deposited in the gen-bank with the following accession numbers: hun during 2012, 63 stool specimens were screened for astvs using broad-range astv-specific primer set. of these, seven tested positive; 4 out of 25 diarrheic animals (16%) and 3 out of 38 non-diarrheic animals (8%). the difference between groups was not statistically significant (chi-square value, 1.003; p = 0.316). a subset (n = 37) of samples was selected from 33 animals for viral metagenomics to detect virus diversity in canine fecal specimens. among these 37 samples 26 specimens contained eukaryotic origin viral sequences (fig. 1) . by viral metagenomics (table 1) , astv rna was initially identified in 11 out of 37 fecal specimens (29.7%). however, the proportion of astv specific reads ranged from 1 out of 18711 ( < 0.1%) to 1627 out of 1635 (99.5%) eukaryotic viral sequence reads, respectively, and five samples that had very low astv sequence read numbers (range, 1-4) were not considered for further processing. other enteric viruses detected in the stools included parvovirus (in 20 samples), coronavirus (in 14 samples), rotavirus (in 11 samples), picornavirus (in 7 samples), gyrovirus (in 1 sample), adenovirus (in 1 sample) and calicivirus (in 1 sample). overall, by either the pan-astrovirus rt-pcr or by the viral metagenomic screening, we identified astv rna in 17/63 (26.9%) samples. interestingly, some samples negative in rt-pcr generated astv-specific reads by deep sequencing, whilst, in turn, some samples containing astv-specific reads tested negative by the panastrovirus rt-pcr, indicating a weak correlation between the two methodologies (table 1) . for further analysis the complete, coding complete and partial genome sequence was determined for four, one and one astv strains, respectively. in general, the length of the coding sequences ranged between 6441 and 6474 nucleotide (nt), with a gc content of 45.19-45.48%. the total genome length, considering the 5 and 3 utrs, ranged between 6535 and 6587 nt. the 5 utr was sequenced completely for strains hun/2012/2, hun/2012/115, hun/2012/126 and hun/2012/135, and ranged between 42 and 60 nt in length. the 3 utr was sequenced completely for all the six strains and ranged between 49 and 100 nt in length. the genome of strain hun/2012/115 was 6569 nt long, excluding the polya-tail. the 5 utr and 3 utr were, respectively, 45 nt and 83 nt long. the genome of strain hun/2012/135 was 6587 nt long and it was organized similarly to strain hun/2012/115, even if the 3 utr was 2 nt longer. the genome of strain hun/2012/2 was 6576 nt long and it differed for the longer 5 utr region (60 nt) from the other canine astvs. despite several attempts, for strain hun/2012/6, it was not possible to generate the actual 5 utr sequence. the genome of this strain was organized in an identical manner to the canine astv strain gbr/2014/gillingham. the genome length of the astv strain hun/2012/126 was 6535 nt long, excluding the polya-tail. the 5 utr was 42 nt in length, with an additional in-frame atg codon located 18 nt upstream (bases 25-27) of the atg codon of orf1. the 3 utr was 49 nt long. only the partial genome (3187 nt in length) of strain hun/2012/8 was determined. the virus possessed an early stop codon in the orf2, resulting in a longer (100 nt) 3 utr ( table 2) . the coding sequences showed the typical organization of astv genome. there were three overlapping orfs, coding the non-structural and structural proteins. the predicted genome organization of the viruses is shown in fig. 2 and summarized in table 2 . the complete orf1a was 2670 nt long for all canine astvs, except strain hun/2012/126, and codes for a 889 aa long polyprotein. for strain hun/2012/126, an in-frame start codon is located 18 nt upstream of the orf1 start codon (the length of the orf is 2667/2685 depending on the start). a ribosomal frameshift signal, the heptameric (slippery) aaaaaac sequence, was recognized at nt 2622-2628 (2619/2637 in hun/2012/126 depending on the start used), with the orf1b initiating after the slippery sequence. the predicted 95 kda nsp1a protein, encoded by orf1a, contained conserved domains typical for the astv serine protease between aa 439 and 583 aa. five transmembrane domains were identified on the predicted orf1a protein at residues aa151-168, aa240-266, aa287-313, aa319-339 and aa351-380. a possible coiled coil structure was also found at residues aa595-616 and aa635-656 (fig. 2) . upon sequence comparison, strains hun/2012/2, hun/2012/6, hun/2012/115 and hun/2012/135 displayed 96-99% nt identity to each other and 93-98% nt to the canine strains uk/2014/lincoln and uk/2014/gillingham, whilst strain hun/2012/126 displayed only 74% nt identity to the other canine astvs. identity to non-canine astvs was markedly lower (33-51% nt) (table 3) . the complete orf1b was 1530 nt long in all canine astvs, except the partially sequenced strain, hun/2012/8, where a 770 nt long portion was determined. the orf1b begins with the last nucleotide of the slippery heptamer and with a −1 frameshift with respect to orf1a. also, the downstream stem loop structure was highly conserved among canine astvs (caddy and goodfellow, for the orf1a and orf2, the orf length is calculated considering the presence of an additional start codon. this value is also calculated for the orf1b/orf2 overlap. a partial sequence. b an additonal atg codon is present 18 nt upstream (bases 25-27) of the orf1a initiation codon. c based on the additional orf1a start codon, the orf1a-predicted polyprotein could be 6 aa longer; -not available. 85-86% nt (87-88% aa) to the other canine astvs. strain hun/2012/8 displayed the highest identity to a mink astv (68% nt and 70% aa), whilst identity to other canine astvs was lower (56-58% nt and 50-52% aa) ( table 3) . the length of orf2 is variable among the canine astv strains. in all but one strains analysed in this study, the orf1b and orf2 overlapped by 8 nt. in strain hun/2012/8 the overlapping was 11 nt in length. in addition, there was a second in-frame atg start codon, located 180 nt upstream of the start codon homologous to other mamastrovirus genomes. only in strain hun/2012/8, this additional in-frame start codon was located 141 nt upstream of the first start codon. during the rna replication, orf2 is expressed from a sgrna (walter and mitchell, 2003; mendez et al., 2014) . the sgrna promoter sequence has a highly conserved nucleotide sequence motif and is present upstream of the orf2 start codon (mendez et al., 2014) . the highly conserved nucleotide stretch upstream of orf2, atatggaggggaggaccaaaaaattgcgatggc, believed to be part of a promoter region for synthesis of sgrna, was completely conserved in the sequence of canine astv strains except for the sequence of strain hun/2012/8, that was ctttggaggggaggaccaaagcagcgctatggc. the orf2 was 2292/2472 nt long for strain hun/2012/115 and hun/2012/135, 2295/2475 nt long for strain hun/2012/2, 2325/2505 nt long for strain hun/2012/6, 2298/2478 nt for strain hun/2012/126 and 2328/2469 nt for hun/2012/8 ( table 2 ). the terminal nt stretch of orf2 and the adjacent 3 utr (s2m) is highly conserved among most astvs and similarities have been observed in the sequence and folding of the 3 utr of viruses from other viral families, suggesting that it is relevant for astv genome replication (monceyron et al., 1997) . the conserved s2 m sequence ccgcggccacgccgagtaggaacgagggtacag overlaps the 6 aa c terminus of the capsid (srghae), that is highly conserved in several mammalian astvs, including the canine astvs (fig. 2) . however, in strain hun/2012/8 the highly conserved s2m stretch was found downstream of the orf2 termination codon, in the 3 utr. as a consequence, the 6 aa c terminus of strain hun/2012/8 was tlstkh. upon sequence comparison, the hungarian canine astvs were found to differ markedly. strains hun/2012/2 and hun/2012/135 were highly related to each other (92% nt and 96% aa identity) and to chinese castvs (91-96% nt and 95-99% aa identity) as well as to the uk strain gbr/2014/lincoln (91-95% nt and 95-99% aa). strain hun/2012/6 displayed the highest identity (91-95% nt and 96-98% aa) to three italian strains (ita/2005 -3, ita/2005 -6, ita/2005 and to the uk strain gbr/2014/gillingham. strain hun/2012/126 displayed the highest identity (97% nt and 98% aa) to the italian strain ita/2010/zoid. strain hun/2012/115 displayed the highest identity (79% nt and 79% aa) to the italian strain ita/2010/zoid, while sequence identity to other canine astvs ranged from 72 to 75% at the nt level and 75-80% at the aa level. interestingly, strain hun/2012/8 displayed the highest identity (63% nt and 64% aa) to a mink astv (table 3) . phylogenetic trees were constructed using the nucleotide alignments of orf1a, orf1b and orf2 (fig. 3) . the orf1a and orf2 alignments were based on the full-length orf sequences and also included a selection of sequences of mammalian astv strains. the orf1b alignment included 415 nt at the 3 end (138 aa at the c terminus of the protein). in all phylogenetic trees, the majority of canine astvs formed a distinct monophyletic group. in the orf1a-based tree (fig. 3a) , only five canine astv strains generated in this study and two astv strains from uk could be included, as information on the orf1a is not available for other canine astvs. although monophyletic, the canine astvs segregated in two distinct branches, with strain hun/2012/126 in the basal position. when enlarging the number of analysed sequences, in the orf1b (fig. 3b) , this pattern of segregation was confirmed. four strains (hun/2012 /2, hun/2012 /6, hun/2012 /115, and hun/2012 were grouped with the majority of canine astvs identified in european and extra-european countries, whilst strain hun/2012/126 clustered with the highly virulent strain ita/2010/zoid. interestingly, strain hun/2012/8 was distantly related to all other canine astvs and it was grouped with astvs identified in minks and in californian sea lions. the unique evolutionary relationship of this canine strain was confirmed in the orf2 (capsid)-based tree (fig. 3c) . this gene is considered as the fundamental target for classification and characterization of astvs. when observing in detail the large monophyletic group of canine astvs which classified into species 5 (schultz-cherry, 2013), it was possible to identify clearly sub-clustering patterns, with at least 6 sub-clusters. all chi-nese strains, the uk strain gbr/2014/lincoln and the hungarian strains hun/2012/2 and hun/2012/135 formed a unique group, with more than 91% nt identity in the full-length orf2. a second group included three italian strains (ita/2005 -3, ita/2005 -6, and ita/2005 , the uk strain gbr/2014/gillingham and strain hun/2012/6, with more than 91% nt identity to each other. a third group encompassed the virulent strain ita/2010/zoid and strain hun/2012/126 (97% nt identity to each other). other canine strains (gbr/2014 /braintree, gbr/2014 /huntingdon, hun/2012 were intermingled between these three main sequence groups and, as supported by sequence comparison (table 3) , they clearly appeared as distinct genetic sub-lineages. astvs have been reported repeatedly in dogs across the world, although the association with the disease, prevalence rates and information on the genetic diversity are still largely unknown. in this study, we could neither confirm nor refute an association between astv infection and diarrhea in dogs. furthermore, we observed discrepancies between results obtained by the panastrovirus pcr assay and those derived from viral metagenomics. however, none of these methods were developed for routine diagnostic procedures and we do not have information about the actual sensitivity of these assays. at present it seems more appropriate to consider these methods, originally developed to detect viral diversity, as independent approaches that help understand the viral community and diversity in clinical/pathological specimens. thus, our prevalence data for astv must be interpreted with caution, even if the detection rates of astvs fell in the ranges reported in the literature. for example, canine astv rna has been detected in 6-27% of dogs with enteritis and 0-19% of aysmptomatic dogs in studies in italy ( (takano et al., 2015) , france (grellet et al., 2012) and uk (caddy and goodfellow, 2015) . even if those studies differed in the number, provenience and age of the animals enrolled in the investigations, in most studies the detection rates were found to differ significantly between symptomatic and asymptomatic dogs, suggesting a possible role of astvs as enteric pathogens of dogs. also, the onset and evolution of the clinical signs have been found to correlate with the patterns of virus shedding and with seroconversion in two dogs naturally infected by canine astvs (martella et al., 2012) . yet, experimental studies will be needed to demonstrate firmly the association of canine astvs with gastroenteritis. sequencing of the orf1a, orf1b and orf2 regions revealed significant sequence variation in the castvs detected in this study. five strains were grouped with other canine astvs identified worldwide, but further separated into multiple capsid (orf2) sub-clusters. by parallelism with what is observed in human astvs (mastv species 1), that are classified antigenically in 8 serotypes, it is possible to speculate that the genetic heterogeneity observed in canine astvs might account for a significant antigenic diversity (caddy and goodfellow, 2015) . weak antigenic cross-reactivity has been observed in immune electron microscopy between strain ita/2008/bari and ita/2010/zoid virus particles and the sera of convalescent dogs (martella et al., 2012) . however, isolation in vitro of these viruses is fastidious and only one strain, ita/208/bari, has been adapted to grow in vitro thus far (martella et al., 2011a,b) , thus hampering a precise evaluation of the antigenic relationships among different canine astvs. interestingly, one hungarian strain, hun/2012/126, resembled a highly virulent canine astv, ita/2010/zoid, associated with enteric disease in an adult and young dog in italy (martella et al., 2012) . the question whether some canine astv strains may differ in their biological properties in terms of fitness or virulence remains open. even more interestingly, one of the canine astvs identified in hungary, hun/2012/8, was found to be genetically distantly related from all other canine astvs identified worldwide. although we were successful to determine only the partial (3.2 kb) 3'end of the genome, the virus displayed unique genetic signatures, such as a longer orf1b/orf2 overlap (11 nt), and a longer 3 utr. also, this strain markedly varied from all the other canine astvs in the highly conserved promoter region for synthesis of subgenomic rna, located upstream of the orf2 (walter and mitchell, 2003; mendez et al., 2014) . conversely, all other canine astvs analysed in this study displayed a conserved genomic architecture, which was shared with all currently canine astvs for whom the complete or partial (orf1b and orf2) genome sequence is available (martella et al., 2011a (martella et al., ,b, 2012 takano et al., 2015) . in addition, upon sequence comparison and phylogenetic analysis, strain hun/2012/8 differed markedly from other canine astvs concerning the partial orf1b and the full-length orf2 (table 3) . according to the 9th ictv report (http://talk.ictvonline.org/files/ictv official taxonomy updates since the 8th report/m/vertebrate-official/ 4178), for mastvs the mean aa genetic distances (p-dist) range from 0.378 to 0.750%, and from 0.006 to 0.312%, respectively, between and within groups (species). using our set of orf2 sequences, the p-dist of strain hun/2012/8 was calculated to range between 0.350 and 0.772. accordingly, this strain appears distantly related from any other known mammalian astv and may represent a novel astv species. it remains to be established whether strain hun/2012/8 is actually a canine virus, or it is the result of inter-species transmission from an unidentified animal source. although astvs are regarded as viruses with a robust host-species restriction, several pieces of evidences in mammals (including humans) indicate that the host species barrier may be permeable in some occasions, and that heterologous viruses may stably adapt to the new host (finkbeiner et al., 2008 (finkbeiner et al., , 2009 nagai et al., 2015) . moreover, occasional transmission of astvs is thought to have occurred between avian and mammalian species in both directions (sun et al., 2014; pankovics et al., 2015) . in summary, data about canine astvs is currently scarce. nonetheless, the genetic heterogeneity among circulating astv strains is evident representing some challenge for laboratory diagnosis. this diversity may also be challenging for future prophylaxis strategies, complicating the design of future vaccines, as it is currently unknown whether antigenic cross-reaction exists among and cross-protection is elicited by different canine astv strains. further efforts are needed to better understand the biology of canine astvs and large-scale structured surveillance programs should be initiated to elucidate the relative veterinary importance of various canine astv types. the authors declare no conflicts of interest. the study was supported by the momentum program awarded by the hungarian academy of sciences. s.m. was a recipient of the jános bolyai fellowship (awarded by the hungarian academy of sciences). f.j. was supported by támop (4.2.4.a/2-11-1-2012-0001) and únkp-16-4-iii, new excellence program of the ministry of human capacities. g.k. received funding from únkp-16-3-iii, new excellence program of the ministry of human capacities. the funders had no role in study design, data collection, analysis and interpretation, or the decision to submit the work for publication. detection of a novel astrovirus in brain tissue of mink suffering from shaking mink syndrome by use of viral metagenomics nineteen new species in the genus mamastrovirus in the astroviridae family complete genome sequence of canine astrovirus with molecular and epidemiological characterisation of uk strains novel astroviruses in insectivorous bats astrovirus infections in humans and animals-molecular biology, genetic diversity, and interspecies transmissions viral genome sequencing by random priming methods 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human astrovirus cell entry enteric viral infections of sheltered dogs in hungary candidate new rotavirus species in sheltered dogs molecular characterisation of the 3'-end of the astrovirus genome full genome analysis of bovine astrovirus from fecal samples of cattle in japan: identification of possible interspecies transmission of bovine astrovirus detection of a mammalian-like astrovirus in bird, european roller (coracias garrulus) astrovirus research. essential ideas, everyday impacts, future directions detection and characterization of avastrovirus associated with diarrhea isolated from minks in china detection of canine astrovirus in dogs with diarrhea in japan mega6: molecular evolutionary genetics analysis version 6.0 genetic characterization of a new astrovirus detected in dogs suffering from diarrhoea electron microscopic demonstration of viruses in feces of dogs with diarrhea astrovirus infection in children astrovirus-like, coronavirus-like, and parvovirus-like particles detected in the diarrheal stools of beagle pups isolation and characterization of canine astrovirus in china key: cord-329183-s0zrvn9o authors: graham, robert i.; rao, shujing; sait, steven m.; attoui, houssam; mertens, peter p.c.; hails, rosemary s.; possee, robert d. title: sequence analysis of a reovirus isolated from the winter moth operophtera brumata (lepidoptera: geometridae) and its parasitoid wasp phobocampe tempestiva (hymenoptera: ichneumonidae)() date: 2008-04-10 journal: virus res doi: 10.1016/j.virusres.2008.02.005 sha: doc_id: 329183 cord_uid: s0zrvn9o a reovirus was isolated from operophtera brumata (obrv) and its parasitoid wasp phobocampe tempestiva. each of the 10 dsrna genome segments of obrv was sequenced and shown to contain a single open reading frame (orf). conserved motifs ([+ve] 5′-aaataaa … (g)/(t)aggtt-3′) were found at the termini of each segment, with the exception of seg-6 and seg-8, where the 5′ termini were 5′-aacaaa…-3′. the putative proteins encoded by each segment were compared with those of other members of the family reoviridae. phylogenetic comparisons to published sequences for the rna-dependent rna polymerase genes from other reoviruses indicated that obrv is most closely related to members of the genus cypovirus. however, unlike the cypoviruses, obrv has a double-layered capsid structure. when the protein encoded by obrv seg-10 was expressed (by inserting the open reading frame into a baculovirus expression vector) no ‘occlusion bodies’ were observed in the recombinant baculovirus infected insect cell cultures. this suggests that unlike the cypoviruses, seg-10 of obrv does not contain a polyhedrin gene. further phylogenetic comparisons also identified relationships between seg-2 and seg-10 of obrv, and genes of diadromus pulchellus idnoreovirus 1 (dpirv1), suggesting that obrv represents a new species from the genus idnoreovirus. numerous dna and rna viral pathogens can infect insect species, including baculoviruses (cory and myers, 2003; fuxa, 2004) , entomopoxviruses (arif and kurstak, 1991) , ascoviruses , polydnaviruses (kroemer and webb, 2004) and reoviruses (mertens et al., 2005a) . the reoviridae is a large family containing 15 recognised genera of double-stranded rna (dsrna) viruses. these include several insect transmitted viruses that cause important diseases of animals or plants, which are classified within the genera orbivirus, seadornavirus and coltivirus, or fijivirus, oryzavirus and phytoreovirus, respectively (mertens et al., ଝ the nucleotide sequence data reported in this paper have been submitted to genbank and have been assigned the accession numbers dq192235-dq192244. * corresponding author. 2005a). however, many of the reoviruses can only infect invertebrates and are classified within the genera cypovirus (green et al., 2007; mertens et al., 2005b; shapiro et al., 2005) , idnoreovirus (mertens et al., 2005c) or dinovernavirus (attoui et al., 2005) . at least 14 other reoviruses that infect insects, crustaceans or arachnids also remain unclassified (renault et al., 2005) , reflecting differences from the recognised genera in their morphology and genome features. five reoviruses have previously been isolated from species of hymenoptera, which appear to be non-pathogenic in their wasp hosts (renault et al., 2005) . three of these, microplitis croceipes reovirus-like particles (mcrvlp) (hamm et al., 1994) , hyposoter exiguae idnoreovirus (heirv) (mertens et al., 2005c; stoltz and makkay, 2000) , and opius concolor reovirus-like particles (oprvlp) (jacas et al., 1997) , use their wasp hosts as vectors, and do not appear to be involved in lepidopteran host defence suppression (renault et al., 2005) . all three of these viruses were found in association with other viruses which facilitate the pathogenicity of the parasitoid against the wasp's lepidopteran host. for example, stoltz and makkay (2000) observed heirv and a dna polydnavirus, h. exiguae polydnavirus (hepdv) replicating within the same cells of the parasitoid h. exiguae. a fourth virus, diadromus pulchellus idnoreovirus-1 (dpirv-1), has a commensal relationship with its host d. pulchellus, and is detected in most populations of this wasp species. dpirv-1 is always associated with d. pulchellus ascovirus-4 (dpav-4), which is indispensable for suppressing the immune system of the parasitoid's host. although dprv-1 has not been shown to directly increase the parasitic success of d. pulchellus, it may retard the replication of dpav-4 within the lepidopteran host cells. this prevents rapid cell lysis, thereby enabling normal development of the wasp larva (renault et al., 2005) . the fifth virus, d. pulchellus idnoreovirus-2 (dpirv-2; renault et al., 2003) , has a mutualistic relationship with d. pulchellus. its injection by parasitoid females during oviposition is essential for suppressing the immune system of the parasitised lepidopteran host (renault et al., 2005) . the winter moth, operophtera brumata l., is a polyphagous univoltine pest. out-break populations of o. brumata larvae have recently been observed on heather moorland on orkney, in northern scotland (graham et al., 2004 (graham et al., , 2006 kerslake et al., 1996) . three reoviruses were isolated from populations of winter moth on orkney, which were identified on the basis of their dsrna genome profile after gel electrophoresis (graham et al., 2006) . sequence analysis confirmed two of these viruses as belonging to the genus cypovirus (graham et al., 2007) . however, a third virus, that was initially identified as o. brumata reovirus (obrv), was isolated from both o. brumata and an ichneumonid parasitoid wasp phobocampe tempestiva, that is known to attack the winter moth. obrv is a non-occluded reovirus which has double-shelled virions ∼55 nm in diameter. virus isolated from o. brumata and p. tempestiva contained a genome composed of 10 segments of doublestranded rna, with a total size of approximately 25.3 kb (graham et al., 2006) . we describe the complete sequence analysis of genome segments 1-10 (seg-1 to seg-10) of obrv. for further characterisation and a comparison with known cypoviruses, the protein encoded by the open reading frame (orf) of seg-10, was expressed in insect cells using a recombinant-baculovirus vector-system. o. brumata larvae were collected on calluna vulgaris heather at 15 sites on the orkney isles (graham et al., 2004) . p. tempestiva were collected as they emerged from parasitised o. brumata larvae. purification of non-occluded virus particles was achieved using a method modified from stoltz and makkay (2000) . approximately 1 g of pooled insect tissue was homogenised manually in 1 ml of sterile te buffer (10 mm tris, 0.1 mm edta, ph 8.0). insect debris was removed by centrifugation at 500 × g for 10 min. the supernatant was loaded onto a two-step sucrose density gradient (50% sucrose [w/w] and 10% sucrose [w/w]) and centrifuged at 60,000 × g for 90 min (sw41 rotor, beckman). the virus band was collected at the density interface and dialysed in te buffer for 4 h. the sample was further pelleted at 15,000 × g for 5 min to remove cell debris. the supernatant was collected and centrifuged at 60,000 × g for 1 h. this virus pellet was resuspended in 200 l te buffer overnight at 4 • c and stored at −20 • c. nucleic acid was purified using the dneasy tissue kit (qiagen). dsrna was finally eluted in 150 l of te into sterile 1.5 ml microtubes, and stored at −20 • c. the full-length genome of o. brumata reovirus was cloned using the flac method described previously (maan et al., 2007; shapiro et al., 2005) . briefly, an anchor-primer 5 p-gac ctc tga gga ttc taa ac/isp9/tcc agt tta gaa tcc-oh 3 , (isp9 is a c9 spacer) was ligated to purified viral genome dsrna using t4 rna ligase (promega). the reaction was incubated at 8 • c overnight, heat-denatured and added to a reverse transcription (rt) reaction (amv reverse transcriptase, promega; 37 • c for 50 min). a pcr was subsequently performed, with reactions (100 l) containing the following components: 1 l (5 u) enzyme (extaq, takara), 4 l dntps (300 mm), 10 l 10× buffer (25 mm taps [ph 9.3], 50 mm kcl, 2 mm mgcl 2 , 1 mm dtt), 3 mm primer (5 -ggg atc cag ttt aga atc ctc aga ggt c-3 ), and 1 l of cdna (an initial 20 l volume of cdna was added to 200 l water before 1 l was used in pcr). conditions were as follows: (i) 94 • c for 5 min, 1 cycle; (ii) 94 • c for 20 s, 62 • c for 20 s, 68 • c for 4.5 min, 26 cycles; (iii) 70 • c for 10 min, 1 cycle. the product was examined using 1% agarose gel electrophoresis, bands excised, ligated into pgem-t easy vector (promega) and transfected into electrocompetent dh10b cells (invitrogen). selected clones were sequenced with m13 primers using a 3730 dna analyzer (abi). initial sequence data were used to design primers for subsequent direct sequencing of the viral segments. while several segments were sequenced from multiple clones, most were done using both clone and pcr dna as the sequencing templates. sequence data was assembled and analysed using pregap4 (staden), gap4 (staden), and bioedit (hall, 1999) . phylogenetic analysis was carried out using clustalw (thompson et al., 1994) and mega3.1 (kumar et al., 2001) packages. nucleotide and deduced amino acid sequences were compared with data available in genbank using the ncbi's online blast program (http://www.ncbi.nlm.nih.gov/blast) or a local blast program implemented in the dnatools package (version 5.2.018, s.w. rasmussen: valby data center, denmark) using a local database constructed from all available sequences of the reoviridae. hydrophobicity of segment 10 was analysed as described by kyte and doolittle (1982) using bioedit. protein motifs were examined using prosite (hulo et al., 2004) . oligonucleotides were designed to amplify a product comprising the complete orf of genome segment 10 for obrv (obrvseg10forf, 5 cct ggc cga att gtg agt a3 and obrvseg10rorf, 5 ggc tcc att tcg cat aca tc3 ). the subsequent product was purified (pcr purification kit, qiagen), ligated into pgem-t vector and used to transform e. coli (dh5␣) (invitrogen) via heat shock. m13 forward and reverse primers were used to read across the insert to ensure the correct sequence was present. the cloned orf was ligated into the transfer vector pbacpak8 (clontech), using the compatible restriction sites saci and bglii to derive pbacpak8.rvs10. cloned orf inserts were examined for the correct sequence using primers bac1 (5 acc atc tcg caa ata aat aag3 ) and bac2 (5 aca acg cac aga atc tag cg3 ). spodoptera frugiperda-21 cells (sf-21) were inoculated with a sample containing 500 ng of pbacpak8.rvs10 dna, 100 ng of flashbac tm dna (oxford expression technologies) and an equal volume of lipofectin (king and possee, 1992) . cells were incubated at 28 • c. the virus stock was amplified in secondary cultures of the same cells and the infectivity determined by plaque assay (king and possee, 1992) . cell cultures (10 6 cells per 35 mm dish) were inoculated with virus (10 pfu per cell), incubated at 28 • c and photographed at intervals. at 72 h p.i., the cells were harvested and proteins were analysed by sds-page, running samples for 16 h in 12% polyacrylamide gels. following electrophoresis, proteins were stained with coomassie brilliant blue and photographed. the full sequences of obrv seg-1 to seg-10 were determined. the complete genome is 25,194 bp long and the corresponding sequences have been deposited in the genbank database (see table 1 for accession numbers). each genome segment contains a single orf, starting with the codon 'aug'. analysis of the noncoding regions (ncrs) showed that all of the segments share seven conserved nucleotides at the 5 ends and five conserved nucleotides at the 3 ends, except for s6 and s8 (table 1) . seg-1 of obrv is 4170 nt in length (table 1) , with a single large orf between nt 33 and 4109 (stop codon tga), coding for a predicted protein of 1358 aa (155.7 kda), which is identified as vp1. blastp searches showed vp1 to be homologous to the rna-dependant rna polymerases (rdrp) from lymantria dispar cpv-14 (21% identity, e-value = 1 × 10 −23 ); fiji disease virus (24% identity, e-value = 9 × 10 −19 ); mal de rio cuarto virus (24% identity, e-value = 1 × 10 −19 ); bombyx mori cpv-1 (20% identity, e-value = 2 × 10 −15 ); cryphonectria parasitica reovirus (23% identity, e-value = 2 × 10 −14 ); o. brumata cpv-19 (21% identity, e-value 2 × 10 −14 ); and colorado tick fever virus (27% identity, e-value = 1 × 10 −13 ); indicating that vp1 is the rdrp of obrv. a leucine-zipper motif was found between aa 1310 and 1331, which has been suggested as a dna-binding structure (landschulz et al., 1988) . seg 2 of obrv is 3780 nt in length (table 1) , with an orf between 78 and 3701 nt (stop codon tga) coding for a predicted protein of 1207 aa (137 kda) that is identified as vp2. the use of local blastp identified a cognate protein from dpirv-1 (encoded by the 4.230 kb genome segment: accession number x82049), with 20% amino acid identity (42% aa similarity) over the full-length of the protein. seg-3 of obrv is 3595 nt long, with a single orf between nt 16 and 3501 (stop codon taa: table 1 ), coding for vp3 which is 1161 aa long (133 kda). tentative homology was detected, between aa 582 and 766, with fijivirus segment 4 (27% identity, e-value = 0.007). the use of the local blast program also identified aa identities to partial sequences of the proteins encoded by seg-3 of rice dwarf virus (rdv-genus phytoreovirus) (29% identity, 55% similarity) 55% similarity: amino acids 1-70 of rdv p3), and african horse sickness virus (ahsv-genus orbivirus) (31% identity, 47% similarity: amino acids 149-220 of ahsv vp3). both of these proteins form the subcore shell (t2 layer, made of 120 copies) of their respective virus particles. the protein encoded by seg-3 of obrv may therefore also form the sub-core layer of the virus. further analysis predicted the existence of three leucine zipper motifs at aa 446-467, 878-899 and 885-906. seg-4 of obrv is 3362 nt long, with a single large orf between nt 15 and 3290 (table 1 : stop codon tag). the encoded protein (vp4) has a predicted size of 1091 aa (122.7 kda). a local blast search showed 32% aa identity (46% similarity) to a partial sequence of rdv p2 (encoded by seg-2) (aa 879-980 of rdv: compared to aa 809-900 of obrv). the p2 of rdv is an outer coat protein that is essential for vector transmission. seg-5 of obrv consists of 2106 nt (table 1) , with a single orf between nt 31 and 1893 (stop codon taa) coding for a vp5 protein that is 620 aa long (69.2 kda). no homology was found with other virus sequences. the only tentative homology detected was between aa 85 and 218, with 'copper amine oxidase' of the common garden pea pisum sativum (29% identity, e-value = 3.5). seg-6 of obrv consists of 1935 nt (table 1) , with a single orf between nt 63 and 1847 (stop codon tag), coding for a vp6 protein that consist of 594 aa (68.4 kda). partial sequences of vp6 matched with vp6 of rice ragged stunt virus (rrsv-genus oryzavirus) (aa 357-435 of rrsv, compared to aa 348-417 of obrv: aa identity 26%, aa similarity 47%). amino acid identity of 41% (e-value = 5.6) was also detected with aa 379 and 417 of the protein encoded by seg-9 of oat sterile dwarf virus (genus fijivirus). seg-7 of obrv is 1606 nt long (table 1) , with a single long orf between nt 18 and 1517 (stop codon taa), which codes for the vp7 protein, with a predicted length of 499 aa (57.1 kda). blastp searches showed no homology with the proteins of any virus previously sequenced. tentative homology was seen between aa 339 and 442 with trypanosoma brucei hypothetical protein tb106k154000 (32% identity, e-value = 2.1). seg-8 of obrv is 1584 nt long, with a single orf between nt 99 and 1502 (table 1) coding for a predicted vp8 protein that is 467 aa long (51.6 kda). tentative homology was observed between 52 and 119 aa with the apicomplexan theileria parva abc transporter protein (31% identity, e-value = 1.4). seg-9 of obrv is 1547 nt long, with a single large orf between nt 77 and 1390 (table 1) , coding for a predicted vp9 protein that is 437 aa long (49.6 kda). a local blast search identified a partial match with aa 85-150 of vp6 of colorado tick fever virus, ctfv (29% identity, 49% similarity). seg-10 of obrv is 1509 nt long, with a single large orf between nt 22 and 1425 (stop codon tag, table 1 ), coding for vp10 with a predicted length of 467 aa (53.6 kda). tentative homology was detected between aa 298 and 395 of vp10, with the surface antigen d15 from the bacterium ralstoni metallidurans ch34 (29% identity, e-value = 2.4). a local blast analysis showed a match over the whole length of the protein with an unassigned protein of dpirv-1 (accession number x82045) with aa identity of 20% and a number of conserved motifs. the hydrophobicity of obrv vp10 showed no direct similarity to hydrophobicity plots or secondary structures obtained for the polyhedrin proteins of different cypoviruses (echeverry et al., 1997 ). an analysis of the predicted amino acid sequence indicated that, unlike the cypovirus polyhedrin proteins, vp10 of obrv is not especially rich in tyrosine (4.49%), aromatic residues (phenylalanine, tryptophane), or basic amino acids (lysine and arginine). however, it was particularly rich in leucine (10.04%), which is an amino acid periodically involved in signalling motifs (hurley, 2003) , which could be a factor in cell necrosis-inducing activities (sawada et al., 2000) . the rdrp is the most conserved reovirus protein and can be used for phylogenetic comparisons across the family as a whole. a tree was constructed using rdrp sequences from representatives of the different genera of the family reoviridae, including that of obrv (fig. 1) . unfortunately, no rdrp sequences are available for any of the idnoreoviruses. obrv is clearly distinct from the other reoviruses where the rdrp sequence is known. vp10 of obrv was synthesised using a baculovirus expression system. recombinant-baculoviruses were inoculated onto sf-21 cells, and virus infection (cpe) observed at 72 h p.i. (fig. 2) . however, no polyhedra-like occlusion bodies (ob) were observed as a result of vp10 expression. sds-page analysis of the baculovirus-infected cell lysates showed the presence of a ∼54 kda protein (fig. 2) . obcpv-19 polyhedrin was synthesised as a positive control (as previously described, graham et al., 2007) . the presence of ob was detected at 72 h p.i. (fig. 2) . sds-page analysis of the infected insect cell lysates confirmed the presence of the obcpv-19 polyhedrin at ∼29.2 kda (fig. 2) . a reovirus (obrv) was isolated from the lepidopteran o. brumata and the ichneumonid wasp p. tempestiva. obrv was more abundant in the parasitoid host (100% of wasp adults were infected) than in its lepidopteran host (of which only 11.5% were infected, graham et al., 2006) . recent studies have suggested that three types of relationship may occur between certain reoviruses and parasitoid wasps; non-pathogenic, commensal viruses (occur and replicate in wasp populations, and may be found in association with other types of viruses); putative mutualistic viruses (found in association with other viruses, but which do not appear to play a major role in host/parasitoid relationships); mutualistic viruses (not associated with other viruses, and contribute significantly to parasitoid success) (renault et al., 2005) . it is difficult to be certain of the exact nature of the relationship of obrv with p. tempestiva without further study. five hymenopteran reoviruses have been previously described (renault et al., 2005) . an interesting feature of these viruses is that four have never been found alone in their wasp host; they are always associated with another virus type. herv is associated with concolor (hamm et al., 1994; rabouille et al., 1994; jacas et al., 1997; stoltz and makkay, 2000) . in its hymenopteran host, obrv was predominantly found alone, apart from two individuals additionally infected with a cypovirus (graham et al., 2006) . the 'independent' nature of obrv suggests a replication strategy similar to that of the other cypoviruses or idnoreoviruses, both of which infect insect species. the observation of obrv in seemingly non-parasitised o. brumata indicates the ability of this virus to persist in this host without the need for the wasp (although wasp probing may have occurred without oviposition). the presence of obrv in adult winter moth also suggests transmission of the virus through lepidopteran life stages (graham et al., 2006) . this shows similarity with infections caused by a cypovirus from d. pulchellus (renault et al., 2003) , which was able to infect and multiply in lepidopteran gut cells after oviposition (renault et al., 2003) , and assisted development of wasp larvae by down-regulating the immune response of the host. interestingly, obrv isolated from female wasps appears to contain a genome of 11 segments (graham et al., 2006) . studies on the hymenopteran d. pulchellus showed that the idnoreovirus dpirv-1 isolated from diploid wasps (female or sterile diploid male wasps) also contained an extra genomic segment of dsrna when compared to virus isolated from haploid wasps (fertile males; rabouille et al., 1994) . this extra segment is thought to provide additional nucleic information, necessary for the development of eggs laid by the female parasitoid in the lepidopteran host (rabouille et al., 1994) . unfortunately, during the course of this study, insufficient viral dsrna was available from female p. tempestiva to sequence the 11th segment. as more reoviruses are being isolated from insects, and specifically from parasitic wasps, it is becoming clear that some may play a close and important role in ensuring the success of the parasitoid host, and that complex relationships have developed to increase the fitness of both parasitoid and virus. all 10 genome segments of obrv were cloned and sequenced, showing that in each case they contain a single large orf. seg-1 encodes a putative rna-dependent-rna-polymerase. the dsrna viruses use a viral rdrp to synthesis mrna, functioning both as a transcriptase and replicase. the viral mrna molecules can associate with other viral proteins to form large complexes. the viral rdrps can subsequently use these mrnas as templates for the synthesis of complementary −ve strands, reforming the dsrna genome of the virus. within the family reoviridae, rdrp aa identity of over 30% groups members within the same genus, while members of distinct genera have aa identities lower than 20% (attoui et al., 2000; rao et al., 2003) . however, two exceptions to this rule have been discussed: these include aquareoviruses and orthoreoviruses, which clearly have a common ancestor with an aa identity of 42%; and rotavirus b which has only 22% identity to other rotaviruses. amino acid identity values of between 19 and 27% were observed when comparing obrv rdrp with the rdrps of the "turreted reoviruses", while the "non-turreted reoviruses" gave only 11 to 13% aa identities. these data indicate that obrv does not belong to any of the existing genera of the reoviridae family for which rdrp sequences are available. a comparison of the obrv seg-10 sequence with those in genbank, generated no matches with other insect viruses. although obrv seg-10 is 1509 bp in length, some species of cypovirus also have a large seg-10 > 1 kb. for example, seg-10 of choristoneura fumiferana cpv-16 and antheraea mylitta cpv-4 are ∼1.5 and ∼1.2 kb, respectively, although they only encode proteins of ∼260 aa in length (echeverry et al., 1997; sinha-datta et al., 2005) , corresponding to the expected size of their respective polyhedrin proteins. expression of the orf confirmed that obrv seg-10 encodes a protein with a molecular mass of ∼54 kda, which did not form occlusion bodies in infected sf-21 cells. further analysis on the function of this protein remains to be done. the double-layered capsid, the non-occluded nature of obrv (graham et al., 2006) , phylogenetic analyses of the rdrp, and the expression analysis of vp10 all indicate that that obrv was not a member of the genus cypovirus.however, similarities were detected between vp2 from obrv and vp2 protein from an idnoreovirus (dpirv-1), over the whole length of the molecule. vp10 of obrv also showed similarities to vp10 (encoded by seg-10) of dpirv-1 protein again over the whole length of the molecule. although the lack of sequence information from this genus (particularly the rdrp), makes it difficult to make further comparisons, the data presented here, together with the detection of an eleventh genome segment in female wasps (graham et al., 2006) , suggest that obrv represents a new member (a new species) within the genus idnoreovirus, which contains other non-occluded insect reoviruses. in this case the isolate would belong to the species 'idnoreovirus-6' and should be identified as 'operophtera brumata idnoreovirus-6' (obirv-6). by a nerc non-thematic grant (ne/d008077/1) awarded to rdp, sms and rsh. the entomopoxviruses complete sequence determination and genetic analysis of banna virus and kadipiro virus: proposal for assignment to a new genus (seadornavirus) within the family reoviridae expansion of family reoviridae to include nine-segmented dsrna viruses: isolation and characterisation of a new virus designated aedes pseudoscutellaris reovirus 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on taxonomy of viruses idnoreovirus, reoviridae a member of the reoviridae (dprv) has a ploidy-specific genomic segment in the wasp diadromus pulchellus (hymenoptera) comparison of the amino acid sequences of rna-dependant rna polymerases of cypoviruses in the family reoviridae the cypovirus diadromus pulchellus rv-2 is sporadically associated with the endoparasitoid wasp d. pulchellus and modulates the defence mechanisms of pupae of the parasitized leek-moth, acrolepiopsis assectella commensal and mutualistic relationships of reoviruses with their parasitoid wasp hosts inhibition of l-leucine methyl ester mediated killing of thp-1, a human monocytic cell line, by a new anti-inflammatory drug morphological and molecular characterization of a cypovirus (reoviridae) from the mosquito uranotaenia sapphirina (diptera: culicidae) molecular cloning and characterisation of antheraea mylitta cytoplasmic polyhedrosis virus polyhedrin gene and its variant forms co-replication of a reovirus and a polydnavirus in the ichneumonid parasitoid hyposoter exiguae clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice this project was funded through a nerc non-thematic research studentship allocated to the centre for ecology and hydrology and key: cord-286473-sl5zy8nj authors: gomaa, m.h.; barta, j.r.; ojkic, d.; yoo, d. title: complete genomic sequence of turkey coronavirus date: 2008-05-12 journal: virus res doi: 10.1016/j.virusres.2008.03.020 sha: doc_id: 286473 cord_uid: sl5zy8nj turkey coronavirus (tcov), one of the least characterized of all known coronaviruses, was isolated from an outbreak of acute enteritis in young turkeys in ontario, canada, and the full-length genomic sequence was determined. the full-length genome was 27,632 nucleotides plus the 3′ poly(a) tail. two open reading frames, orfs 1a and 1b, resided in the first two thirds of the genome, and nine additional downstream orfs were identified. a gene for hemagglutinin-esterase was absent in tcov. the region between the membrane (m) and nucleocapsid (n) protein genes contained three potential small orfs: orf-x, a previously uncharacterized orf with an associated putative trs within the m gene (apparently shared among all group iii coronaviruses), and previously described orfs 5a and 5b. the tcov genome is organized as follows: 5′ utr – replicase (orfs 1a, 1b) – spike (s) protein – orf3 (orfs 3a, 3b) – small envelop (e or 3c) protein – membrane (m) protein – orf5 (orfs x, 5a, 5b) – nucleocapsid (n) protein −3′ utr – poly(a). tcov genome structure and sequence was most similar, but distinct from, avian infectious bronchitis virus (ibv). this is the first complete genome sequence for a tcov and confirms that tcov belongs to group iii coronaviruses. turkey coronavirus (tcov) is associated with highly contagious gastroenteritis in young poults. first identified in 1951 (peterson and hymass, 1951) , tcov causes high morbidity, some mortality, and poor long-term growth of the affected birds, resulting in significant economic losses in the turkey industry. outbreaks have been reported in different areas in the usa including minnesota, north carolina, and indiana as well as in quebec, canada (dea et al., 1986) . turkey coronavirus was subsequently determined to be the causative agent of blue comb disease during an investigation of an outbreak of acute, highly contagious enteritis in a flock of young turkeys (nagaraja and pomeroy, 1997) . in addition to turkeys, tcov can infect a variety of avian hosts including chickens, pheasants, sea gulls, and quail (deshmukh and pomeroy, 1974) . recently, tcov was incriminated as one of the most important causative agents of poult enteritis and mortality syndrome teixeira et al., 2007) . coronaviruses are divided into three groups (i, ii, and iii) based on the genome structure and organization (holmes and lai, 1996) . the coronavirus genome is a single-strand positive-sense rna with a 5 cap and poly (a) tail at the 3 end. two large open reading frames (orfs) occupy the 5 proximal two thirds of the genome and are involved in polyprotein processing, genome replication, and subgenomic rna synthesis, and the 3 one third of the genome codes for structural proteins. orf1 consists of two overlapping orfs (orf1a and orf1b) that are translated into 1a and 1a/1b polyproteins by a ribosomal frame-shifting mechanism. orf1a encodes 2 proteases; papain-like cysteine protease (plp) and picornavirus 3c-like chymotrypsin protease (3clp). both proteases cleave the polyproteins into at least 16 cleavage products (sawicki et al., 2007) . an intergenic consensus sequence of about seven bases is found immediately upstream of each gene, which plays an important role for subgenomic rna synthesis (sawicki et al., 2007) . most coronaviruses code for four major structural proteins; spike (s) glycoprotein, membrane (m) protein, small envelope (e) protein, and nucleocapsid (n) proteins, in addition to hemagglutinin esterase (he) glycoprotein in some group ii coronaviruses. the genome structure and organization are not known for any tcov, and this lack of genetic information has made it difficult to develop effective detection and control measures for tcov. the goal of the present study was to complete the full genomic sequence of tcov and establish a better understanding of the virus at the molecular level. clinical specimens consisting of intestinal tracts were collected from turkey poults during an outbreak of diarrhea in a turkey farm in ontario, canada. the tissue samples were processed by washing several times with phosphate buffer saline (pbs) and cut into small pieces prior to grinding using a sterile mortar and pestle. coarse particles and tissue debris were removed by centrifugation at 3000 × g for 30 min at 4 • c. the supernatants were filtered through 0.22-m filters (millipore, bedford, ma). virus was isolated from the filtrates by inoculating embryonated turkey eggs. allantoic fluids were collected 3 days post-inoculation then clarified by centrifugation 3000 × g for 30 min at 4 • c and the supernatant were filtered through 0.22-m filters and stored at −80 • c until use. complementary dna (cdna) was synthesized using 10.5 l of the first strand mixture (invitrogen, carlsbad, ca) containing 0.2 g of random hexamers and 2 g of total rna isolated from the intestinal specimens. the mixture was incubated at 70 • c for 10 min and then quick-chilled on ice for 5 min. rt master mix was composed of 4 l 5× rt buffer (invitrogen), 2 l 10 mm dtt, 2 l 10 mm dntps (amersham, piscataway, nj), 1 l superscript ii reverse transcriptase (invitrogen), and 0.5 l rnase inhibitors (invitrogen). this rt master mix was added to 10.5 l of the first strand mixture and then incubated at 42 • c for 2 h. the reaction was terminated by heating at 95 • c for 10 min then chilling on ice for 5 min. fifty microliters pcr reactions included 2 l of cdna and 48 l of the master mix composed of 5 l 10× buffer (100 mm tris-hcl, 500 mm kcl), 1.5 l of 15 mm mgcl 2 , 1.5 l of 10 mm dntps, 2 pmol of upstream and downstream primers, two units of taq high fidelity dna polymerase (invitrogen), two units of amplitaq gold (roche molecular systems, inc., usa) plus 38 l nuclease free water. pcr was performed for 35 cycles as follow; 95 • c for 1 min, 65 • c for 1 min, 55 • c for 30 s, 72 • c for 3 min, followed by final extension at 72 • c for 10 min. the pcr products were analyzed by 1% agarose gel electrophoreses and visualized by staining with ethidium bromide and uv illumination. thirteen overlapped pcr fragments spanning the entire viral genome were amplified using specific primer sets (table 1) . the pcr products were purified using the qia quick pcr purification kit (qiagen, valencia, ca) and cloned in pgem-t easy (promega, madison, wi). transformants were screened by restriction enzyme digestion and sequencing using primers specific for t7 and sp6 promoter. the sequences were analyzed using the sequencher 4.5 sequence analyses program, and a single contiguous sequence comprising the entire tcov genome was constructed. prediction for orfs was conducted using vector nti advanced 10 (invitrogen), and the sequences were analyzed using lasergene dna star (version 7, lasergene corp, madison, wi). the pairwise nucleotide identity was determined using vector nti advanced 10 and multiple sequence alignments were generated using clustal-w (thompson et al., 1997) . comparative analyses of tcov with other coronaviruses were conducted using the coronavirus database (covdb, huang et al., 2007) . cdna clone representing the 5 end of the tcov-mg10 genome had been synthesized according to the 5 race system for rapid amplification of cdna ends (invitrogen). the antisense primer had been designed based on the available tcov-mg10 sequence (5 -cgccaggtgttattttgtca) then cdna was synthesized as mentioned before. the cdna was purified using qiagen column purification kits. tailing of the cdna was done using dctp and dtd. pcr had been done to amplify the dc-tailed cdna with the abridged anchor primer together with the designed primer (5 -gttgtcactgtctattgtatg) according to the instructions of the kits. the 3 end of tcov genome had been done using the 3 race system for rapid amplification of cdna ends (invitrogen) according to the instruction of the kite. cdna was synthesized using the adaptor primer then the cdna was amplified with pcr using the tcov-mg10 specific primer (5 ctatcgccagggaaatgtct 3 ) and the universal amplification primer according to the instruction of the kits. the obtained pcr products had been cloned and sequenced in both directions as mentioned earlier. the obtained sequence aligned with the rest of the genomic sequence and run through the poly(a) tail. phylogenetic relationships among coronaviruses were investigated using the following complete genomes representing groups i, ii, and iii: bcov-ent (nc 003045), hcov-nl63 (dq445912.1), hcov-oc43 (nc 005147.1), ibv-m41 (ay851295), ibv-p65 (dq001339.1), ibv-cal99 (ay514485), ibv nc (nc 001451), sarscov-bj202 (ay864806.1), hcov-229e (nc 002645.1), mhv-jhm (nc-006852.1) and fipv wsu-79/1146 (nc 007025.1). whole genome sequences were aligned using clustalw (thompson et al., 1997) and subsequently optimized by eye using the geneious software (version 3.0.6, biomatters ltd, auckland, new zealand) (kumar et al., 2004) . aligned nucleotide sequences were analyzed using paup (version 4.0), and maximum likelihood (hky85 model with transition/transversion ratio of 2) and maximum parsimony (transversion/transition ratio of 2) search criteria were used with branch and bound search strategies (swofford, 1991) . bootstrap supports for the resulting trees were determined using 100 replicate heuristic tree searches in both parsimony and likelihood analyses using the same search criteria. for more detailed ingroup analysis of relationships among the group iii coronaviruses, amino acid sequences for the spike glycoprotein (s), envelope protein (e, also known as 3c) and the nucleocapsid (n) protein were aligned using clustalw and subsequently analyzed using maximum parsimony (branch and bound search algorithm) followed by bootstrap analysis using a heuristic search method. the following additional group iii coronavirus sequences used for the comparative sequence analyses: tcov-gh s gene (ay342356), tcov-gi s gene (ay342357), quail coronavirus italy/elvia/2005 s1 gene (ef446155.1), tcov-nc95 n gene (af111997), tcov-minnesota n gene (af111996), tcov-indiana n gene (af111995). all ingroup translation products (s, e and n proteins) from the whole genomes of the same group i and ii coronaviruses used above were included in these analyses. the group i and ii coronaviruses were considered functional outgroups for determining relationships among the group iii coronaviruses. the tcov full genomic sequence described in this report was deposited in the genbank database with accession number eu095850. a tcov was isolated from an outbreak in an ontario turkey farm and designated tcov-mg10. subsequently, the full-length genomic sequence of tcov-mg10 was determined by sequencing of overlapping pcr fragments in both directions. the sequences were assembled into one contiguous sequence to represent the entire viral genome. the sequence of 27,632 nucleotides was obtained, plus the polyadenylation tail at the 3 end. the entire genome has a gc content of 38.3%. the tcov genome contained two large slightly overlapping orfs in the 5 two thirds of the genome and multiple additional orfs in the 3 one third of the genome (fig. 1) . both termini were flanked with untranslated regions (utrs). the tcov genome was similar overall in its coding capacity and genomic organization to those of other coronaviruses. eleven orfs were identified in the genome (table 2) . gene 1 was 19,806 nucleotides comprising orf1a and orf1b, located between nucleotides 529 and 20,333. this gene contained motifs common in all coronaviruses including ribosomal frameshifting and slippery sequences, as orf1b is translated in the −1 frame. the typical coronavirus structural genes encoding the spike (s), small envelope (e, also known as 3c), membrane (m), and nucleocapsid (n) proteins were identified following gene 1 (table 2, fig. 1 ). the tcov genome had polycistronic genes 3 and 5 interspersed between the s and e genes, and between m and n genes, respectively. in ibv, gene 3 is believed to be tricistronic consisting of 3a, 3b and 3c, where 3c has been shown to encode the small envelope (e) protein. gene 5 contained a coding potential for two products, 5a and 5b, of unknown function. a third orf of 282 nucleotides was located between the m gene and gene 5a. this gene, designated orf-x, contained a coding potential for a protein of 94 amino acids that had no structural or sequence homology with any known protein. blast searches failed to identify any protein homologs, but identified highly similar nucleotide sequences from other tcov isolates as well as from numerous ibv isolates. orf-x had a relatively distant but highly conserved putative transcription regulatory sequence (trs). in summary, the genome organization for tcov was determined as follows: 5 utr-gene 1 (orf1a, 1b)fig. 1 . turkey coronavirus (tcov) mg10 genome organization. the full-length tcov genome is 27,632 nucleotides excluding the polyadenylation tail at the 3 end. center: diagrammatic representation of the genome organization shows the predicted genes and their relative sizes and positions along the tcov genome. s, spike glycoprotein gene; 3a, 3b, and 3c (e), tricistronic gene 3; m, membrane protein gene; orf-x, unique orf conserved among group iii coronaviruses; 5a and 5b, bicistronic gene 5; n, nucleocapsid protein gene; utr, untranslated region. scales indicate relative positions of the various genes within the genome in kilobases. top: expanded representation of the two orfs (orf1a and orf1b) comprising the polycistronic gene 1 and the likely cleavage products and cleavage sites after proteolytic processing of the 1a/1b polyprotein. bottom: expanded representation of the s gene indicating the signal peptide (sigp), putative cleavage site (s1/s2), endodomain (clear bars), endodomain (hatched bar) and a short transmembrane region (solid bar). s-gene 3-(orfs 3a, 3b, e)-m-gene 5 (orfs x, 5b, 5c)-n-utr-3 ( fig. 1) . the 5 terminus of the tcov genome was characterized by the presence of a 528 nucleotides long utr with relatively higher gc content of 50.4% compared with the genome as a whole. the tcov 5 utr showed a high degree of sequence similarity with that of most ibv isolates such as ibv-nc (98% identity). following 5 utr, gene 1 of 19,806 nucleotides was located. gene 1 included two slightly overlapping orfs, 1a and 1b. orf1a was 11,853 nucleotides in size enabling it to code for a protein of 3951 amino acids, and orf1b was 7992 nucleotides in length for a coding potential of 2664 amino acids. those two orfs overlapped by 40 nucleotides. orf1b did not contain a typical aug translational initiation codon but instead started with gaa at position 12,342 ( fig. 2a) . the heptanucleotide slippery sequence (uuuaaac) was present which is conserved among all coronaviruses (fig. 2b) , and therefore the ribosomal frame-shifting mechanism seemed to be applicable to tcov and orf1b was believed to be translated by the ribosomal frame-shifting mechanism as a fusion with orf1a to make the polyprotein of 1a/1b. for most coronaviruses, the polyprotein is processed into 16 cleavage products, while the ibv polyprotein is likely cleaved into 15 products (ziebuhr et al., 2000) . as with other coronaviruses, the tcov polyprotein was also believed to undergo proteolytic processing by viral proteinases. the tcov replicase protein was similar to that of ibv in its processing patterns. since the potential cleavage sites were conserved for both viruses (table 2) , the tcov polyprotein was assumed to be processed in the similar manner. two main proteases used by coronaviruses have been identified; plp (papain-like proteinase) which produces 2 or 3 n-terminal products of the polyprotein and 3clp (3-c like protease) which produces the central and the c-terminal region of the polyprotein by cleaving 11 sites (ziebuhr et al., 2000) . similar to sars-cov, ibv encodes only one plp, whereas other coronaviruses code for two plps. in the case of ibv, plp equivalent to plp2 of other coronaviruses cleaves the polyprotein at the qs dipeptide to produce the n-terminal 100 kd proteins, and tcov plp seemed to function in a similar fashion. tcov 3clp was mapped in nsp5. based on the sequence comparisons between tcov and ibv, the replicase cleavage products and their putative functions were predicted (fig. 1, table 2 ). it has been suggested that nsp1 does not exist in ibv (ziebuhr et al., 2007) . due to the high degree of similarity between tcov and ibv in the n-terminal region of the polyprotein 1a/1b, tcov also seemed not to contain nsp1. thus, for tcov, nsp2 through nsp11 would be produced from orf1a, while nsp12 through nsp16 would be produced from orf1b. the n-terminal most cleave product nsp2 was predicted to locate between nucleotide positions 529 and 2549. the sequence analysis for nsp1/2 reveals 44%, 55%, and 90% identity to bcov, hcov-229e, and ibv, respectively. plp1 was suggested to have been lost during the ibv virus evolution (ziebuhr et al., 2001) , and our sequence data also supports that tcov contained only plp2. therefore, plp2 would be responsible for cleavage of the n-terminal part of the polyprotein at two sites between nsp2/nsp3 (ag/gk) and nsp3/nsp4 (ag/gi). plp2 was identified in nsp3. nsp3 was the largest subunit of the replicase cleavage products and was highly conserved among tcov-mg10 and ibv-baudette's strain, as both viruses share 89% identity on the nucleotide level. in contrast, 3clp would cleave 1a/1b polyprotein at 11 sites and generate nsp5 through nsp16 (table 2 ). the motif for 3clp was found in nsp5. the 1a/1b polyprotein contained the motifs found in other nidoviruses (fig. 1, table 2 ). an earlier study identified a 87 kd (p87) protein encoded in this region (lim and liu, 1998) , and the p87 homolog was also found in tcov-mg10 as a 673 amino acids protein in the same region. 3clp was located in nsp5 of 307 amino acids. nsp5 was similar by 52%, 44%, and 92% at the nucleotide sequence level and by 39%, 44%, and 93% at the amino acids level to hcov-229e, bcov, and ibv, respectively. this region was believed to play a critical role for orf1b processing since the deletion of nsp5 in ibv resulted in the unprocessing of 1b protein (liu et al., 1997) . nsp3, nsp4 and nsp6 were predicted to carry a hydrophobic transmembrane domain which may play an important role in the transcription/replication process as recently discussed for other coronaviruses (sawicki et al., 2007) . nsp10 was rich in cysteine and histidine and was predicted to contain a metal binding domain as well as ntp binding helicase domain as with other coronaviruses (gorbalenya et al., 1989) . nsp11 was predicted to be a small peptide of 23 amino acids in length, which is likely the c-terminal most cleavage product of 1a. nsp12 contained the rnadependant rna polymerase (rdrp) activity, which would likely be involved in the genome replication and transcription (liu et al., 1994) . the rdrp motif was highly conserved among all coronaviruses, and tcov-mg10 also showed a high degree of sequence identity for rdrp by 64%, 62%, 94% at the amino acid level to hcov-229e, bcov, ibv, respectively. nsp13 has previously been suggested to play a role for genome replication by unwinding double-strand rna (gorbalenya et al., 1989) . nsp14 was assumed to possess the exonn domain. this domain may be associated with rna metabolism such as proofreading ability and recombination. both coronaviruses and toroviruses contain one exonn motif while roniviruses contain two copies of the exonn motif (snijder et al., 2003) . nsp15 contained the motif for nendou activity. in sars-cov and hcov-229e, nendou cleaved the double-strand rna at the uridylate-containing sequence, and this activity was essential for rna synthesis and progeny virus production (ivanov et al., 2004; posthuma et al., 2006) . nsp16 contained a motif for 2 omethytransferase (mt) in other coronaviruses , and tcov also contained nsp16 as the most c-terminal cleavage product of 1ab. tcov orf1a showed a 46% sequence identity to both bcov and hcov-oc43, which are group ii coronaviruses, and a 90% identity to ibv-nc, ibv-m41, and ibv-p65 which are group iii coronaviruses (table 3) . tcov orf1b was more conserved than orf1a, as tcov orf1b showed a 59% identity to groups i and ii coronaviruses, and 93% identity to ibv which is a group iii coronavirus. as with other coronaviruses, tcov was characterized by the presence of 4 major tructural genes, located in the 3 one-third of the genome: spike (s) glycoprotein, small envelope (e) protein, membrane (m) protein, and nuclocapsid (n) protein genes (fig. 1) . the s protein gene was located immediately downstream from orf1b for a predicted protein of 1,226 amino acids. the s gene was fecov 46 43 59 40 37 43 38 hcov-229e 60 45 59 40 40 43 40 hcov-nl63 50 45 60 41 42 44 39 bcov-nc 46 46 59 45 41 50 44 hcov-oc43 48 46 59 44 42 51 44 mhv-jhm 46 43 58 43 43 51 42 sars-cov 45 41 59 44 31 45 41 ibv-beaudette 94 90 93 57 90 94 92 ibv-p65 87 90 93 57 88 92 92 ibv-m41 87 90 93 57 88 92 93 tcov-mg10 100 100 100 100 100 100 100 tcov-g1 n/a b n/a n/a 96 n/a n/a n/a tcov-gh n/a n/a n/a 96 n/a n/a n/a tcov-nc95 n/a n/a n/a n/a n/a n/a 94 tcov-min n/a n/a n/a n/a n/a n/a 94 tcov-ind n/a n/a n/a n/a n/a n/a 93 a fecov, feline coronavirus; hcov, human coronavirus; bcov, bovine coronavirus; mhv, mouse hepatitis virus; sars-cov, severe acute respiratory syndrome coronavirus; ibv, infectious bronchitits coronavirus; tcov, turkey coronavirus. b n/a: data not available. the most variable gene in the tcov genome as compared to that of other coronaviruses. the s gene showed only 40% and 44% identities to that of group i and group ii coronaviruses, respectively, while the similarity to different strains of ibv was 57% (table 3 ). the tcov s gene showed the highest sequence identity to that from other tcov isolates, tcov-gh and tcov-gi, by 96% for both. the sequence variability was mainly due to the hypervariable and the receptor binding regions in the s protein. the high degree of sequence identity for s among tcov isolates suggests that tcov is less heterogenic as with ibv than other coronaviruses (cavanagh, 2005) . tcov s was slightly larger than ibv s as tcov s was 3678 nucleotides, while ibv s was 3453 nucleotides in size. the coronavirus s protein is responsible for receptor binding and virus-host cell membrane fusion. for groups ii and iii coronaviruses, the s protein is cleaved into two subunits; the n-terminal s1 product and the c-terminal s2 product. tcov s was also presumed to be cleaved into s1 (542 amino acids) and s2 (684 amino acids) with the putative cleavage site characterized by presence of basic amino acids (arg-arg-ser) between 542 and 543 ( fig. 1 ) as for ibv s (cavanagh, 2007) . the receptor-binding domain in s1 is not well-identified for ibv and thus was not possible to predict for tcov. tcov s was likely highly glycosylated as it contained 24 potential n-linked glycosylation sites. tcov s contained three hydrophobic transmembrane domains; two ectodomains and one endo-domain. tcov gene 3 was thought to be tricistronic (orf 3a, 3b, and 3c). the small membrane (e) protein of 99 amino acids was potentially encoded by orf3c. both orf 3b and e genes overlapped by fig. 3 . illustration of conservation of the orf-x amino acid sequence and the putative trs found upstream of the orf-x gene among a number of group iii coronaviruses. tcov and ibv share a highly conserved 94 amino acids hypothetical protein, designated as orf-x. a highly conserved trs motif (gtcaacaa) for orf-x is found 288 nt upstream, within the m gene, in all group iii coronaviruses. the beaudette strain of ibv and strains derived from it such as ibv-p65 have a 49 nt deletion in orf-x but the remaining sequence aligns unequivocally with all other group iii coronaviruses when this deletion is taken into account. 1 n/a, no sequence available for this region; 2 single nucleotide (a) from position 446 (af072911.1) was deleted to maintain reading frame; 3 17 amino acid deletion (51 nucleotides for 17 codons) in these sequences. 16 nucleotides. the tcov e gene showed a high degree of sequence identity to ibv e by 90%. the e protein was reported to be a viroporin which played a role in the membrane permeability in sars-cov and porcine reproductive and respiratory syndrome virus, another member of nidoviruses (wilson et al., 2004; lee and yoo, 2006) . the membrane (m) protein gene was 699 nucleotides in size and was able to make a protein of 223 amino acids. the m gene seemed to be highly conserved within group iii coronaviruses since tcov m showed a 94% nucleotide identity to ibv m. the m protein contained a single putative n-linked glycosylation site at amino acid position 4 as well as 3 potential sites for o-linked glycosylation. it remains to be determined whether these sites are functional for tcov. the nucleocapsid (n) protein gene was 1,227 nucleotides with a coding capacity of 409 amino acids. the tcov n gene showed a 93% sequence identity to that of various ibv strains. the n protein was shown to be a serine phosphoprotein in other coronaviruses and arteriviruses (alexander et al., 2005; wootton et al., 2002) . the tcov n protein contained 20 serine residues but no tyrosine residue. gene 3 (orf3) is possibly tricistronic as with other coronaviruses. orf3 was able to code for two non-structural proteins and the small envelope (e) protein. orf3a and 3b were 171 and 192 nucleotides in length capable of coding for 57 and 64 amino acid proteins, respectively. orf5 is potentially bicistronic to code for 5a and 5b proteins. orf5a was 195 nucleotides in length for 65 amino acids while orf5b had a potential for 82 amino acids. orfs 5a and 5b overlapped by three nucleotides, while orf5b and the downstream n gene overlapped by 57 nucleotides. the presence of gene 3 and gene 5 were highly suggestive that tcov was related to group iii coronaviruses. a recent study for the role of gene 3 and gene 5 for ibv replication showed that deletion mutant viruses succeeded in replication in a similar manner to the wild-type virus, suggesting that those genes were non-essential for ibv replication (casais et al., 2005) . in contrast, orf5a and orf5b were not found in mammalian coronaviruses, and thus the presence of gene 5 may be considered a characteristic feature of avian coronaviruses including ibv and tcov. tcov was characterized by the presence of an additional orf, designated orf-x. orf-x was 282 nucleotides in length with 33.3% gc contents. this orf was located upstream of gene 5 and started immediately following the m gene. this gene was able to encode a hypothetical protein of 94 amino acids. a blast search for this orf using the amino acid sequence found no homology to described proteins; however, at the nucleotide level, this orf was strongly conserved among group iii coronaviruses including all ibv strains and all available sequences for turkey coronaviruses in the genbank database. for several ibv strains, this region contained a wellidentified orfs including initiation (aug) and stop codons (fig. 3) . interestingly, a sequence, that might be the putative trs (gucaa-caa) for this particular orf was found 288 nucleotides upstream of the initiation codon for orf-x within the m gene (table 4) ; this putative trs was conserved at the same relative location in virtually all ibv sequences in the genbank database spanning the putative trs region (fig. 3) . further studies are warranted determine the significance of this apparently expressed protein found only in group iii coronaviruses as far as is known (fig. 3) . a 3 utr of 352 nucleotides was present immediately downstream the n gene of the genome. it has been previously shown that the 3 utr of both tcov indiana and minnesota strains were 502 nucleotides long, while tcov-nc95 strain has a 3 utr of 349 nucleotides which lack the first 153 nucleotides at the 5 end (breslin et al., 1999) . also, tcov-mg10, like ibv and some other coronaviruses, contained a conserved stem-loop structure in the 3 utr as illustrated in fig. 5 . the tcov genome contained putative trss located upstream of the start codon of each gene (table 4 ). although the distance between the putative trs and the downstream initiation codon varied among the various genes in the tcov genome, the particular distance for each gene was similar in both ibv and tcov. in all cases, the putative trs contained a conserved aacaa motif. the leader trs (cuuaacaa) was found in the 5 utr at genomic positions 57-64 which is 465 nucleotides upstream of the orf1a initiation codon. the trs for s gene, cugaacaa, differed from the leader trs by replacement of u at the 3rd position with g, and was located 53 nucleotides upstream from the s gene start. the m gene trs was identical to the leader trs and was located 77 nucleotides upstream of the m gene start. in the same manner, the n gene trs fig. 4 . phylogenetic trees for tcov and other coronaviruses. aligned nucleotide sequences of complete coronavirus genomes representing coronavirus groups i, ii, and iii were utilized to construct maximum likelihood (ml) and maximum parsimony (mp) trees using the paup software package followed by bootstrap analysis using a heuristic search method. for each tree, the bootstrap support for each branch is indicated for each branch and the horizontal lengths of branches are proportional to the amount of hypothesized evolutionary change. all trees are rooted using the group i coronaviruses as a functional outgroup. the ln likelihood for the ml tree and the consistency index (ci) and retention index (ri) are provided for the mp trees. for ingroup analyses of relationships among the group iii coronaviruses, aligned amino acid sequences for the spike glycoprotein (s), envelope (e) and nucleocapsid (n) proteins were analyzed using maximum parsimony (branch and bound search algorithm). hcov, human coronavirus; fipv, feline infectious peritonitis virus; bcov, bovine coronavirus; mhv, murine hepatitis virus; ibv, infectious bronchitis virus; tcov, turkey coronavirus. alignment of highly conserved stem-loop (s2m) sequences present in the 3 utr of a variety of coronaviruses. this highly conserved region, flanked by highly divergent sequence (not shown), is found in tcov, ibv and other avian coronaviruses. the s2m sequence is also found in the atypical group iii coronavirus sars-cov and the recently sequenced coronavirus isolated from an asian leopard cat. was identical to the leader trs and located 93 nuclotides from the n gene start. phylogenetic reconstruction of the whole genomes of 12 coronaviruses using maximum likelihood and maximum parsimony produced well-supported trees (fig. 4) that placed tcov-mg10 as a sister taxon to the four ibv isolates included in the analysis. bootstrap support for both analyses was 100% at each node indicating significant support for the branching order. group i coronaviruses (fipv, hcov-nl63, hcov-229e) formed a monophyletic clade as did the group ii coronaviruses (sars-cov, mhv, bcov, hcov-oc43). sars-cov was basal to the other group ii coronaviruses in the whole genome analyses. the solid support for the monophyly of tcov-mg10 with four ibv isolates supports the conclusion that this tcov should be classified as a member of the group iii coronaviruses. analyses of aligned amino acids sequences for the e, n, and s proteins of various coronaviruses produced phylogenetic trees that largely reflected the nucleic acid-based whole genome trees (fig. 4) . the n protein produced a well-supported tree that had a largely unresolved polychotomy consisting of five tcov isolates (three usa isolates, one uk isolate, and isolate mg10 from canada) and the four ibv isolates used in the whole genome analyses. tcov-mg10 was most closely related to tcov-nc95 and these two isolates formed a well-supported monophyletic group based on the n protein; however, this protein did not contain sufficient information to infer relationships reliably among group iii coronavirus. similar results were obtained with a large number of ibv sequences and five tcov sequences with the m protein (data not shown). the e protein analysis placed tcov-mg10 as the sister taxon to the three ibv isolates used in the analysis; however, there was only a single tcov available for that analysis so no conclusions was drawn regarding the utility of this protein for inferring evolutionary relationships among these group iii coronaviruses. unlike the analyses based on aligned s or n protein amino acid sequences, group ii coronaviruses did not form a monophyletic group in the analysis based on aligned e protein sequences; sars-cov did not group with the other three group ii coronaviruses included in this analysis. using available sequences for the s protein, three tcov isolates (gh, g1 and mg10) formed a monophyletic group closely related to a quail coronavirus (qcov) isolate from italy. together, these isolates formed the sister group to a number of ibv isolates. the phylogenetic reconstruction based on the s protein, unlike other analyses based on the amino acid sequences of the n, e or m proteins (data not shown), produced a well-supported clade containing only tcov strains and suggests that the s protein may be a more useful molecule for inferring relationships among the group iii coronaviruses. the family coronaviridae is included in the order nidovirales along with the arteriviridae and roniviridae families. coronaviruses are divided into three groups (i, ii, and iii) based on the genome structure and organization (holmes and lai, 1996; lai and cavanagh, 1997) . group i coronaviruses include porcine epidemic diarrhea virus (pedv), tgev, canine coronavirus (ccov), feline infectious peritonitis virus (fipv), hcov-229e, and a newly identified hcov-nl63, whereas group ii includes murine hepatitis virus (mhv), bcov, hcov-oc43, rat sialodacryoadenyleitis virus (sadv), canine respiratory coronavirus (crcov), and equine coronavirus (ecov). group iii coronaviruses are ibv as well as the newly discovered pheasant coronavirus. group iii coronaviruses are characterized by modification of gene 3 to a tricistronic structure that codes for genes 3a and 3b as well as the e gene, and the insertion of an additional unique sequence designated gene 5. the e gene is common in all coronaviruses but is incorporated into the tricistronic gene 3 located between s and m genes only in the group iii coronaviruses as far as is known. in contrast, gene 5 located between the m and n genes had only been reported previously from ibv. in tcov-mg10, gene 5 has two small orfs (orf5a and orf5b) that code for products of 65 and 82 amino acids, respectively; this is identical in length to the gene 5 products of ibv. the presence of gene 5 has been suggested as a genetic maker for group iii coronaviruses . in vitro work with ibv has demonstrated that gene 5 is not essential for virus replication in cell culture (casais et al., 2005) . whether or not gene 5 is unnecessary for in vitro replication of tcov is unknown. since first identified in 1951 (peterson and hymass, 1951) and despite the economic importance to the turkey industry, tcov has remained one of the least characterized among all known coronaviruses. early studies suggested that this virus could be a group ii coronavirus along with bcov and hcov-oc43 based on serology and partial sequences for n and m genes. (verbeek and tijssen, 1991) reported that the tcov n gene was 100% identical to bcov n gene. this suggestion was supported by a study using the minnesota strain of tcov that demonstrated hemagglutination activity of tcov for rabbit erythrocytes (dea et al., 1990) . a he gene is found in many group ii coronaviruses. despite these early confounding observations, , using antibodies to discriminate among coronaviruses, suggested that tcov was more closely related to ibv than bcov (dea and tijssen, 1989; dea et al., 1990) , which is in agreement with our genomic data. our completion of the full genome sequence of a field isolate of a tcov shows clearly that the tcov genome structure and sequence are much closer to ibv than any other coronavirus. like ibv, the genome structure of tcov-mg10 is: 5 utr -replicase (orf1a and orf1b) -spike (s) protein -orf3 (orfs 3a and 3b) -small envelop (e or 3c) protein -membrane (m) protein -orf-x -orf5 (orfs 5a, and 5b) -nucleocapsid (n) protein -3 utr-poly(a), in order (fig. 1) . the non-structural protein gene immediately downstream of orf1b and the further downstream he gene that are both commonly found in group ii coronaviruses were entirely absent in tcov. our data did not support the presence of any gene similar to he and we concluded that tcov does not contain the he gene. as is obvious from table 3 , tcov-mg10 and other tcov isolates demonstrate much higher sequence similarity to strains of ibv that they do for any other coronavirus. for example, the n gene of tcov was only 44% identical to bcov and hcov-oc43 but had 92% identity to the n gene of ibv. this apparent discrepancy with previous sequencing results (e.g. verbeek and tijssen, 1991) may be explained by several reasons. one possibility is genome recombination between tcov and bcov. it is also possible that a cell line commonly used for coronavirus cultivation, hrt-18, may harbor a latent infection with one of the human coronaviruses which was then activated upon infection with another coronavirus. laboratory contamination of bcov is also a possibility. our data shows a high degree of sequence identity between ibv and tcov in the replicase, e, m, and n genes with greater than 90% sequence identity for each (table 3 ). the s gene is most variable among ibv strains and between ibv and tcov, perhaps reflecting the role that the s protein has in determining receptor binding in coronaviruses (cavanagh, 2005) . despite the relatively large genetic variation in the s gene of various ibv strains, tcov also showed a relatively higher sequence identity to ibv (up to 57%) while the sequence identity to the s gene of bcov was only 45%. in contrast to ibv, our limited study indicates that tcov s is relatively conserved among isolates, suggesting that the tcov s genes might be less varied than the ibv s genes. ibv and tcov have distinct clinical presentations in infected hosts. ibv causes respiratory disease in chickens whereas tcov causes enteric disease in young turkeys. perhaps the tropisms exhibited by ibv and tcov reflect the sequence variation of the s glycoprotein and the resulting differences in receptor affinities. the tcov s gene is larger than that of most ibv isolates. a study is required to understand the functional difference in s for ibv and tcov, which may have a great impact on the antigenic properties and tissue tropism of both ibv and tcov as well as the development of control measures against them. the presence of gene 3 and gene 5 is a unique characteristic for group iii coronaviruses as those genes do not exist in mammalian coronaviruses. as those genes are highly conserved in avian coronaviruses, they might serve as a cis acting elements essential for virus replication. based on the overall structure of the genome and sequence similarities to ibv, we conclude that tcov should be classified as a group iii coronavirus. our sequence data revealed the presence of a novel orf in tcov located upstream of gene 5 and downstream of the m gene. this orf is unique to tcov and ibv, the only group iii coronaviruses for which sequence data in this region of the genome are available. orf-x was strongly conserved among group iii coronaviruses including most ibv strains and all available sequences for turkey coronaviruses in genbank as well as the tcov-mg10 isolate. eight (e.g. beaudette and p65 strains) of 40 or more ibv sequences for orf-x have a 49 base deletion in comparison with other ibv strains; all of the strains with this deletion were laboratory-adapted, cell cultured viruses originating with the beaudette strain of ibv, suggesting that this deletion may be an artifact of cultivation outside of the natural host. the maintenance of this long sequence within a coronavirus genome seems highly unlikely if this was not a functional gene. finding a highly conserved putative trs, 288 bp upstream of the initiation codon for orf-x within the m gene, reinforces the notion that this gene is functional in both ibv and tcov despite the unusually long distance between the putative trs and the initiation codon. this region may be a good marker for group iii coronaviruses. further studies are being carried out to further characterize this particular orf and determine its role, if any, in virus replication. among coronaviruses, only sars-cov contains an orf immediately upstream of the n gene, which is referred to as gene 8. gene 8 plays an important role in the sars-cov replication and induction of apoptosis of its host cells (chen et al., 2007) . the function of the small orf found in tcov remains unknown and the lack of any homology with any known protein makes inferring its function difficult. the 3 utr is believed to be involved in genome replication of coronaviruses (williams et al., 1993) , despite its apparent ability to possess quite variable sequence and sequence lengths. this variation within available 3 utr sequences of tcovs is the same as for ibv strains. the 3 utr of ibv strains (beaudette, kb8523 and cu-t2) are 503-505 nucleotides in length in contrast to 320 nucleotides for ibv strain m41 (boursnell et al., 1985) . the 3 utr of tcov-mg10 was highly conserved with a 94-98% nucleotide identity to most published ibv and tcov 3 utr sequences. in contrast, the identity to bcov was only 45%. some viruses such as ibv, human astrovirus, and turkey astrovirus were reported to contain a stem loop-like motif (s2m) in the 3 utr (jonassen et al., 1998) . this motif appeared to be conserved among those different viruses, suggesting that it might have resulted by rna recombination between different viruses (monceyron et al., 1997) . tcov was found to contain the same motif in the 3 utr (fig. 5) . the presence of the s2m motif in the 3 utr may also suggest that ibv and tcov share a common ancestor. interestingly, sars-cov, but not other group ii coronaviruses, shares the presence of this conserved s2m motif (see fig. 5 ). sequence analyses of the 3 utr from 19 different ibv strains revealed the presence of two distinct regions: region i was highly variable and located immediately downstream of the n gene while region ii was highly conserved and located upstream of the poly(a) tail (dalton et al., 2001) . gobel et al. (2007) reported the presence of the octamer motif (5 -ggaagagc-3 ) within the 3 utr hypervariable region that was highly conserved among all coronaviruses. tcov-mg10 had two copies of the octamer motif in its 3 utr; the first copy between nucleotides 25,690 and 25,697, and the second copy between nucleotides 27,553 and 27,560. the role of this motif for coronavirus replication is unknown, but (gobel et al., 2007) suggested that it might play a role in the coronavirus replication cycle. in summary, this is the first completion of the full-length tcov genomic sequence. this study should lead to a better understanding of the molecular biology of tcov and perhaps contribute to our understanding of poult enteritis mortality syndrome (pems) affecting young turkey flocks thought to result from co-infection of tcov with turkey astrovirus. by completing the first genome of a tcov, we have established the genome organization and coding strategy for the virus that unequivocally establishes that tcov is a group iii coronavirus, closely related to ibv and other avian coronaviruses. in addition, we have identified a putatively functional gene (orf-x) shared among all sequenced ibv and tcov strains that may be a shared feature of all group iii coronaviruses. severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice poult enteritis-mortality syndrome (spiking mortality) of turkeys sequence of the nuclocapsid genes from two strains of avian invectious bronchits virus sequence analysis of turkey coronavirus nucleocapsid protein and 3 untranslated region identifies the virus as a close relative of infectious bronchitis virus gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication coronaviruses in poultry and other birds detection of a coronavirus from turkey poults in europe genetically related to infectious bronchitis virus of chickens. avian path coronavirus 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open reading frame (orf) 1b of the coronavirus infectious bronchitis virus is processed by orf1a products proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10 kilodalton polypeptide and determination of its cleavage sites molecular characterization of the 3 end of the astrovirus genome coronaviral enteritis of turkeys (bluecomb disease) antibiotic in treatment of unfamiliar turkey disease site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle a contemporary view of coronavirus transcription unique and conserved features of genome and proteome of sars coronavirus, an early split-off from the coronavirus group 2 lineage paup: phylogenetic analysis using parsimony, version 3.1 computer program distributed by the illinois natural history survey detection of turkey coronavirus in commercial turkey poults in brazil the clustal x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools sequence analysis of the turkey enteric coronavirus nuclocapsid and membrane protein genes: a close genomic relationship with bovine coronavirus analysis of a hypervariable region in the 3 non-coding end of the infectious bronchitis virus genome sars coronavirus e protein forms cation-selective ion channels phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein human coronavirus 229e papin like protease have overlapping specificities but distinct functions in viral replication virus-encosed proteinasees and proteolytic processing in the nidovirales the autolytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogus papin-like proteinase that cleave the same peptide bond this study was supported by nserc canada. mg is a scholarship recipient of the arab republic of egypt. we would like to thank janet swinton for technical assistance. key: cord-312489-ywep0c08 authors: andoh, kiyohiko; suenaga, kiyotaka; sakaguchi, masashi; yamazaki, kenichi; honda, takashi title: decreased neutralizing antigenicity in ibv s1 protein expressed from mammalian cells date: 2015-10-02 journal: virus res doi: 10.1016/j.virusres.2015.06.019 sha: doc_id: 312489 cord_uid: ywep0c08 we evaluated the antigenicity of recombinant infectious bronchitis virus (ibv) s1 protein expressed in mammalian cells. recombinant s1 was expressed as a secreted protein fused with a trimerization motif peptide, then purified using ni sepharose. the purified protein was analyzed by western blotting, mixed with oil adjuvant, and administered to 29-day-old specific-pathogen-free chickens. six weeks after immunization, anti-ibv neutralizing titer and anti-s1 elisa titer were determined; immunized chickens then were inoculated with ibv via the trachea and ciliary activity was observed. results showed that the recombinant s1 protein was highly glycosylated, and the neutralizing antigenicity of recombinant s1 protein was lower than that of inactivated virus. however, anti-s1 elisa indicated that the recombinant s1 protein induced antibodies against s1. these results suggest that the recombinant s1 may retain non-neutralizing epitopes but have unnatural glycosylation pattern and conformation, resulting in lacking neutralizing conformational epitopes. in conclusion, the neutralizing antigenicity of recombinant s1 protein expressed from mammalian cells was decreased, and was not sufficient to induce neutralizing antibodies. infectious bronchitis virus (ibv) belongs to the order nidovirales, family coronaviridae, genus gammacoronavirus, and causes respiratory disease and pathology in the kidney and gonads of chickens and other birds (boltz et al., 2004; cavanagh, 2007) . ibv is an economically important disease in the poultry industry, and several vaccines have been used to prevent the spread of ibv. ibv shows extensive antigenic variation, reflecting mutation of the spike protein gene (cavanagh et al., 1988 (cavanagh et al., , 1997 wang and huang, 2000) . vaccines targeting individual ibv serotypes yield poor cross-protection; therefore, various attenuated and inactivated multivalent vaccines (derived from several different serotypes) are used (deguchi et al., 1998; sjaak de wit et al., 2011) . the spike protein (s) is an envelope glycoprotein that forms a dimer or trimer, and has been shown to play an important role in viral infection (cavanagh et al., 1986; ignjatovic and galli, 1994; wickramasinghe et al., 2011) . s is highly glycosylated, and based on its amino acid sequence, the spike protein is predicted to contain 21 to 35 n-glycosylation sites. s has two main functions: to attach the virus to the host cell receptor, and to activate fusion of the virion membrane with the host cell membrane (casais et al., 2003; wickramasinghe et al., 2011) . the s protein is the most important antigen in inducing neutralizing antibodies against ibv, and the n-terminal s1 region is especially important (cavanagh et al., 1986; ignjatovic and galli, 1994; kant et al., 1992; koch et al., 1990; promkuntod et al., 2014) . the s1 domain forms the bulbous head of the spike protein, and several virus neutralization (vn) epitopes have been reported to reside within the first and third quarter of the s1 sequence (cavanagh et al., 1988; kant et al., 1992; koch et al., 1990; sjaak de wit et al., 2011) . thus, analysis of the antigenicity of the s1 domain is expected to be critical to the development of effective anti-ibv vaccines. to prevent ibv infection, several recombinant subunit vaccine developments have been attempted using the recombinant s1 protein or other proteins. immunization with recombinant s1 protein expressed from baculovirus has been shown to provide in chicken effective protection against ibv infection (song et al., 1998) . other groups also have reported that immunization with recombinant s1 http://dx.doi.org/10.1016/j.virusres.2015.06.019 0168-1702/© 2015 elsevier b.v. all rights reserved. epitope peptide expressed from escherichia coli (e. coli) protected chickens against ibv infection (yang et al., 2009a (yang et al., , 2009b . furthermore, viral vectored vaccines co-expressing the s1 protein and host cytokines have been reported to induce anti-s1 antibodies (chen et al., 2010; shi et al., 2011; tomley et al., 1987; wang et al., 2009; zeshan et al., 2011; zhang et al., 2012) . however, in these reports of protection, recombinant antigen did not provide perfect protection and antibody titers were evaluated only by hemagglutination inhibition (hi) or enzyme-linked immunosorbent assay (elisa); the respective authors did not indicate whether these antigens retained their native neutralizing antigenicity (song et al., 1998; yang et al., 2009a yang et al., , 2009b . therefore, it remains unknown whether recombinant s1 protein, without the s2 domain, completely retains its conformational epitopes and neutralizing antigenicity. to address these issues, we analyzed the recombinant s1 expressed from mammalian and avian cells by western blotting and analyzed the neutralizing antigenicity of recombinant s1 protein and compared titers against those of inactivated virus antigen. for the immunization experiment, recombinant s1 protein was expressed as the secreted protein with a trimerization motif because some researchers have reported that secreted recombinant s1 protein, when expressed fused to the trimerization motif peptide, retains the ability to bind the cell receptors (promkuntod et al., 2013; wickramasinghe et al., 2011) and it seemed to be suited to the vaccine antigen. the present work employed the tm86 ibv strain, an isolate of genotype jp-ii that originally was recovered from a field chicken; this strain subsequently has been used as a vaccine strain (ariyoshi et al., 2010; mase et al., 2004) . tm86 was propagated in specific-pathogen-free (spf) chicken embryonated eggs. ibv tm86 adapted to chicken kidney (ck) cells was used for the vn test and elisa of the antigen. ck cells and chicken embryo fibroblast (cef) cells were incubated in eagle's medium (emem) supplemented with 10% tryptose phosphate broth and 5% heat-inactivated fetal bovine serum (fbs; hyclone), along with 100 units of penicillin and 100 g of streptomycin per ml, and cells were grown at 37 • c in 5% co 2 incubators. 293t cells and 293 cells lacking nacetylglucosaminyltransferase i (293 gnti − , reeves et al., 2002) were propagated in dulbecco's modified eagle's medium (dmem) supplemented with 10% fbs along with 100 units penicillin and 100 g of streptomycin per ml, and cells were grown at 37 • c in 5% co 2 incubators. a segment of the s1-encoding gene, coding for the protein from the n-terminus to the cleavage site between s1 and s2, was amplified from the spike gene of ibv strain tm86 (accession no. ab120655) by reverse transcriptase polymerase chain reaction (rt-pcr). the primer set used for rt-pcr was 5 -caaattattggtcagagatgttgg-3 (s1.1) and 5 -gaatcattaaacagactttttaggtct-3 (s2r1). after amplification, the dna encoding the signal sequence of s1 (mlvkslflvtll-falcs) was replaced with a dna sequence encoding the signal sequence of marek's disease virus (mdv)-glycoprotein a (ga) (mltprvlralgwtglfflllspsnvl). furthermore, dna sequences encoding a 6× his-tag peptide sequence, with or without those encoding a t4 phage fibritin coiled-coil trimerization motif (gsgyi-peaprdgqayvrkdgewvllstflg), were inserted in-phase and downstream of the recombinant s1-encoding gene. the resulting fig. 1 . schematics of the ibv s1 segment expressed as a secreted protein. to construct the secreted s1 expression plasmid, the nucleotide sequence encoding the s1 domain lacking the cleavage sequence (rrfrr) was amplified from the ibv tm86 genome. to increase expression levels of the recombinant s1 protein, the nucleotide sequence encoding the signal sequence was replaced with a nucleotide sequence encoding mdv ga. to facilitate purification and multimerization, the resulting sequence was cloned upstream and in-phase with nucleotide sequences permitting expression as c-terminal fusions to a 6× his-tag peptide sequence with or without a t4 phage fibritin coiled-coil trimerization motif. loci encoded the recombinant s1 protein with c-terminal fusions to the indicated domains ( fig. 1) . the constructed sequence was cloned into the sal i site of the expression plasmid pcaggs, which contains a cag promoter (modified chickenˇ-actin promoter with cytomegalovirus immediate-early (cmv-ie) enhancer), rabbitˇglobin gene sequence including polyadenylation signal, and simian virus 40 (sv40) ori (niwa et al., 1991) . the constructed plasmids were named pcaggs-s1-t4-his and pcaggs-s1-his, respectively. for transfection, the plasmid was purified using a qiaprep spin miniprep kit (qiagen). the mouse mabs against ibv s1 or s2 region were established in our institute. it was confirmed that these mabs recognize nonconformational epitopes by western blotting under denaturing condition. it was also confirmed that these mabs do not show neutralizing activity in vn test. before examination, the mabs were purified using protein g column. 293t, 293 gnti − , and cef cells were transfected with either of the constructed plasmids using polyethylenimine (pei). transfection using pei was performed according to the protocol established by boussif et al. (1995) . briefly, 88 g of the plasmid was mixed with 440 l of pei (2 mg/ml) and then transfected into approximately 2 × 10 7 of 293t cells. transfected cells were incubated in opti-mem (life technologies) and the supernatant of transfected cells was harvested at 72-96 h post-transfection. the recombinant protein was purified using ni sepharose (ge healthcare) according to the manufacturer's protocol. sds-page was carried out under denaturing and nondenaturing conditions. under denaturing conditions, the purified protein was mixed with 2× sample buffer (100 mm tris-hcl (ph 6.8), 4% sds, 20% glycerol, 0.2% bromphenol blue) containing 200 mm dithiothreitol (dtt) and boiled for 5 min at 95 • c. in contrast, non-denaturing sds-page was performed according to the protocol reported by bender et al. (2005) . briefly, the purified protein was mixed with 2× sample buffer containing 0.4% sds (in the absence of a reducing agent) and loaded directly onto a gel (without boiling of the sample). the protein was separated by 5-20% polyacrylamide gradient gel (e-pagel, atto) and transferred to a polyvinylidene difluoride (pvdf) membrane (millipore). the membrane was incubated in 5% skim milk (wako) in t-pbs buffer (phosphate-buffered saline, ph 7.2 (pbs) containing 0.05% tween 20) for 60 min at 37 • c, and then incubated with a mab against s1 protein in 5% skim milk in t-pbs buffer. next, the membrane was incubated with a peroxidase-conjugated second antibody, goat anti-mouse igg (h + l) (jackson). the reacted protein was visualized using a tmb substrate kit (invitrogen). the immunization experiment consisted of 4 groups of 5 or 10 chickens. the spf chickens (layer-type) used for this study were maintained in our institute and 10 chickens were immunized with the purified recombinant s1 protein, which was his-tagged with trimerizaton motif, or inactivated virus, respectively. five chickens were immunized with pbs-mock vaccine (mock group) and 10 chickens were left untreated to serve as an unvaccinated control group. light liquid paraffin, sorbitan monooleate, and polysorbate 80 in a volume ratio of 9:36:4:1 was used as oil adjuvant. recombinant s1 protein was mixed with oil adjuvant to make an s1 suspension at a concentration of 20 g/dose. the ibv tm86 strain was inactivated using formaldehyde and mixed with oil adjuvant to generate a suspension harboring inactivated virus at a virus concentration of 10 7.0 eid 50 /dose. chickens were inoculated intramuscularly. at 6 weeks post-inoculation, blood was collected from each animal, and the resulting sera were used to determine antibody titers. each chicken then was challenged with 10 3.5 eid 50 ibv tm strain administered via the trachea. animals were sacrificed at 4 days post-challenge and ciliostasis was assessed by a slightly modified version of the previously reported protocol (cook et al., 1999) . briefly, tracheas were removed aseptically from euthanized chickens. cilia were observed microscopically and the cessation of ciliary movement was considered to be a sign of symptoms. sera were serially diluted two-fold with emem in a microplate and mixed with 200 tcid 50 of ck-adapted ibv tm strain. after incubation for 1 h at 37 • c, ck cells were inoculated with ibv and incubated at 37 • c in 5% co 2 incubators. vn titer was defined as the reciprocal of the highest dilution showing no cytopathic effect (cpe). the mab against ibv s1 protein was diluted to 2 g/ml with pbs and 50 l was added to each well of a 96-well microplate (maxisorp; nunc, denmark). after incubation overnight at 4 • c, plates were washed three times with t-pbs. next, 300 l of t-pbs containing 5% skim milk was added and incubated at room temperature (rt) for 1 h. after washing three times with t-pbs, 50 l of s1 antigen was added to each well and incubated at rt for 1 h. for the recombinant s1 elisa, supernatant of 293t cells transfected with the expression plasmid pcaggs-s1-his was used. for the native spike elisa, lysate of ibv-infected ck cells was used as the antigen. ck cells were infected with ibv at a moi of 0.2 and extracted 72 h post-infection by treatment with ripa buffer (containing 1% tritonx-100, 1% sodium deoxycholate, 0.05 m tris-hcl (ph 8.0), 0.1 m nacl, and 1 mm edta). antigens were diluted with dilution buffer (t-pbs containing 5% skim milk) and added to wells. the levels of recombinant and native s1 protein used for the elisa test were selected as the respective concentrations that yielded absorbances (following reaction with the positive serum) of 1.0 to 1.5. the contents of the wells were washed with t-pbs; an aliquot (50 l) of sera (diluted 1:100 with dilution buffer) was dispensed to each well, and plates were incubated at rt for 1 h. in the recombinant s1 elisa, primary sera were pre-treated with 1 g/ml of purified his-tagged ibv e protein at 4 • c overnight to remove antibodies against the his-tag peptide. the contents of the wells were washed with t-pbs; an aliquot (50 l) of peroxidaseconjugated anti-chicken immunoglobulin (donkey anti-chicken igy (h + l) (jackson)) was dispensed to each well, and plates were incubated at rt for 30 min. the contents of the wells were washed with t-pbs; an aliquot (100 l) of tmb substrate kit (dako) was dispensed to each well, and plates were incubated at rt for 15 min. after incubation, the enzymatic reaction was stopped by addition of 100 l/well of 1 m sulfuric acid. the absorbance was measured using a spectrophotometer (versamax), with a 450-nm and 650nm filter. a hyperimmune serum against ibv, which was prepared by immunization with attenuated and inactivated vaccines, was used as the positive serum. separate work (data not shown) using chicken antisera against ibv e, m and n proteins and anti-s2 mouse mab (described above) confirmed that native s1 elisa does not react with antibodies raised against ibv component proteins s2, e, m, or n. antisera against ibv e, m and n were prepared from chickens immunized with purified recombinant proteins expressed from e. coli and it was confirmed that these antisera reacted to ibv component by elisa (data not shown). all statistical analyses were performed with ezr (saitama medical center, jichi medical university), which is a graphical user interface for r (the r foundation for statistical computing) (kanda, 2013) . the constructed expression plasmids, pcaggs-s1-t4-his and pcaggs-s1-his, were transfected into cells and the recombinant protein was expressed as secreted protein. the secreted s1 protein was purified using ni sepharose and analyzed by western blotting. the result of western blotting under denaturing condition showed that recombinant s1 protein expressed from 293t cells ran as a broad band of nominal molecular weight ranging from 100 to 150 kda. the size range of this band was increased compared to that of native s1 protein (derived from ibv propagated in chicken embryonated eggs), with the native protein running on the denaturing gel as a single sharp band at a nominal size of approximately 100 kda (fig. 2) . recombinant s1 protein derived from 293 gnti − cells, which lack the ability to synthesize complex n-glycans, ran on the denaturing gel as a sharp band at a nominal molecular weight of 75 to 100 kda, with a size smaller than that observed for recombinant protein expressed from 293t cells (fig. 2) . recombinant s1 protein expressed in cef cells (cells that are derived from the natural host for ibv) also ran under denaturing conditions as a slightly broader and larger band than native s1 obtained from ibv virions; notably, the protein produced in cef cells ran as a sharper band than protein produced in 293t cells (fig. 3) . these results indicated that recombinant s1 protein might have an unnatural glycosylation pattern. using western blotting under non-denaturing conditions, we examined the conformation of recombinant s1 protein produced and secreted from 293t cells. we observed that recombinant s1 protein lacking the fibritin motif existed in monomeric, dimeric, and trimeric conformations. on the other hand, recombinant s1 protein with the fibritin motif existed primarily in the trimeric conformation (fig. 4) . for the immunization experiment, the purified recombinant s1fibritin protein expressed from 293t cells was mixed with adjuvant to generate a s1 inoculum at a concentration of 20 g/dose. vn titer induced by the recombinant s1 protein was lower (p < 0.05) than that induced by the inactivated virus antigen (fig. 5) . to compare the efficacy against ibv infection, ciliostasis was evaluated via ibv challenge in animals previously immunized with s1 or with inactivated ibv virus; as a control, unvaccinated and mock-vaccinated . the asterisk indicates statistical significance (p < 0.05) and the error bar indicates standard deviation. comparison between two groups was performed using t test. protection defined by ciliostasis in the trachea. protection a recombinant s1 4/10 inactivated virus 10/10 unvaccinated control 3/10 pbs-mock 0/5 a birds showing active ciliary movement/total. chickens also were subjected to ibv challenge. following challenge, all (10/10) chickens immunized with inactivated virus antigen retained ciliary movement. in contrast, post-challenge ciliostasis was observed in 60% (6/10) of animals immunized with recombinant s1, 70% (7/10) of unvaccinated animals, and all (5/5) mock-vaccinated chickens (table 1 ). these results indicated that immunization with recombinant s1 protein was not sufficient to provide protection from subsequent ibv infection. to analyze the antigenicity of the recombinant s1 protein, sera obtained from the immunization experiment were analyzed by elisa, using recombinant or native s1 protein as antigen. recombinant s1 elisa showed that sera from chickens immunized with recombinant s1 protein had a slightly high, but not significant (p = 0.14), titer than sera from chickens immunized with inactivated virus (fig. 6a) . native s1 elisa detected similar titers in the sera of animals immunized with recombinant s1 protein compared to those in sera of chickens immunized with inactivated virus (fig. 6b ) (control experiments (data not shown) demonstrated that mock and unvaccinated control groups did not differ from each other in vn and elisa tests of anti-s1 activity.) these results indicated that while recombinant s1 protein retained antigenicity (the ability to induce antibodies against s1 protein), the resulting antibodies was decreased its neutralizing activity, in contrast to those induced by inactivated virus. 6 . indirect sandwich elisa using recombinant s1 protein (a) and the lysate from ibv-infected cells (b). primary sera were collected from immunized chickens and diluted 1:100. in recombinant s1 elisa, primary sera were pre-adsorbed with purified recombinant his-tagged protein to remove the antibodies against the histag peptide sequence. reactivities are shown as the s/p ratio of absorbance at 450 nm and 650 nm. comparisons between three groups were performed using steel-dwass test and n.s. indicated not significant. this study showed that recombinant s1 protein exhibited decreased ability to induce a neutralizing antibody response, although the recombinant s1 induced non-neutralizing antibodies. the elisa using recombinant s1 used sera that already had been adsorbed with his-tag peptides, precluding a role for the purification (his-tag) domain in the induced antibody response. in the elisa using native s1, the native protein may have contained noncleaved spike protein, thus including both s1 and s2 domains as part of the antigen. however, we found (data not shown) that inclusion ("spiking") of s2 protein did not affect the results of the native s1 elisa. this observation is consistent with other reports suggesting that the s2 region contains fewer neutralizing epitopes than does the s1 region; the literature indicates that most epitopes are located in the s1 region (cavanagh et al., 1986; ignjatovic and galli, 1994; kant et al., 1992; koch et al., 1990; promkuntod et al., 2014) . these results excluded the possibility that the other viral components affect the titer in elisa. thus, although recombinant s1 protein induced antibodies, this recombinant protein was decreased the ability to induce neutralizing antibodies, suggesting the recombinant s1 expressed in mammalian cells may change its character. in a separate test, we observed that antigen from inactivated virus that was denatured by boiling (5 min, 95 • c) did not induce neutralizing antibodies (data not shown). this result suggested that the correct conformation is important for s1 protein to induce neutralizing antibodies. in the present study, the recombinant s1 protein used for immunization was expressed in mammalian cells, rather than in avian cells; the expression efficiency of recombinant proteins in avian cells was decreased compared to that in mammalian cells, and expression in avian cells was insufficient to obtain recombinant proteins in the quantities needed for the experiments. the present work also used recombinant s1 expressed as secreted protein because secretion permitted accumulation of recombinant protein to higher levels and made it easy to purify recombinant protein. these modifications, using mammalian cells and expressing recombinant protein as the secreting form, may affect the conformation of the s1. western blotting using denaturing conditions indicated that recombinant s1 protein expressed in 293t cells exhibited increased (and less uniform) sizes compared to the native protein. in contrast, recombinant s1 protein expressed in 293 gnti − cells (which lack n-acetyl-glucosaminyltransferase i activity and so do not generate complex n-glycans) ran as a sharper band of a size similar to that of native s1 protein. together, these data indicated that the glycosylation pattern of recombinant s1 protein might differ from that of native s1, and that this difference reflects the level and nature of n-glycosyl modifications. we hypothesize that the native spike protein is typically decorated with high-mannose or hybridtype n-glycans, and/or that the complex-type glycans decorating the native spike proteins are few in number and are homogeneous. it is known that glycosylation characteristics differ between species, and glycosylation affects the conformation of glycoproteins (helenius, 1994) . therefore, glycosylation pattern differences may affect the conformation and antigenicity of the s1 protein. furthermore, glycans attached on the surface of the envelope protein are known to mask antigenic sites, thereby permitting evasion of the host immune system (sun et al., 2011; zhang et al., 2015) . we infer that recombinant s1 protein antigenic sites also may be masked by glycans. from these hypotheses, there is the possibility that s1 expressed in other expression systems, which have different glycosylation character, also changes its antigenicity. other reports suggest that the s2 region of the ibv spike protein is important for the correct conformation of s (callison et al., 1999; promkuntod et al., 2013) . therefore, the recombinant s1 protein secreted in this study might assume a non-native conformation. we hypothesize that expression of the s1 domain without the s2 region results in the assumption of an unnatural conformation, resulting in unnatural glycosylation patterns, or that unnatural glycosylation of s1 protein causes the recombinant peptide to assume an unnatural conformation. recombinant s1 protein may expose hidden epitope(s) that are normally latent in the protein. however, some researchers have reported that secreted recombinant s1 protein, when expressed fused to the trimerization motif peptide, binds the cell receptors (promkuntod et al., 2013; wickramasinghe et al., 2011) . these reports suggested that the secreted s1 protein retains receptor binding ability. in addition, expression of other viral envelope proteins (specifically, human immunodeficiency virus (hiv) gp120 and influenza virus hemaglutinin (ha)) as secreted proteins have been reported to induce neutralizing antibodies (pancera et al., 2005; wei et al., 2008) . these reports indicated that secreting envelope proteins with a trimerization motif is an effective method for creating vaccine antigens. additionally, cavanagh (2007) reported that ibv s1 protein was sufficient for the induction of neutralizing antibodies (thought s1 was not sufficient for protection against ibv infection). furthermore, cytotoxic t-lymphocyte (ctl) activity induced by immunization of nucleocapsid protein (np) has been shown to be important for virus clearance (collisson et al., 2000; seo and collisson, 1997) . however, in the present study, while immunization with recombinant ibv s1 (expressed as a secreted protein fused to the fibritin motif) induced antibodies, the response mainly consisted of non-neutralizing antibodies. therefore, recombinant s1 protein (expressed as a secreted protein in mammalian cells) may be insufficient for use as a vaccine antigen. recently, virus-like particle (vlp) -based antigens have been tested as new candidates for subunit vaccines against ibv (cavanagh, 2003; lv et al., 2014) . receptor binding domain (rbd) peptides also have been tested as vaccine candidates against severe acute respiratory syndrome (sars) coronavirus (jiang et al., 2012) . these alternative approaches to antigen production may overcome the challenges observed in the present work. in conclusion, we demonstrated that recombinant s1 protein (expressed as a secreted protein in mammalian cells) was decreased the ability to induce neutralizing antibodies in chicken. we infer that the recombinant protein lacked conformational epitopes as a result of changes in glycosylation and changes in conformation compared to the native s1 protein. further research will be required to identify factors necessary to establish a recombinant s1 antigen that retains native neutralizing antigenicity. classification of ibv s1 genotypes by direct reverse transcriptase-polymerase chain reaction (rt-pcr) and relationship between serotypes and genotypes of strains isolated between 1998 and 2008 in japan herpes simplex virus glycoprotein b binds to cell surfaces independently of heparan sulfate and blocks virus entry avian infectious bronchitis virus: a possible cause of reduced fertility in the rooster a versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine infectious bronchitis virus s2 gene sequence variability may affect s1 subunit specific antibody binding recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism coronavirus ibv: virus retaining spike glycopolypeptide s2 but not s1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection amino acids within hypervariable region 1 of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes relationship between sequence variation in the s1 spike protein of infectious bronchitis virus and the extent of cross-protection in vivo severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus coronavirus avian infectious bronchitis virus construction and immunogenicity of a recombinant fowlpox vaccine coexpressing s1 glycoprotein of infectious bronchitis virus and chicken il-18 cytotoxic t lymphocytes are critical in the control of infectious bronchitis virus in poultry breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes influence of inoculation site of combined oil-adjuvanted vaccine on the antibody response in chickens how n-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum the s1 glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens roadmap to developing a recombinant coronavirus s protein receptor-binding domain vaccine for severe acute respiratory syndrome investigation of the freely available easy-to-use software "ezr" for medical statistics location of antigenic sites defined by neutralizing monoclonal antibodies on the s1 avian infectious bronchitis virus glycopolypeptide antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions production and immunogenicity of chimeric virus-like particles (vlps) containing the spike (s1) glycoprotein of infectious bronchitis virus (ibv) phylogenetic analysis of avian infectious bronchitis virus strains isolated in japan efficient selection for high-expression transfectants with a novel eukaryotic vector soluble mimetics of human immunodeficiency virus type 1 viral spikes produced by replacement of the native trimerization domain with a heterologous trimerization motif: characterization and ligand binding analysis contributions of the s2 spike ectodomain to attachment and host range of infectious bronchitis virus mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous n-glycosylation by a tetracycline-inducible n-acetylglucosaminyltransferase i-negative hek293s stable mammalian cell line specific cytotoxic t lymphocytes are involved in in vivo clearance of infectious bronchitis virus evaluation of recombinant fowlpox virus expressing infectious bronchitis virus s1 gene and chicken interferon-␥ gene for immune protection against heterologous strains infectious bronchitis virus variants: a review of the history, current situation and control measures induction of protective immunity in chickens vaccinated with infectious bronchitis virus s1 glycoprotein expressed by a recombinant baculovirus glycosylation site alteration in the evolution of influenza a (h1n1) viruses expression of the infectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice relationship between serotypes and genotypes based on the hypervariable region of the s1 gene of infectious bronchitis virus protection of chickens against infectious bronchitis by a recombinant fowlpox virus co-expressing ibv-s1 and chicken ifngamma comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian h5n1 influenza virus binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity multivalent dna vaccine enhanced protection efficacy against infectious bronchitis virus in chickens the protective immune response against infectious bronchitis virus induced by multi-epitope based peptide vaccines protective immune responses induced by in ovo immunization with recombinant adenoviruses expressing spike (s1) glycoprotein of infectious bronchitis virus fused/co-administered with granulocyte-macrophage colony stimulating factor protection conferred by a recombinant marek's disease virus that expresses the spike protein from infectious bronchitis virus in specific pathogen-free chicken hemagglutinin glycosylation modulates the pathogenicity and antigenicity of the h5n1 avian influenza virus the authors wish to thank other members of the laboratory for technical assistance, and thank mr. mcculloch for proof reading of the manuscript. the authors declare that they have no conflict of interest. key: cord-280643-n8qjorqk authors: wu, kai-lang; zhang, xue; zhang, jianlin; yang, yongbo; mu, yong-xin; liu, mo; lu, lu; li, yan; zhu, ying; wu, jianguo title: inhibition of hepatitis b virus gene expression by single and dual small interfering rna treatment date: 2005-04-26 journal: virus res doi: 10.1016/j.virusres.2005.04.001 sha: doc_id: 280643 cord_uid: n8qjorqk rna interference (rnai) has been successfully applied in suppression of hepatitis b virus (hbv) replication. to circumvent the problem that mutation in hbv genome may result in resistance when sirna is further developed as an anti-viral drug, in this study, we established a dual small interfering rna (sirna) expression system, which could simultaneously express two different sirna molecules that can specifically target two genes. to test the effectiveness of this system, we applied this new approach to express simultaneously two different 21-bp hairpin sirna duplexes that specifically attack the hbs and hbx genes of hbv, respectively, in bel-7402 and hepg2.2.15 cells. results indicated that dual sirna could simultaneously inhibit the expression of hbs and hbx gene by 83.7% and 87.5%, respectively, based on luciferase assays. in addition, dual sirna molecules were able to significantly reduce the amount of hbv core associated dna, which is considered as an intracellular replicative intermediate, and the viral dna in culture supernatant. therefore, this dual sirna system provides a more powerful tool for the study of gene function and implicates a potential application in the treatment of viral infection. rna interference (rnai) is a natural process of eukaryotic cells by which double-stranded rna initiates and directs the degradation of homologous mrna (hannon, 2002) . this rna silencing mechanism was first described in caenorhabditis elegans and drosophila melanogaster (fire et al., 1988) . it has many similarities to the posttranscriptional gene silencing in plants. specific inhibition of cellular mrna by rnai can be triggered in mammalian cells by the introduction of synthetic 21-to 23-nucleotide double-stranded small interfering rna (sirna) (elbashir et al., 2001; paul et al., 2002) or, alternatively, by the transcrip-tion of sirna from a dna construct driven by the rna polymerase cassette (brummelkamp et al., 2002) . these findings open up a new field for the analysis and control of the processes of gene expression, and perhaps pathogen infection. the replication of a growing number of human pathogenic viruses in cell culture was shown to be inhibited by rnai, including poliovirus (coburn and cullen, 2002) , hiv-1 (jacque et al., 2002; lee et al., 2002) , flock house virus (fhv) (dector et al., 2002) , rous sarcoma virus (hu et al., 2002) dengue virus (adelman et al., 2002) , hepatitis c virus (hcv) (kapadia et al., 2003) replicons, influenza virus (ge et al., 2003) , hepatitis b virus (hbv) (hamasaki et al., 2003; mccaffrey et al., 2003) , hpv (jiang and milner, 2002) . recently, it was reported that rnai could also induce transcriptional silencing of sars coronavirus (he et al., 2003) . in most above studies, synthetic 21-nucleotide doublestranded sirnas were applied. however, vector based rnai techniques were used more frequently in recent studies. each vector expresses unique sirna that can degrade a specific target. eight genotypes (a-h) of hbv have been described. the number of hbv carriers worldwide has been estimated to be more than 400 million. these individuals have a 15-25% risk of developing liver diseases such as liver cirrhosis and hepatocellular carcinoma (kao and chen, 2002) . although a few drugs were developed against hbv infection, the success rate of these treatments, however, is low and frequently infections reoccur (carreno et al., 1992; lai et al., 1997) . the fact that rnai can be applied for blocking the replication of hbv in several reports provided insights into the field of controlling infectious human hepatitis. nevertheless, mutations in hbv genome may result in viral resistance to sirna. it has been reported that hiv-1 can escape from rnai-mediated inhibition due to nucleotide change in the genome (das et al., 2004) . one strategy to circumvent the problem is to choose target in the relatively conserved dna sequence. the other approach is to produce multiple sirnas that target different sites or genes on the viral genome. we here established a system that can express two sirna duplexes simultaneously and target the s and x genes of hbv, respectively. to study the effects of dual rnai on hbv gene expression in a cell culture model, we used a derivative of the human hepg2 hepatoma cell line, hepg2.2.15, which has been stably transformed with several copies of the hbv genome and used as an in vitro model for hbv replication. the effects of dual sirna system on hbv gene expression were investigated in this study. two human hepatoma cell lines, bel-7402 and hepg2.2.15 were maintained in dulbecco's modified eagle medium (gibco/brl) supplemented with 100 units/ml penicillin, 100 g/ml streptomycin and 10% heat-inactivated fetal bovine serum at 37 • c under 5% co 2 . cells were seeded onto 24-well plates at a density of 1.0 × 10 5 or 4.0 × 10 5 cells per 24-well plate or 6-well plate and grown to the confluence reaching approximately 60% at the time of transfection. cells were transfected with 0.1 or 0.4 g of plasmid pcmv-hbs together with 0.45 or 1.2 g psliencer-2.1-u 6 -sirna, using sofast tm transfection reagent (xiamen sunma biotechnology co. ltd., china) according to the protocol provided by the manufacturer. the cells were harvested 48 h after transfection. full-length hbv genomic dna (subtype ayw) was cloned into the hindiii and saci sites of pbluescript (stratagene) to generate the plasmid pblue-hbv. hbs gene was cloned into the hindiii and saci sites of vector pcmv-tag2a (stratagene) fig. 1 . schematic diagrams of luciferase fusion genes, sirna targeting sites and dual sirna expression cassettes. (a) diagram of the two reporter fusion vectors, which contain targeted sequences of hbs or hbx gene and the luciferase report gene driving by the cmv promoter. (b) locations of rnai targeted sites and structure of the hbv genome. downward arrows indicate the locations of rnai target sites within the four hbv transcripts. the 3.5-kb transcript is the pregenomic rna that serves as the template for hbv viral dna replication. the hbv open reading frames are shown below aligned with the hbv mrnas. pol, polymerase; core, hbcag; s1, large presurface antigen; s2, mid pre-surface antigen; s, hb-sag; x, x gene. the numbers above the arrows indicate the sirna target sites. 1 = hbs 1 sirna, 2 = hbs 2 sirna, 3 = hbs 3 sirna, 4 = hbs 4 sirna, 5 = hbs 5 sirna, 6 = hbx 1 sirna, 7 = hbx 2 sirna, 8 = hbx 3 sirna. (c) diagram of dual sirna expression cassettes. to yield plasmid pcmv-hbs. hbx gene was cloned into pcmv-tag2a at ecori and xhoi sites to generate pcmv-hbx. two-pair of primers 5 -ctgcgagatctatggagagc-tcacatcaggattc-3 (sense), 5 -gttaggtcgacaa-tgtatacccaaagacaaaaagaa-3 (antisense) or 5 -gatcatacgcgtaagcttttcatttattgatcat-3 (sense), 5 -gtcggggcttcattcactcgtctagaac-tgat-3 (antisense) were used to amplify the hbs and hbx gene, respectively. the pcr products were then cloned into sali and bglii sites of plucf to generate plasmid plucf-hbs and plucf-hbx (fig. 1a) , in which the hbs or hbx were fused in frame with the luciferase gene and the expression of the fusion gene was drove by the cmv promoter (fig. 1a ). five regions of the hbs gene and three regions of the hbx gene were selected as the targeted sequences of sirna in this study (fig. 1b) . to construct single sirna expression vector, two 64nt primers, each containing a 19nt target sequence in the sense and antisense forms from different regions of the hbs gene or hbx gene as indicated below, were systhesized (invitrogen): 5 -gctcccgcgtgtcttggcc-3 (hbs 1 sirna); 5 -ggtggacttctctcaattt-3 (hbs 2 sirna); 5 -gccaaaattcgcagtccc-3 (hbs 3 sirna); 5 -gttgctgtaccaaacctt-3 (hbs 4 sirna); 5 -gctcagtttactagtgcca-3 (hbs 5 sirna); 5 -gcacttcgcttcacctctg-3 (hbx 1 sirna); 5 -gcaatgtcaacgaccgacc-3 (hbx 2 sirna); 5 -gtttaaagactgggaggag-3 (hbx 3 sirna). sense and antisense primers were then cloned into psilence-2.1-u 6 plasmid (amibion) at bamhi and hindiii sites after annealing according to the manufacturer's instructions. to generate the dual sirna expression plasmid, two primers 5 -gctgatgacgtcagtggaaagacgcg-3 -(sense) and 5 -tcagcgaattcacgccaagcttttcc-3 (antisense) were designed to amplify a dna fragment containing u6 promoter and hbx 2 sirna expression cassette from recombinant plasmid psilencer-2.1-u6-hbx 2 . the pcr product was then cloned into aatii and ecori sites of plasmid psilencer-2.1-u6-hbs 2 to generate recombinant plasmid psilencer-2.1-u6-hbsx, which carries two independent sirna expression cassettes (fig. 1c ). bel-7402 cells were co-transfected with reporter plasmids and sirna expression plasmids. cells were washed with pbs and lysed with luciferase cell culture lysis reagent (promega). ten microliters of the cell lysates and 100 l of luciferase assay substrate (promega) were mixed and fluorescence intensity was detected by the luminometer (turner t20/20). assays were performed in triplicate, and expressed as means ± s.d. relative to vector control as 100%. bel-7402 cells and hepg2.2.15 cells were transfected with sirna expression plasmids, the level of hbsag protein in culture media from transfected cells were then determined by enzyme-linked immunosorbent assay using a hbv diagnostic kit (shanghai kehua biotech co. ltd.). assays were performed in triplicate independent experiments. bel-7402 cells and hepg2.2.15 cells were transfected with sirna expression plasmids, total rna were then extracted from transfected cells by trizol reagent (invitrogen) according to the method described in the manufacturer's manual. reverse transcription were performed with total rna as the template. the cdnas were synthesized with hbs or hbx gene specific primers, 5 -gcggggtttttcttgttgac-3 (sense), 5 -ctacgaaccactgaacaaat-3 (antisense) or 5 -cctgcgcgggacgtcctttg-3 (sense), and 5 -cagtctttgaagtatgcctc-3 (antisense). to assay the effect of sirnas on hbv replication, intracellular core-associated hbv dna was extracted by the method described previously (pugh et al., 1988) . briefly, 1 × 10 5 transfected hepg2.2.15 cells were lysed and centrifuged at 25 • c. magnesium chloride was added to the supernatant. dna not protected by hbv core was treated digested with deoxyribonuclease (dnase i). then the lysates were treated with proteinase-k and, after phenol/chloroform extraction; core-associated hbv dna was recovered by ethanol precipitation, and quantified by real time-pcr (rt-pcr) as described by the manufacturer (pg biotech, shenzhen, china). the hbv dna in the supernatants was also quantified following the procedure provided by the manufacturer (pg biotech, shenzhen, china). primers used in rt-pcr were: p1, 5 -atcctgctgctatgcc-tcatctt-3 and p2, 5 -acagtggggaaagcccta-cgaa-3 . the probe was 5 -tggctagtttactagtgc-cattttg-3 . pcr reaction was carried out and analyzed by a pe gene amp 7700 (perkin-elmer, usa). to efficiently screen sirna molecules, selected targeting dna sequences were fused in frame with that of luciferase gene, in which luciferase activity was supposed to represent the level of hbs or hbx mrna expression. cells were co-transfected with plucf-hbs or plucf-hbx and eight single sirna expression vectors, respectively. luciferase activities were then determined from those transfected cells. result showed that hbs 1 sirna, hbs 2 sirna and hbx 2 sirna strongly inhibited luciferase activities by 81.5%, 80.5%, and 76.5%, respectively, comparing to that of vector control ( fig. 2a and b) . these results indicated that the three sirnas could efficiently degrade the mrna of hbs-luciferase or hbx-luciferase fusion gene. to evaluate the effects of dual sirna expression plasmid on the inhibition of hbs-luciferase or hbx-luciferase fusion gene expression, cells were co-transfected with plucf-hbs or plucf-hbx and the dual sirna expression plasmid phb-sxsirna. result from luciferase activity assays indicated that there was a further reduction in luciferase activity by dual fig. 2 . quantitative analysis of luciferase activity in cells after transfected with sirna expression plasmids. (a) bel-7402 cells were co-transfected with plucf-hbs plasmid and psliencer-2.1-u 6 -sirna (hbs 1 sirna, hbs 2 sirna, hbs 3 sirna, hbs 4 sirna, hbs 5 sirna) plasmids; psliencer-2.1-u 6 plasmid was used as a control. (b) bel-7402 cells were co-transfected with plucf-hbx plasmid and psliencer-2.1-u 6 -sirna (hbx 1 sirna, hbx 2 sirna, hbx 3 sirna) plasmids; psliencer-2.1-u 6 vector was used as a control. (c) bel-7402 cells were co-transfected with plucf-hbs and psliencer-2.1-u 6 -sirna (hbs 2 sirna, hbx 2 sirna, hbsxsirna) plasmids; psliencer-2.1-u 6 was used as vector control. (d) bel-7402 cells were co-transfected with plucf-hbx and psliencer-2.1-u 6 -sirna (hbx 2 sirna, hbsxsirna, hbs 2 sirna); psliencer-2.1-u 6 was used as control. forty-eight hrs after transfection, cells were lysed and luciferase activities were determined by luminometer. sirna duplexes (hbsxsirna) comparing to that of single sirna expression vectors (hbs 2 sirna or hbx 2 sirna). the reduction rate of luciferase activity caused by hb-sxsirna was 83.7% to hbs and 87.5% to hbx, respectively ( fig. 2c and d) . to evaluate the influence of rnai on hbs gene expression, bel-7402 cells were transfected with psilence2.1-u6-sirna, pcmv-hbs or hbsxsirna and hepg2.2.15 cells were transfected with psilence2.1-u6-sirna or hbsx sirna. hbsag concentrations in the culture media of transfected and control cells were measured 2 days after transfection by elisa using hbv diagnostic kit. results showed that hbsag level was decreased in the bel-7402 cells after transfection with hbs 1 sirna, hbs 2 sirna or hbsxsirna with reduction rate of 91.5%, 88.5% ,and 83.7%, respectively ( fig. 3a and d) . in hepg2.2.15 cells, transfection with hbs 1 sirna, hbs 2 sirna, or hbsxsirna reduced hbsag level by 75.4%, 85.7%, and 87.6%, respectively ( fig. 3b and c) . in addition, transfection with hbx 2 sirna reduced the level of hbsag production by 65.3% in hepg2.2.15 cells (fig. 3e) . to determine whether sirnas specificly degrade hbs or hbx mrna, we used semi-quentitation rt-pcr analyses to determine the levels of hbs or hbx mrna in two different cell lines, bel-7402 ( fig. 4a and b) and hepg2.2.15 ( fig. 4c and d) , 2 days after transfection. results indicate that the levels of hbs mrna were significantly decreased by the treatment of hbsxsirna (fig. 4a, lane 1) , hbs 1 sirna (fig. 4a, lane 2) , hbs 2 sirna (fig. 4a, lane 3) in bel-7402. the levels of hbx mrna were also decreased by the treatment of hbsxsirna (fig. 4b, lane 1) , hbx 2 sirna (fig. 4b, lane 2) , but not by that of psliencer-2.1-u 6 or untreated cells (fig. 4b, lanes 3 and 4) . similar results were also obtained in hepg2.2.15 cell lines under the same conditions of sirna treatments ( fig. 4c and d) . results showed that the levels of hbs mrna were dramatically reduced in hepg2.2.15 cells after the treatment of hbsxsirna (fig. 4c, lane 6) , hbs 1 sirna (fig. 4c, lane 7) and hbs 2 sirna (fig. 4c, lane 8) , respectively. the levels of hbx mrna in hepg2.2.15 cells were also reduced by the treatment of hbsxsirna (fig. 4d, lane 2) , hbx 2 sirna (fig. 4d, lane 3) , but not by that of psliencer-2.1-u 6 or untreated cells (fig. 4d, lanes 1 and 4) . in addition, our results showed that the inhibition effects of dual sirna, hbsxsirna on the levels of hbs and hbx mrna (fig. 4a (lane 1) , b (lane 1), c (lane 6), and d (lane 2)) were more sever than that of single sirna (fig. 4a, (lanes 2 and 3) , b (lane 2), c (lane 7 and 8), and d (lane 3)). to determine the effectiveness of sirnas on viral dna replication, hbv core associated dna (as an intracellular replicative intermediate) and hbv dna were extracted from hepg2.2.15 cells transfected with hb-sxsirna, hbs1sirna, hbs2sirna, hbx2sirna, and vector, respectively. the levels of hbv core associated dna and hbv dna were determined by real time pcr. results indicated that the levels of hbv core associ-ated dna were significantly decreased in the cells transfected by hbsxsirna, hbs 1 sirna, hbs 2 sirna, and hbx 2 sirna with reduction rate of 90.2%, 85.7 %, 81.3%, and 60.4%, respectively, compared with that of vector control (fig. 5a) . in hepg2.2.15 cells, transfection with hb-sxsirna, hbs 1 sirna, hbs 2 sirna and hbx 2 sirna reduced the level of viral dna in supernatants media by 88.7%, 82.6%, 78.4%, and 58.3%, respectively (fig. 5b) . it has been attracted considerable attentions in the use of rnai as therapeutics to treat a variety of diseases, including tumors and viral infections. hamasaki et al. (2003) demonstrated that rnai could attenuate the replication of hbv genome in cell culture. shlomai and shaul (2003) used a similar approach to inhibit the replication and expression of hbv in hepg2.2.15 cell line, in which all hbv proteins could be expressed. recently, mccaffrey et al. (2003) went further in this field by showing that rnai were function well in transgenic mice. these reports demonstrate that sirna treatments can be used to suppress hbv in cell cultures and animal models as well as provided insights into the application of controlling infectious human hepatitis. in this study, we applied a different approach by designing a pair of 64nt primers that contain a specific 19nt target sequence from hbv genome to create recombinant psilencer-u6 plasmid. primers were annealed and cloned into bamhi-hindiii sites of the psilencer2.1-u6 vector. in order to construct a useful tool to choose the most effective sirna molecules, we created a quick screening vector plucf by fusing the targeted sequence and the reporter luciferase gene together to produce recombinant plucf plasmid, which could express hbs-luciferase or hbx-luciferase fusion mrnas. therefore, we can initially select the suitable sirna duplexes rapidly by simply analyzing the activities of lucifearse. by using this approach, we have identified two sirna molecules (hbs 1 sirna and hbs 2 sirna) having significant impact on the hbs-luciferase fusion gene expression and one sirna duplex (hbx 2 sirna) having effects on the expression of hbx-luciferase fusion gene. this provides a quick approach to select effective sirna in the study of gene expression and function analysis. to further study the effects of selected rnai molecules on hbv gene expression and viral replication in a cell culture models, we used a derivative of the human hepg2 hepatoma cell line, hepg2.2.15, which has been stably transformed with several copies of the hbv genome and used as an in vitro model for hbv replication. the effects of dual sirna system on hbv gene expression and viral replication were studied thoroughly by the analyzing the levels of viral protein production through enzyme-linked immunosorbent assay and the levels of viral rna expression by semi-quantitated rt-pcr analysis. all results indicated that hbs 1 sirna, hbs 2 sirna, and hbx 2 sirna had significant reduction effects on viral mrna expression, and viral protein production. the fact that mutation in hbv genome may result in resistance if sirna molecules were further developed as antiviral drugs raised our concerns. our strategies to address such potential problems are to choose targets in the relatively conserved dna sequences and to generate multiple sirna molecules that can target different sites or genes on the viral genome. to test our approach, in this study we established a system that can simultaneously express two sirna duplexes from a single vector that can attack the s and x genes of hbv, respectively. results from luciferase activity assay, enzyme-linked immunosorbent assay and semiquantitated rt-pcr analysis were consistently showed that the dual sirna molecules had synergetic effects or more efficient on the targeted viral protein production and hbs and hbx gene expression comparing to that of the single sirna molecules. more importantly, dual sirna could simultaneously inhibit the expression of hbs and hbx gene by 83.7% and 87.5%, respectively. therefore, this dual sirna system could provide a more powerful tool for the study of gene function and could be used as a potential application in the treatment of viral infection. in the last 20 years, hbv infection affects millions of people each year worldwide. current therapies of hbv infection including immune modulators such as interferon alfa, or nucleoside analogs such as lamivudine have provided some degree of cures, but the efficiency of treatment was limited. as a potential therapy, sirna seems to be a hopeful alternative strategy. we believe that our approach presented in this study could be broadly used. for example, it could be used to generate more than two sirnas duplexes that could silent more genes in order to study the interactions of genes and their functions. such strategies of constructing multiple-sirna vectors can confront the evading mechanism of virus infections. obviously, this cocktail approach would be benefit to application of sirna therapy in viral infections, especially to those viruses with high mutation rate. in addition, this approach could also used to deal with two or more viruses, which are especially useful in the treatment of co-infections by two or more pathogens, such as hbv-hiv and hcv-hiv. we are in the process of testing these approaches. rna silencing of dengue virus type 2 replication in transformed c6/36 mosquito cells transcribing an inverted-repeat rna derived from the virus genome asystem for stable expression of short interfering rnas in mammalian cells long-term follow-up of hepatitis b chronic carriers who responded to interferon therapy potent and specific inhibition of human immunodeficiency virus type 1 replication by rna interference human immunodeficiency virus type 1 escapes from rna interference-mediated inhibition rotavirus gene silencing by small interfering rnas duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells potent and specific genetic interference by doublestranded rna in caenorbabditis elegans rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription short interfering rna-directed inhibition of hepatitis b virus replication rna interference inhibition of sars-associated coronavirus infection and replication by rna interference inhibition of retroviral pathogenesis by rna interference modulation of hiv-1 replication by rna interference selective silencing of viral gene expression in hpv-positive human cervical carcinoma cells treated with sirna, a primer of rna interference global control of hepatitis b virus infection interference of hepatitis c virus rna replication by short interfering rnas lamivudine is effective in suppressing hepatitis b virus dna in chinese hepatitis b surface antigen carriers: a placebocontrolled trial expression of small interfering rnas targeted against hiv-1 rev transcripts in human cells inhibition of hepatitis b virus in mice by rna interference effective expression of small interfering rna in human cells duck hepatitis b virus(dhbv) particles produced by transient expreesion of dhbv dna in a human hepatoma cell line are infectious in vitro inhibition of hepatitis b virus expression and replication by rna interference this study was supported by the research funds from the ministry of education and wuhan university. key: cord-303111-iv4lzpev authors: almazán, fernando; sola, isabel; zuñiga, sonia; marquez-jurado, silvia; morales, lucia; becares, martina; enjuanes, luis title: reprint of: coronavirus reverse genetic systems: infectious clones and replicons() date: 2014-12-19 journal: virus res doi: 10.1016/j.virusres.2014.09.006 sha: doc_id: 303111 cord_uid: iv4lzpev coronaviruses (covs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. the large size of the cov genome and the instability of some cov replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense rna viruses. to overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last 13 years. in this report, we briefly review and discuss the different reverse genetic systems developed for covs, paying special attention to the severe acute respiratory syndrome cov (sars-cov). coronaviruses (covs) are enveloped rna viruses mainly responsible for respiratory and enteric infections in animals and humans (lai et al., 2007; masters, 2006) . historically, cov infection in humans has been associated with mild upper respiratory tract diseases caused by the human covs (hcovs) hcov-229e and hcov-oc43 (masters, 2006) . however, the identification in 2003 of a novel life-threatening cov causing the severe acute respiratory syndrome (sars-cov) redefined historic perceptions (stadler et al., 2003) . more recently, three novel hcovs associated with respiratory diseases have been identified, including hcov-hku1 associated with chronic pulmonary disease (woo et al., 2005) , hcov-nl63 that causes upper and lower respiratory tract disease in children and adults worldwide (van der hoek et al., 2004) , and the recently emerged (april 2012) middle east respiratory syndrome cov (mers-cov), which has been associated with acute pneumonia and occasional renal failure (zaki et al., 2012) . these findings have potentiated the relevance of covs as important human pathogens and highlight the need of reverse genetic systems to facilitate the genetic manipulation of the viral genome to study fundamental viral processes, to develop vaccine candidates and to test antiviral drugs. covs belong to the coronaviridae family within the order nidovirales (de groot et al., 2012) . they contain the largest known rna genome among rna viruses, consisting in a plus-sense, 5 -capped and polyadenylated rna molecule of 27-31 kb in length. the first two-thirds of the genome encode the replicase gene, which comprise two overlapping open reading frames (orfs), orf 1a and orf 1b, the latter being translated by a ribosomal frameshift mechanism. translation of both orfs results in the synthesis of two polyproteins that are processed by viral proteinases to release the replication-transcription complex components. the final one-third of the genome includes the genes encoding the structural proteins s, e, m, and n, as well as the genus specific proteins characteristic of each cov, which are expressed from a nested set of 3 coterminal subgenomic mrnas masters, 2006; ziebuhr, 2005) . until recently, the study of cov genetics was broadly restricted to the analysis of temperature-sensitive (ts) mutants baric, 1992, 1994; lai and cavanagh, 1997; schaad and baric, 1994; stalcup et al., 1998) , defective rna templates which depend on replicase proteins provided in trans by a helper virus (izeta et al., 1999; narayanan and makino, 2001; repass and makino, 1998; williams et al., 1999) , and recombinant viruses generated by targeted recombination (masters, 1999; masters and rottier, 2005 reverse genetic system devised for covs at a time when it was not clear whether the construction of full-length infectious cdna clones would ever be technically feasible. targeted rna recombination, originally developed for mouse hepatitis virus (mhv), takes advantage of the high rate of homologous rna recombination in covs (baric et al., 1990; kusters et al., 1990; makino et al., 1986 ). in this system, a synthetic donor rna expanding the last 10 kb of the genome is transfected into cells infected with a recipient parental virus presenting some characteristics that can be selected against (ts phenotype or host range-based selection). mutant recombinant viruses are then identified by counterselection of the recipient parental virus and purified. despite its value, targeted rna recombination presents clear limitations, such as the inability to easily manipulate the replicase gene and to study lethal mutations due to the requirement for virus passage. therefore, the development of reverse genetic approaches based on full-length cdnas, which do not have these limitations, should provide a tremendous encouragement to the study of cov biology. however, the large size of the genome (around 30 kb), the instability of some cov replicase gene sequences when they were propagated as cloned cdna in bacteria, and the difficulty to synthesize full-length transcripts in vitro have hampered the generation of cov full-length infectious cdna clones. recently, these problems were overcome employing three creative nontraditional approaches based on the use of bacterial artificial chromosomes (bacs) (almazan et al., 2000) , in vitro ligation of cdna fragments (yount et al., 2000) , and vaccinia virus as a vector for the propagation of cov full-length cdnas (thiel et al., 2001a) . in this report, we review and discuss these three different approaches developed for building cov infectious cdnas by using sars-cov as a model, and how these cov reverse genetic systems have now been extended to the generation of cov replicon rnas. the first cov full-length infectious cdna clone was generated for transmissible gastroenteritis virus (tgev) using the bac approach (almazan et al., 2000; gonzalez et al., 2002) . in this system, the full-length cdna copy of the viral genome is assembled in the bac plasmid pbelobac11 (wang et al., 1997) , a synthetic low-copy-number plasmid based on the escherichia coli f-factor (shizuya et al., 1992) that presents a strictly controlled replication leading to one or two plasmid copies per cell. this plasmid allows the stable maintenance in bacteria of large dna fragments from a variety of complex genomic sources (adler et al., 2003; shizuya et al., 1992) and minimizes the toxicity associated with several cov sequences when amplified in high-copy-number plasmids. the full-length cdna is assembled under the control of the cytomegalovirus (cmv) immediate-early promoter that allows the expression of the viral rna in the nucleus by the cellular rna polymerase ii (dubensky et al., 1996) , and it is flanked at the 3 -end by a poly(a) tail, the hepatitis delta virus (hdv) ribozyme and the bovine growth hormone (bgh) termination and polyadenylation sequences to produce synthetic rnas bearing authentic 3 -ends of the viral genome. this dna-launched system ensures capping of the viral rna and allows the recovery of infectious virus from the cdna clone without the need of an in vitro transcription step. using this approach, a bac clone carrying an infectious genome of the sars-cov urbani strain was generated in three steps ( fig. 1 ) . the first step was the selection of appropriate restriction sites in the viral genome that were absent in the bac plasmid. in case that no adequate restriction sites were available in the viral genome, new restriction sites could be generated by the introduction of silent mutations. in the second step, the intermediate bac plasmid pbac-sars-cov 5 -3 was generated as the backbone to assemble the full-length cdna clone. this plasmid contained the 5 -end of the genome under the control of the cmv promoter, a multicloning site containing the restriction sites selected in the first step, and the 3 -end of the genome followed by a 25-nt synthetic poly(a), the hdv ribozyme and the bgh termination and polyadenylation sequences. finally, the full-length cdna clone (pbac-sars-cov fl ) was assembled by sequential cloning of five overlapping cdna fragments (sars-1 to sars-5) into the multicloning site of the intermediate bac plasmid (fig. 1) . the overlapping cdna fragments flanked by the appropriate restriction sites were generated by standard reverse transcription pcr (rt-pcr). the assembled sars-cov bac clone was fully stable in e. coli and infectious virus was rescued after transfection of susceptible cells . the bac approach presents several advantages, such as the high stability of exogenous sequences, unlimited production of the cdna clone, high efficiency of cdna transfection into mammalian cells, and intracellular expression of the viral rna. furthermore, the manipulation of bac clones is relatively easy and essentially similar to that of a conventional plasmid with slight modifications owing to the large size of the bac clones and the presence of this plasmid in only one or two copies per cell (shizuya et al., 1992) . besides standard protocols for the manipulation of conventional plasmids, the bac clones could be easily and efficiently modified into e. coli by homologous recombination using a two-step procedure that combines the red recombination system and counterselection with the homing endonuclease i-scei (jamsai et al., 2003; lee et al., 2001; tischer et al., 2006; zhang et al., 1998) . in a first step, a linear marker construct containing the desired modification and i-scei recognition site is inserted via red recombination into the target site using positive selection. in a second step, the induced i-scei cleaves at its recognition site creating dna double strand breaks. then, the adjoining duplicate sequence previously introduced is used as the substrate for a second intramolecular red recombination, resulting in the loss of the previously introduced marker. this novel approach results in an accurate and highly efficient method to introduce insertions, deletions or point mutations in bac clones without retention of unwanted foreign sequences. in addition to sars-cov and tgev, the bac approach has been successfully used to engineer infectious clones of hcov-oc43 (st-jean et al., 2006) , feline infectious peritonitis virus (fipv) (balint et al., 2012) , and the recently emerged mers-cov (almazan et al., 2013) . in the last case, a combination of synthetic biology and the use of bacs allowed the generation of a mers-cov infectious clone only four months after the first mers-cov outbreak, illustrating the power of the bac approach. recently, modified bac approaches have been used to generate full-length cdna clones of the sars-cov strains frankfurt-1 (pfefferle et al., 2009 ) and tor2 related clinical isolate cv7 (tylor et al., 2009) , assembled in a bac under the control of the t7 rna polymerase promoter. in the case of the frankfurt-1 strain, infectious virus was rescued after transfection of the full-length transcripts derived from the in vitro transcription of the linearized bac construct. this approach combines plasmid-based handling of the infectious clone with direct delivery of genome-like rna into the cytoplasm, circumventing transcription of the infectious clone in the nucleus driven by the cmv promoter, and avoiding the possibility of splicing. however, although some splicing could occur during the nuclear expression of the viral genome, the efficiency of this phenomenon is very low and does not affect the recovery of infectious virus (almazan et al., 2000) . in contrast, in the case of the cv7 isolate, infectious virus was recovered in situ from cells transfected with the bac clone and infected with a modified vaccinia ankara expressing t7 rna polymerase. in this system an in vitro transcription step is also avoided. this reverse genetic approach has been successfully used to study the role of specific viral proteins in viral replication and pathogenesis and for the generation of genetically attenuated viruses that are potential vaccine candidates for sars-cov and other covs (almazan et al., 2013; dediego et al., 2007; enjuanes et al., 2008; fett et al., 2013; lamirande et al., 2008; netland et al., 2010) . this method, originally applied to tgev (yount et al., 2000) , involves the assembly of full-length cdnas from a panel of contiguous cdna fragments that span the entire viral genome, flanked by native or engineered specific restriction sites with characteristics that allow the systematic and precise assembly of a full-length cdna by in vitro ligation. the assembled full-length cdna, containing a t7 rna polymerase promoter at the 5 -end and a poly(a) tail at the 3 -end, is in vitro transcribed to generate capped full-length transcripts that are used together with capped n gene transcripts to efficiently recover infectious virus after transfection of susceptible cells. in this approach, some fragment boundaries were arranged in such a way that interrupt genomic regions that were unstable when propagated as cloned cdna in bacteria. following this strategy, a genome-length cdna of the sars-cov urbani strain was generated by in vitro ligation (fig. 2 ) (yount et al., 2003) . initially, a panel of six contiguous cdna fragments spanning the entire sars-cov genome was generated by standard rt-pcr using specific primers that introduced unique bgli restriction sites at the 5 and 3 -ends of each fragment without altering the amino acid coding sequences of the virus. bgli is a class ii restriction enzyme that cleaves a symmetrical palindromic sequence (gccnnnn↓nggc) but leaves different asymmetric 3-nt overhangs that do not randomly self-assemble and only anneal with the complementary 3-nt overhang generated at an identical bgli site. these cdna fragments were systematically and unidirectionally assembled into a full-length cdna by in vitro ligation and, after in vitro transcription, genome-length transcripts were used to recover infectious virus. this in vitro assembly technique combined with synthetic biology was further used successfully to engineer infectious clones of a bat sars-like cov (becker et al., 2008) and the recently identified mers-cov (scobey et al., 2013) . one potential problem of this approach is that several silent mutations have to be inserted in the genome to avoid potential t7 transcription termination signals and to introduce the unique bgli sites used to assemble the full-length cdna clones. to overcome this problem, a variation of the approach was used to engineer the mhv infectious cdna . the strategy is based on the incorporation of type iis restriction enzymes (esp31, sapi, bsai and bsmi, among others) at the ends of the cdna fragments. these enzymes recognize asymmetrical sites and cleave external to the recognition sequences, leaving 1-4-nt variable ends that can be only anneal with the complementary overhang generated at an identical site (esp31 site, cgtctcn↓nnnn). using these type iis restriction enzyme sites, two contiguous cdna fragments could be ligated in vitro with traditional cloning approaches, leaving the restriction site within the viral genome sequence. however, due to the asymmetrical nature of these recognition sites, a simple reverse orientation allows for the insertion of these sites on the ends of two adjacent cdna fragments with the variable overhang generated from the virus sequence (no see'm cloning strategy). upon cleavage and ligation, both restriction sites are removed, leaving the exact viral sequence at the junction. this approach allows the generation of cdna clones without mutating the viral genome sequence. following this modified approach, the full-length cdna clone of mhv was generated from either the sequential (in several steps) or simultaneous in vitro ligation of seven cdna fragments covering the entire mhv genome . the feasibility of this method has been demonstrated later by the successful assembly of full-length infectious clones of infectious bronchitis virus (ibv) (fang et al., 2007; tan et al., 2006; youn et al., 2005) and hcov-nl63 . in this reverse genetic system, inclusion of n transcripts during the transfection process has been shown to enhance virus rescue in all analyzed cases, although its mechanism of action is largely unknown. the in vitro cdna assembly approach is simple and straightforward, allows the rapid mutagenesis of independent cdna fragments in parallel using conventional techniques, and is compatible with bac or vaccinia vectors. furthermore, using the no see'm technology it is possible to mutate any given nucleotide in the viral genome by designing primers that incorporate a type iis restriction site and the mutation of interest in the variable domain of the recognition site. adding the type iis restriction site in the proper orientation, the site is removed during reassembly, leaving only the mutation of interest in the final dna product (donaldson et al., 2007; yount et al., 2002) . this reverse genetic approach has been successfully used to study the role of specific viral proteins in sars-cov replication and pathogenesis, to identify zoonotic vaccine candidate strains (deming et al., 2006) , and to develop a recombination safe vaccine platform using a novel strategy that prevents recombination between wild-type and recombinant viruses by rewiring the viral transcription regulating sequences (trss) . an alternative reverse genetic system to study the biology and pathogenesis of covs is based on the cloning and propagation of cov genomic cdnas in vaccinia virus vectors (eriksson et al., 2008; thiel and siddell, 2005) . this approach was first described for the generation of recombinant hcov-229e (thiel et al., 2001a) , and subsequently applied to successfully engineer full-length infectious clones of mhv (coley et al., 2005) , sars-cov (van den worm et al., 2012), ibv (casais et al., 2001) , and fipv (tekes et al., 2008 (tekes et al., , 2012 . this approach presents some advantages. the cloning capacity of poxvirus vectors, and vaccinia virus in particular, exceeds 26 kb of foreign sequences (smith and moss, 1983) . recombinant vaccinia virus genomes are stable, infectious and replicate efficiently in tissue culture. vaccinia virus vectors designed for the insertion of foreign dna by in vitro ligation (merchlinsky and moss, 1992) have been developed, thus obviating the need for plasmid intermediates containing the entire cdna insert. finally, a vaccinia virus-mediated homologous recombination strategy is available to make accessible for site directed mutagenesis the cov cdna cloned in the vaccinia vector thiel and siddell, 2005) . the generation of a reverse genetic system for sars-cov (isolate hku-39849) (van den worm et al., 2012) will be described to illustrate the vaccinia virus-based system. methodologically, this reverse genetic system can be divided into three phases (fig. 3) . the first step involves the generation of subgenomic cdna fragments that are prepared in large amounts either by amplification as bacterial plasmid dna or by preparative rt-pcr. the cdnas are subsequently ligated in vitro to produce a small number of cdnas, which encompass the entire genome. the specific ligation strategy is determined by the sequence of the particular cov, and mainly consists on naturally occurring or engineered restriction sites. for sars-cov strain hku-39849, five cdna fragments encompassing the whole genome were initially in vitro ligated with each other and with synthetic oligonucleotide linkers, resulting in the generation of two cdnas spanning nucleotides 1-20,288 and 20,272-29,727, respectively (van den worm et al., 2012) (fig. 3) . a crucial step in this process is to modify the cdnas containing the 5 and 3ends of the cov genome in order to finally produce infectious rna molecules. to this end, a transcription promoter sequence for the bacteriophage t7 rna polymerase is located upstream the cov genome and a synthetic poly(a) at the 3 -end of the genome, followed by a unique restriction site that can be used to generate run-off transcripts (fig. 3) . finally, to insert the cov cdna into a single noti site present in the genomic dna of the vaccinia virus vector vnoti/tk, the 5 and 3 -genomic termini of cdna fragments have to contain the restriction sites eagi or bsp120i, which lead to dna ends compatible with noti-cleaved vaccinia virus vector dna. during the cloning steps of sars-cov cdna sequences, some problems arose such as the instability in bacterial plasmids of viral cdna sequences (nt 11,370-11,905) and unexpected rt-pcr introduced mutations and deletions. the incorrect cdna regions were replaced by appropriate sars-cov sequences using vaccinia virus-mediated homologous recombination, as it will be described below. in the second step, in vitro assembled cov cdna fragments representing the full-length genome are cleaved with eagi and then ligated in vitro to the long and short arms of noti-cleaved vnoti/tk vaccinia virus dna (merchlinsky and moss, 1992) . subsequently, the ligation reaction is transfected into cv-1 mammalian cells infected with fowlpox virus as a helper (scheiflinger et al., 1992) to rescue recombinant vaccinia viruses including the cdna of cov genome sequences. because fowlpox virus infection of mammalian cells is abortive, and possible recombination events between both poxviruses are extremely rare, the resulting virus stocks collected from cells contain exclusively recombinant vaccinia viruses (thiel et al., 2001a (thiel et al., , 2001b . in the case of sars-cov, two recombinant vaccinia viruses spanning nucleotides 1-20,288 (sars-cov 5 ) and 20,272-29,727 (sars-cov 3 ) were generated (fig. 3) . dna derived from these vaccinia viruses was cleaved with sfii and bgli restriction enzymes producing compatible ends and then ligated to create a cdna containing a genome-length sars-cov cdna (van den worm et al., 2012) . finally, recombinant covs are rescued by generating genomiclength rna transcripts from the cov cdna insert of the recombinant vaccinia virus. purified recombinant vaccinia virus dna containing the full-length cdna of cov genome is cleaved at a unique site located downstream of the poly(a) sequence, representing the 3 -end of the cov genome. the resulting dna is used as template for in vitro transcription with bacteriophage t7 rna polymerase to generate a capped rna corresponding to the cov genome. these transcripts are then transfected into permissive cells, leading to the rescue of the corresponding cov from the tissue culture supernatant. a limiting step of this procedure is the variable amount and purity of full-length synthetic cov rna from in vitro transcription, leading sometimes to failure to rescue recombinant covs. therefore, an alternative strategy is the transcription of template dna in the permissive cell itself by expressing the bacteriophage t7 rna polymerase from a recombinant fowlpox virus rfpv-t7 (britton et al., 1996; casais et al., 2001) . previous experience in the vaccinia virus reverse genetic system has shown that the n transcript may become essential for the recue of some covs such as ibv (casais et al., 2001) . therefore, to contribute positively to the recovery of covs, different methods involving n protein expression were used. initially, n transcripts were cotransfected with the infectious rna, as described for mhv (coley et al., 2005) . more recently, full-length sars-cov rna synthesized in vitro with the t7 rna polymerase was transfected into bhk cells inducibly expressing the sars-cov n protein (chang et al., 2010) and the transfected cells were then co-cultivated with susceptible cells to rescue eventually the recombinant sars-cov (van den worm et al., 2012) . the requirement of the n protein to establish a productive cov infection from infectious rna or dna has been shown to be variable. several covs such as tgev, sars-cov and mers-cov were efficiently rescued from infectious cdnas engineered as bacs, independently of n protein coexpression (almazan et al., 2000 (almazan et al., , 2013 . similarly, hcov-229e was rescued from vaccinia virus in the absence of n protein expressed in trans (thiel et al., 2001a) . in contrast, n transcripts were essential to rescue ibv from recombinant vaccinia virus (casais et al., 2001) . in an intermediate situation, the rescue of mhv and tgev from full-length cdna constructs assembled in vitro was significantly enhanced by the inclusion of n-coding transcripts (baric and sims, 2005; yount et al., 2002) . these results suggest that cov cdnas engineered as bacs and transcribed by the cellular pol ii to produce infectious rnas represent a more efficient method for virus recovery as compared to the other systems relying on infectious rnas transcribed by t7 polymerase, since these reverse genetic systems depend at different extents on n transcripts. at this moment, it is not clear whether n transcripts, n protein, or both are essential for increased virus recovery yields. nevertheless, previous results have shown that the n protein is involved in cov rna transcription and increases rna synthesis (almazan et al., 2004; schelle et al., 2005; zuñiga et al., 2010) . one important advantage of the vaccinia virus reverse genetic system is that vaccinia virus-mediated homologous recombination (ball, 1987) can be used to modify the cov genomic cdna during its propagation in vaccinia virus. this site-directed mutagenesis strategy has been extensively applied to introduce heterologous sequences in the genome of recombinant covs (van den worm et al., 2012), for gene deletion or modification lei et al., 2013; roth-cross et al., 2009; stokes et al., 2010) or even to repair error sequences (coley et al., 2005; van den worm et al., 2012) . two recombination methods based on the sequential use of the e. coli guanine-phosphoribosyl transferase gene (gpt) as both a positive and a negative selection marker (armesto et al., 2008; britton et al., 2005; eriksson et al., 2008) have been developed. in the first method (falkner and moss, 1988; thiel and siddell, 2005) the cov region of interest in the recombinant vaccinia virus is replaced by the e. coli gpt gene. this gene is encoded in a plasmid dna and flanked by cov sequences to facilitate a double recombination event. vaccinia viruses containing the gpt gene at the expected cov genome position are isolated under gpt-positive selection on mammalian cv-1 cells. in a second step, the introduced gpt gene is replaced by a gene of interest encoded in a plasmid dna and flanked by cov sequences mediating double homologous recombination events. isolation of recombinant viruses with the gene of interest requires gpt-negative selection in hela-d980r cells. in the second method, known as transient dominant selection falkner and moss, 1990; kerr and smith, 1991) , the modified cov cdna region is inserted into a plasmid containing the gpt selective marker under the control of a vaccinia virus promoter. then, the complete sequence of this plasmid is transiently integrated into the vaccinia virus including the cov genome by homologous recombination involving a single crossover event. the recombinant vaccinia viruses expressing the gpt gene are positively selected. finally, the gpt marker is lost in the absence of the selective pressure, by a single homologous recombination event between duplicated sequences. two recombination events can occur with equal frequency leading either to the generation of the unmodified cov sequence or to the generation of a cov cdna with the desired modification. replicons have been developed for several rna viruses as an important complementary resource of virus reverse genetic systems (bartenschlager, 2002; brass et al., 2007; frolov et al., 1996; khromykh, 2000; nakamura et al., 2008; zimmer, 2010) . replicons are self-amplifying nucleic acids that contain all viral proteins and rna signals required for viral rna synthesis, and in some cases reporter genes to facilitate the analyses. as replicons lack structural proteins and no infectious viral particles are produced, they constitute a safe alternative to full-length infectious cdnas, especially for bsl3 viruses. moreover, stable cell lines containing non-cytopathic selectable virus replicons have also been obtained (bartenschlager, 2002) . therefore, replicons represent a safe tool for virus replication studies and antiviral compound screening, and even for the setup of semi-automated high-throughput screening systems. cov replicons have been generated for many covs using all the reverse genetic approaches described above (almazan et al., 2004 curtis et al., 2002; ge et al., 2007; hertzig et al., 2004; thiel et al., 2001b) . these replicons were very useful for the identification of viral and cellular factors involved in cov rna synthesis (almazan et al., 2004; galan et al., 2009; moreno et al., 2008) , and for antiviral drug testing (chen et al., 2005) . in the case of sars-cov, a collection of replicons has been engineered, either using the bac system or by in vitro ligation (ge et al., 2007) . different replicon versions include a variety of reporter genes, such as green fluorescent protein (gfp), firefly luciferase (fluc), or renilla luciferase (rluc), alone or in combination with antibiotic selection genes (ahn et al., 2011; ge et al., 2007; pan et al., 2008; tanaka et al., 2012) . the genomic structure of the rna encoded by cov replicons is very similar in all cases. they contain the 5 and 3 cis acting signals required for viral replication, the large orfs 1a and 1b that encode the replicase non-structural proteins (nsps), and the n gene, which is essential for efficient cov rna synthesis (almazan et al., 2004) . the sars-cov replicon obtained by in vitro ligation also includes a gfp-blar fusion gene, located downstream of the orf 1b, under the transcriptional control of s gene trs (trs-s). this heterologous reporter gene, which confers resistance to blasticidin selection, allowed the generation of the first selectable sars-cov replicon cell line (ge et al., 2007) . several bac-based sars-cov replicons also included heterologous genes located downstream of the orf 1b. rluc was expressed under the control of trs-n (tanaka et al., 2012) , although eventually, the duplication of trs-n sequences could lead to reporter gene loss after several replication cycles. alternatively, fluc-neor fusion gene (conferring neomycin resistance) was expressed under the control of trs-n (ahn et al., 2011) or trs-m (pan et al., 2008) . in this case, antibiotic selection would avoid reporter gene loss and allows the future development of selectable cell lines. cov replicons could also be derived from replication-competent propagation-defective viruses, such as those lacking e gene engineered for tgev (curtis et al., 2002; ortego et al., 2002) or mers-cov (almazan et al., 2013) . in this case, additional safetyguards must be introduced to work in a bsl2 containment facility. in this line, a sars-cov replicon was constructed based on an attenuated sars-cov-e infectious cdna, replacing the structural s gene by gfp reporter gene (wang et al., 2008) . this replicon was useful for antiviral drug testing, although a more comprehensive evaluation of replicon safety should be performed. sars-cov replicons have been used to analyze the molecular basis of viral rna synthesis and viral or cellular factors involved. in this sense, the function of some nsps, such as nsp1, nsp14, nsp15, or nsp16, in sars-cov replication was analyzed avoiding the use of infectious virus chen et al., 2009; tanaka et al., 2012) . moreover, a genome-wide analysis of viral proteins involved in sars-cov rna synthesis (pan et al., 2008) and a quantitative proteomic analysis to determine host cell factors involved in sars-cov replication (zhang et al., 2010) were also performed using replicons. to date, despite the increased efforts since sars-cov outbreak, no antiviral drug approved by fda exists against human covs (barnard and kumaki, 2011; kilianski and baker, 2014) . one of the most promising applications of sars-cov replicons is their use for antiviral drug screening. since sars-cov replicons lack structural proteins, compounds inhibiting virus entry or morphogenesis could not be tested. nevertheless, with some exceptions (kilianski and baker, 2014) , these virus life cycle steps are poor antiviral targets, as virus escape mutants could be easily recovered, especially when virus structural proteins are targeted. on the other hand, replicase proteins or viral rna motifs involved in cov replication are in general more conserved, and their mutation rate could be lower than that of the structural proteins due to higher fitness pressure. in fact, several key enzymatic activities, such as main protease or papainlike protease, have been analyzed as targets for cov antiviral drugs (kilianski and baker, 2014) . additionally, sars-cov replicons have been used for further validation of antiviral compounds or nucleic acids that were previously identified using other in vitro screening methods, such as helicase (adedeji et al., 2012) or frameshifting (ahn et al., 2011) inhibitors. moreover, a selectable sars-cov replicon cell line was used for the development of a semi-automated high-throughput antiviral compound screening system, with minimum manipulation after assay set-up, low compound volume, and automated detection of end-point fluorescence (ge et al., 2008) . interestingly, after a screening of a 7,035 compound library, and selection of 7 drugs for further testing, a good correlation was observed between fluorescence levels and sars-cov rna or protein synthesis, highlighting the system suitability. a limitation of this kind of cell-based screening systems is that the viral or cell drug target is not identified, and further analyses using other experimental approaches are required. the potential risk to public health posed by sars-cov and other covs, and the lack of specific antiviral agents and vaccines, have triggered a global effort to study this family of viruses at the molecular level in order to develop effective strategies to prevent and control cov infections. molecular genetic analysis of the structure and function of rna virus genomes has been profoundly advanced by the availability of full-length cdna clones. in the case of cov, the huge genome size and the instability of specific cov cdna sequences in bacterial systems hindered the development of infectious cdna clones until recently. a tremendous amount of creativity in the cov field resulted in the development of four independent and unique reverse genetic systems that overcame these problems. these reverse genetic systems have been established using non-traditional approaches, which are based on the use of targeted recombination, bacs, in vitro ligation of cov cdna fragments, and vaccinia virus as a vector for the propagation of cov genomic cdnas. the availability of cov full-length infectious clones and recombinant viruses expressing reporter genes constitute important tools for the study of cov replication and transcription mechanisms, virus-host interaction and pathogenesis, and also for the rapid and rational development and testing of 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acute respiratory syndrome coronavirus systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 rewiring the severe acute respiratory syndrome coronavirus (sars-cov) transcription circuit: engineering a recombination-resistant genome isolation of a novel coronavirus from a man with pneumonia in saudi arabia quantitative proteomics analysis reveals bag3 as a potential target to suppress severe acute respiratory syndrome coronavirus replication a new logic for dna engineering using recombination in escherichia coli coronavirus replication and reverse genetics rna replicons: a new approach for influenza virus immunoprophylaxis coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription this work was supported by grants from the ministry of science and innovation of spain (mcinn) (bio2010-16705), the european community's seventh framework programme (fp7/2007(fp7/ -2013 under the project "emperie" (health-f3-2009-223498), and the national institute of health (nih) of usa (2p01ai060699-06a1). key: cord-309205-l8vjtrjq authors: shirato, kazuya; chang, hui-wen; rottier, peter j.m. title: differential susceptibility of macrophages to serotype ii feline coronaviruses correlates with differences in the viral spike protein date: 2018-08-15 journal: virus res doi: 10.1016/j.virusres.2018.06.010 sha: doc_id: 309205 cord_uid: l8vjtrjq the ability to infect and replicate in monocytes/macrophages is a critically distinguishing feature between the two feline coronavirus (fcov) pathotypes: feline enteric coronavirus (fecv; low-virulent) and feline infectious peritonitis virus (fipv; lethal). previously, by comparing serotype ii strains fipv 79-1146 and fecv 79-1683 and recombinant chimeric forms thereof in cultured feline bone marrow macrophages, we mapped this difference to the c-terminal part of the viral spike (s) protein (s2). in view of the later identified diagnostic difference in this very part of the s protein of serotype i fcov pathotypes, the present study aimed to further define the contribution of the earlier observed ten amino acids difference to the serotype ii virus phenotype in macrophages. using targeted rna recombination as a reverse genetics system we introduced the mutations singly and in combinations into the s gene and evaluated their effects on the infection characteristics of the mutant viruses in macrophages. while some of the single mutations had a significant effect, none of them fully reverted the infection phenotype. only by combining five specific mutations the infections mediated by the fipv and fecv spike proteins could be fully blocked or potentiated, respectively. hence, the differential macrophage infection phenotype is caused by the cooperative effect of five mutations, which occur in five functionally different domains of the spike fusion subunit s2. the significance of these observations will be discussed, taking into account also some questions related to the identity of the virus strains used. coronaviruses (covs) are enveloped, positive-strand rna viruses with genomes of about 30 kilobases (kb). originally considered of veterinary relevance primarily, the outbreaks of severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov dramatically established their importance as life-threatening human pathogens as well. while the human coronaviruses (hcovs) mainly target the respiratory system, coronaviruses generally cause respiratory and intestinal tract infections. coronaviruses have been detected in almost all animal species. in cats their occurrence is quite ubiquitous, animals often becoming infected already at a very young age. feline coronaviruses (fcovs) generally cause enteric infections that are very mild or, more typically, pass unnoticed, particularly in older cats. yet, the infections by these feline enteric coronaviruses (fecvs) are not efficiently cleared and often persist in cells of the intestinal mucosa, with virus being shed through the feces for weeks or months, sometimes for over a year (kipar et al., 2010; pedersen et al., 2008a, b; vogel et al., 2010) . besides this harmless enteric pathotype another feline coronavirus occurs that causes severe and generally fatal systemic infection: feline infectious peritonitis virus (fipv). this highly virulent but more sporadically occurring pathotype is able to induce lethal immunopathological disease characterized by disseminated pyogranulomas and severe inflammatory damage to serosal membranes, often accompanied by exudation into body cavities (addie et al., 2009; campolo et al., 2005; de groot and horzinek, 1995; drechsler et al., 2011; haijema et al., 2007; kipar and meli, 2014; lorusso et al., 2017; myrrha et al., 2011; pedersen, 2009) . fcovs also come in two serotypes (pedersen et al., 1984a) . serotype i viruses are most prevalent and responsible for about 70-95% of field infections (addie et al., 2003; benetka et al., 2004; hohdatsu et al., 1992; kummrow et al., 2005) . serotype ii viruses are more rarely observed except in asia, where they may represent up to about 30% of infections hohdatsu et al., 1992) . genetic evidence indicates that serotype ii viruses arise by recombination between serotype i fcovs and canine coronaviruses (ccovs) in doubly-infected animals, as a result of which the feline virus acquires the canine virus spike (s) protein (herrewegh et al., 1998; vennema, 1999) . together with the ccov s-specific antigenic properties the resulting type ii fcov thus also acquires receptor specificity for the aminopeptidase n (apn) protein, a practically important feature as it has enabled investigators to grow and study these type ii viruses in fapn expressing culture cells while such susceptible cell systems were lacking for type i viruses. the s proteins of the two fcov serotypes are very different. yet, interestingly, each serotype exhibits both pathotypes, fecv and fipv (hohdatsu et al., 1991a, b; pedersen et al., 2008a) . rather than being distinctive circulating viruses (brown et al., 2009) , ample and convincing evidence has accumulated indicating that fipv arises from fecv by mutation in individual, fecv-infected cats (chang et al., 2010 (chang et al., , 2012 licitra et al., 2013; pedersen et al., 2009 pedersen et al., , 2012 poland et al., 1996; vennema et al., 1998) . favored by its ability to persist in the gut and by the relatively high mutation rate of rna viruses, genetic changes in the fecv genome enable this enteric biotype to go systemic. monocytes/macrophages are considered to be instrumental in this biotope switch as they appear to become susceptible to infection due to the critical mutation(s) (pedersen, 2014) . infected macrophages subsequently transport the virulent pathotype through the body to the organs where it can cause its devastating pathogenic effects. earlier we investigated this macrophage tropism of fcovs in vitro (rottier et al., 2005) . we used the serotype ii strains fecv 79-1683 and fipv 79-1146. the comparative pathogenicity of these strains was originally described in 1984 (pedersen et al., 1984a) , on the basis of which these viruses have subsequently served as the prototypic representatives of the two fcov pathotypes. consistent with observations made by others (dewerchin et al., 2005; stoddart and scott, 1989) , we found that fipv, in contrast to fecv, infects and replicates effectively in cultured bone marrow derived macrophages. by constructing -using reverse genetics (haijema et al., 2003) -and testing fecv/fipv chimeric viruses we were able to map this macrophage tropism to the viral spike protein. surprisingly, but consistent with both viral strains using the fapn receptor, the property was further mapped to the c-terminal, i.e. membrane-proximal domain of the s protein (s2) (rottier et al., 2005) , which is the domain responsible not for virusreceptor interaction but for viral membrane fusion. the residues responsible for the difference were not further defined. the genomic mutation(s) responsible for the fcov virulence switch have not been identified yet. several genes, including the s gene and the accessory genes 3a, 3b, 3c, 7a, and 7b, have been considered (balint et al., 2012; chang et al., 2010; kennedy et al., 2001; licitra et al., 2013; pedersen et al., 2012; rottier et al., 2005; vennema et al., 1998) . recently, by comparative full-genome analysis of 11 serotype i viruses of each pathotype and by subsequently zooming in on one region of consistent variance by specifically sequencing this region, we detected an amino acid difference that occurred in more than 95% among 183 fipvs and 118 fecvs (chang et al., 2012) . intriguingly, this characteristic difference also mapped to the membrane-proximal s2 part of the s protein. though the biological significance of this difference, particularly its possible effect on the viral monocyte/macrophage infection phenotype, is still unknown, further studies indicated it to relate to systemic spread of the virus in the infected animal (barker et al., 2017; porter et al., 2014) . the identification of this characteristic difference between the serotype i fcov pathotypes in the c-terminal part of the s protein, the very part to which we earlier mapped the macrophage tropism in the serotype ii viruses, prompted us to try to identify the critical tropism determinant(s) in these latter viruses. despite some concerns relating to the precise identity of the fcov strains 79-1683 and 79-1146, to be discussed later, but lacking better options to address this critical issue in the pathogenesis of fip, we continued in the present study with investigating the contributions of the amino acids in the spike s2 domain differing between the prototypic strains to the distinguishing macrophage tropism of these viruses. felis catus whole fetus (fcwf) and murine lr7 cells (kuo et al., 2000) were maintained in dulbecco's modified eagle's medium (dmem) (lonza group ltd., bazel, switzerland) containing 10% fetal calf serum (fcs), 100 iu penicillin/ml and 100 μg streptomycin/ml (all from life technologies, ltd, paisley, united kingdom). the recombinant form of fipv strain 79-1146 (rec 79-1146) and its derivative carrying the s protein of fecv strain 79-1683 (79-1146 /1683s) were generated by targeted rna recombination as described below. all recombinant viruses were propagated and titrated using fcwf cells. the chimeric mfipv, a fipv 79-1146 derivative in which the s protein ectodomain is from the mouse hepatitis coronavirus (mhv, strain a59), was propagated in and titrated on lr7 cells (haijema et al., 2003) . for constructing recombinant mutant fipvs the targeted rna recombination method was performed as described previously (haijema et al., 2003; rottier et al., 2005) and as illustrated in fig. 1a . it makes use of the transcription plasmid pbrdi1 (genbank ay204704), rna transcripts of which consist of a defective fipv 79-1146 genome lacking nucleotides (nt) 701-20101, i.e. they are composed of the genomic 5'terminal 698 nt fused to the 3'-terminal 335-nt proximal end of orf1b and running to the 3' end of the genome (haijema et al., 2003) (numbers based on ay204704 and dq010921 sequences). for the preparation of s gene mutants, derivatives of pbrdi1 were constructed in which the s gene was replaced entirely (fig. 1a) or partially (fig. 1b) by that of fecv strain 79-1683 (rottier et al., 2005) and/or in which more subtle mutations were introduced. nucleotide mutations were inserted in the pbrdi1 plasmid using specific primers and the quikchange xl site-directed mutagenesis kit (agilent, palo alto, ca, usa) following the provider's instructions. donor rna transcripts were synthesized from noti-linearized pbrdi1 using mmessage mmachine t7 (life technologies, carlsbad, ca, usa). briefly, 1 μg of template plasmid was mixed with 2μl of 10×buffer, 2μl of gtp, 10μl of cap/ntp, and 2μl of enzyme mix, and incubated at 37°c for more than 2 h. lr7 cells were infected with mfipv at a multiplicity of infection (m.o.i.) of 2. after 4 h of incubation, cells were trypsinized and washed once with 5% fcs/ dmem and once with pbs (-), and then resuspended in 700μl of pbs(-) in 0.4 cm cuvette for gene pulser (bio-rad, hercules, ca, usa). the rna transcripts were added to the cuvette and pulsed once at 0.85 kv with 50μf using gene pulser. after electroporation, the lr7 cells were added to a monolayer of fcwf cells in a t25 flask. after 24 to 48 h of incubation, supernatants were collected and purified by two rounds of limited dilution on fcwf cells. the resulting virus was subsequently amplified once using fcwf cells and used for the experiments. to confirm the sequences of recombinants, viral rnas were isolated after propagation using a qiaamp viral rna mini kit (qiagen, hilden, germany), cdna was synthesized using random primers and super-scriptii reverse transcriptase (life technologies) and the relevant s gene region was amplified with specific primers and the expand high fidelity pcr system (roche, basel, switzerland). the sequencing analysis was performed by macrogen corp. europe (amsterdam, the netherlands). feline macrophages were grown as previously described (rottier et al., 2005; van der meer et al., 2007) with some modifications. briefly, the stocks of bone marrow-derived mononuclear cells (2 × 10 7 cells/ ml) stored at -150°c were thawed, the cells were washed and resuspended in 10 ml of rpmi1640 (life technologies) containing 10% fcs, penicillin, streptomycin, l-glutamine, and 50 ng/ml of feline gm-csf (987-fl: r&d systems, minneapolis, mn, usa), and then seeded in 96-well plates. after 5 days of incubation, non-adherent cells were removed by aspirating the medium and fresh rpmi1640 medium containing 100 ng/ml of gm-csf was added. after an additional 6 days of incubation, the majority of the cells had differentiated into macrophages (0.5-1.0 × 104 cells/well) and were used for infection experiments. macrophages were inoculated with viruses at an m.o.i. of 10 or 50. after 3 h of incubation, inoculum viruses were removed by washing the cells twice with pbs after which 10% fcs rpmi1640 medium containing penicillin, streptomycin and l-glutamine was added and incubation at 37°c continued. at the indicated hours of post-infection (h.p.i.), culture supernatants were collected and the virus titers were determined on fcwf cells by tcid50 assay. fcwf cells were also used to analyze the replication kinetics of viruses. to this end the viruses were inoculated onto fcwf cells at an m.o.i. of 1. after 1 h of virus adsorption the cells were washed twice with pbs and dmem containing 5% fcs, penicillin and streptomycin was added. cells were incubated further at 37°c and culture supernatants were collected at indicated h.p.i. for titration on fcwf cells. to detect viral antigens, macrophages were infected with recombinant viruses as described above and were fixed at 10 and 24 h p.i. with methanol/acetone. the cells were first incubated with a 1:500 dilution in pbs of ascites 9912, derived from a fipv 79-1146 -infected cat (a gift from dr. h. glansbeek and e. te lintelo), and then stained with fitc-conjugated goat anti-cat igg (55291, mp biomedicals, santa ana, ca, usa) and dapi (d21490, invitrogen). the cell images were captured by evos fl (amg, bothell, wa, usa) and the number of nuclei and fluorescence positive cells were counted by image j software (1.46 r, national institute of health, usa). unpaired t-test was used to detect the statistical significance of the difference. a p value of < 0.05 was considered to be statistically significant. the significance levels were expressed as *, p < 0.01 or **, (haijema et al., 2003; rottier et al., 2005) . these observations confirmed that the poor growth phenotype of strain 79-1683 in feline macrophages maps to the c-terminal part of its s protein. there are eleven amino acid differences in the c-terminal domain of the s proteins of fipv 79-1146 and fecv 79-1683 to which we mapped these viruses' differential ability to infect macrophages (fig. 1c) . due to the rare occurrence of type ii viruses in the field (addie et al., 2003; an et al., 2011; hohdatsu et al., 1992) , only few s protein sequences, all from fipv's, are available for comparison. alignment of these sequences indicated three amino acids in the c terminal s2 domain [974(a), 1096(k), and 1239(p)] to be unique to fecv 79-1683 (fig. 1c) . we started studying the significance of these unique differences by individually replacing each one of these amino acids in the 79-1683 s protein of the chimeric 79-1146 /1683s virus to the corresponding residue of fipv s using site directed mutagenesis. the mutant viruses were generated by targeted rna recombination. they all replicated indistinguishable from their parents in fcwf cells to titers of 10 6 to 10 7 tcid50/100μl at 24 h p.i. (fig. 2a) . in macrophages a slight but significant enhancement of replication was observed only by the substitutions a974 v and k1096q, with titers reaching around 10 2 tcid50/100μl (fig. 2b) . clearly, the growth difference between viruses 79-1683 and 79-1146 in feline macrophages is not caused by a single point mutation in the s protein. thus, we also created double-mutants by combining substitutions. though the resulting three viruses all seemed to exhibit increased replication in macrophages relative to the parental 79-1146 /1683s virus, the effect was only significant for the virus carrying the combined substitutions a974 v and k1096q (fig. 2b ). yet, the titer this virus reached was still much lower than that of fipv rec 79-1146. in a next round of mutagenesis, we combined additional substitutions by including in our analysis also "non-unique" amino acid differences between the s2 proteins of strains 79-1683 and 79-1146. we were particularly interested in the effect of substitutions in the protein's c-terminal domain, in view of the atypical amino acid pattern of strain tokyo/cat/130627 in this domain relative to that of all other fipvs in our sequence collection (see fig. 1c ). thus, we combined substitutions of the three 79-1683 -specific amino acids with substitutions at positions 1407 and 1436. all 9 new viruses grew equally well in fcwf cells to titers of 10 6 to 10 7 tcid50/100μl (fig. 2c) . in macrophages, replication of most viruses appeared to be enhanced to some extent relative to virus 79-1146 /1683s. among the 7 viruses combining 2 substitutions, significant enhancement was observed for the combination of mutations a974 v/i1407 v and p1239s/m1436i (fig. 2d) . in the latter case it is of note that each of these mutations individually had not revealed any effect ( fig. 2b and d) . additional enhancement of replication was achieved when the mutations at these different sites were further combined. thus, the combination of three (positions 974, 1096 and 1407) and five (positions 974, 1096, 1239, 1407 and 1436) amino acid substitutions increased virus yields extra by 1-2 log10 units (fig. 2d) . next, we studied the effects of the reciprocal mutations. thus, corresponding substitutions were made into recfipv 79-1146 and evaluated for their effect on virus replication in macrophages (fig. 3) . as before, all recombinant viruses showed unaffected growth to 10 6 to 10 7 tcid50/100μl in fcwf cells (fig. 3a) . consistent with our earlier observations (fig. 2) , single amino acid substitutions at positions 972 (v to a) and 1094 (q to k) reduced virus multiplication in feline macrophages slightly (fig. 3b) . when combining these substitutions, viruses carrying mutations v972 a/q1094 k and v972 a/q1094 k/v1405i grew statistically less efficient in macrophages than rec 79-1146 thought the difference was only about 1 log unit (fig. 3b) . finally, combining substitutions of all five amino acids resulted in a strong and significant virus growth reduction to a level approximating that of virus 79-1146 /1683s (fig. 3b) . collectively, the observations in figs. 2 and 3 indicate that the combined amino acid substitutions in the c-terminal part of s protein can revert the fcov macrophage infection phenotype. there are six other differences between fipv 79-1146 and fecv 79-1683 in the c-terminal part of their s proteins ( fig. 1c ; note that fipv strains df-2 and 79-1146 have the same origin). among these differences, the a to d substitution at position 1014 in 79-1146 s has been reported not to affect fcov infection of feline macrophages (rottier et al., 2005) . we therefore evaluated the effect of mutations at the remaining five positions in the 79-1146 s protein (fig. 4) . once again, all the resulting recombinant viruses grew to the usual titers of 10 6 to 10 7 tcid50/100μl in fcwf cells (fig. 4a) . of the five single amino acid substitutions, only mutation f1416c caused a significant, about 3 log 10 decrease in virus yield in feline macrophages. consistently, the reciprocal mutation c1418 f introduced into the 79-1146 /1683 virus increased replication significantly as well (fig. 4b) . we also introduced this mutation into two rec fipv 79-1146 viruses already carrying mutations that decreased replication in macrophages (v972 a/q1094 k/v1405i and v972 a/q1094 k/s1237 p/v1405i/ i1434 m) as well as into the corresponding two 79-1146 /1683s viruses that already carried the inverse mutations causing increased replication in macrophages (a974 v/k1096q/i1407 v and a974 v/k1096q/ p1239s/i1407 v/m1436i). however, when studied for its effect, no additional decrease or increase in virus yield was observed (fig. 4c) . finally, we evaluated the effects of the mutations on the susceptibility of macrophages to infection and on virus spread. primary infection (virus entry) was measured by immunofluorescence staining of the cells at 10 h.p.i. and virus spread was measured by staining the cells at 24 h.p.i. feline macrophages were inoculated at an m.o.i. of 10 (note that all virus stocks were titrated on fcwf cells), at which dose about 2% of the cells became antigen-positive at10 h.p.i. after inoculation with fipv rec 79-1146 while almost no cells became positive after inoculation with virus 79-1146 / 79-1683 (fig. 5) , consistent with observations using the parental viruses fipv 79-1146 and fecv 79-1683 (rottier et al., 2005) . for this study we selected three 79-1146 /1683s mutant viruses based on their significantly increased growth phenotypes in macrophages: mutants c1418 f, a974 v/k1096q/i1407 v and a974 v/k1096q/p1239s/i1407 v/m1436i. all three recombinant viruses showed increased primary infection at 10 h as well as increased infection spread in macrophages at 24 h after inoculation (fig. 5a) . the reciprocal substitutions, introduced into fipv rec 79-1146 s, had generally less dramatic effects on virus entry. only the substitution of all five amino acids caused a significantly decreased infection at 10 h.p.i. (fig. 5b) . however, when measured at 24 h.p.i., all three mutant viruses exhibited a significant decrease in secondary spread (fig. 5b) . the results indicate that the observed differences in viral growth in feline macrophages are caused by differences in virus entry and spread (i.e. secondary infection) rather than by differences in virus replication per se. serotype ii fcov strains 79-1683 and 79-1146 grow similarly well in feline culture cells like fcwf and crfk but propagate remarkably different in cultured feline bone marrow macrophages, with strain 791683 multiplying very poorly in these macrophage cultures. interesting as these observations are already by themselves, their impact seems even more relevant considering that the differential ability to infect macrophages is the main hallmark distinguishing the harmless enteric from the lethal systemic fcov pathotype (pedersen, 2009 (pedersen, , 2014 . hence the observations described in this paper are of peculiar interest as the fecv 79-1683 and fipv 79-1146 s trains studied have historically served as the prototypes of both pathotypes, though their precise origin and identity have recently been questioned, as discussed below. earlier we mapped the differential efficiency of macrophage infection by the two prototypic serotype ii fcov strains to the s2 subunit of the viral spike protein. coronavirus spike proteins, trimers of which form the typical virion projections, are class i fusion proteins of which the n-terminal s1 subunit functions in receptor binding and the cterminal s2 subunit in membrane fusion. functional domains within the s2 protein are a proteolytic cleavage site (s2') for fusion activation, located just upstream of a fusion peptide (fp), downstream of which two heptad repeats (hr1 and hr2) occur, followed by a transmembrane (tm) domain and a c-terminal tail. of the 5 amino acid differences in the s2 subunit that distinguish fecv 79-1683 from almost all known fipvs one is located within the putative fp, one in hr1, one in the domain between hr1 and hr2, one in the tm domain and one in the c-terminal tail (fig. 1c) . when analyzing these differences by introducing the relevant mutations into the s2 domains of our chimeric test viruses it appeared that none of the 5 differences individually could account for the difference in macrophage tropism. thus, though some of the single mutations (a974 v, k1096q) significantly enhanced infection mediated by the fecv 79-1683 s protein, the effect of the others was only marginal. reciprocally, none of the single mutations introduced into fipv 79-1146 s ignificantly reduced infection of macrophages. rather, combinations of the 5 mutations were required, and only the collective effect of all 5 mutations elicited the maximal, almost complete phenotype reversal, both ways. as the tropism difference between the two fcov strains results from multiple amino acid differences in multiple functional domains of the s2 subunit, a clear mechanistic interpretation is difficult. since infection by both viruses is mediated by binding of their s1 subunit to the same fapn receptor (rottier et al., 2005) , the individually insignificant differences in their s2 subunit somehow cooperatively determine whether or not feline macrophages, not other feline cells, can be infected. if not at the level of receptor recognition, these differences must act at another level of cell entry, one that manifests itself specifically upon infection of macrophages. considering their distribution across the s2 polypeptide various aspects can then be considered including coreceptor usage, fusion activation and membrane fusion per se. except for the potential involvement of sialic acids and c-type lectins as attachment factors -mediated by the s1 subunit (li et al., 2016) and by s protein-linked sugars (regan et al., 2010; van hamme et al., 2011) , respectively -there are no indications for an involvement of an additional receptor in fcov infection. yet, it cannot be excluded that such co-receptor, interacting with the s2 subunit, is essentially required for infection of feline macrophages and specifically exposed by these cells. similarly, also conditions for membrane fusion per se might be typically different in macrophages as compared to other cells. specific circumstances such as the membrane lipid composition might require adaptations in the s2 protein to enable efficient fusion and infection. activation of coronavirus spike proteins for membrane fusion occurs by proteolytic cleavage at two sites, the s2' site located just upstream of the fp and the s1/s2 site (millet and whittaker, 2015) . cleavage at the latter site is typically mediated by furin-like enzymes. this cleavage seems, however, not essential; a furin cleavage motif is absent in the s proteins of many coronaviruses. interestingly, unlike serotype i fcovs the s proteins of which do have a furin cleavage site (de haan et al., 2008) , such a site is lacking in fcovs of serotype ii. the implications hereof are presently unclear. in the comparable situation of sars-cov, introduction of a furin cleavage motif at the s1/s2 site was shown to enhance cell-cell fusion mediated by the expressed spike protein (belouzard et al., 2009) . proteolytic activation at the s2' site liberates the fusion peptide, which seems critical for efficient initiation of membrane fusion. cleavage is achieved by enzymes occurring at the cell surface or in the endosomes. the repertoire of these proteases can vary significantly among different cell types, which hence potentially represents a host tropism determinant. consistently, the tropism difference between the serotype ii fcov pathotypes was found by regan et al. to be accompanied by distinctive differences in the sensitivities of fecv 79-1683 and fipv 79-1146 to inhibitors of endo-/lysosomal enzymes and endosomal acidification. thus, while infection of various feline cell lines (crfk, ak-d and fc2lu) with fecv was highly dependent on cathepsin b and l as well as on low endosomal ph, infection by fipv was independent on cathepsin l and low ph condition but dependent on cathepsin b activity only. notably, this dependence of fipv on cathepsin b was also found to be critical for infection of primary feline blood monocytes . the primary sequences of the two viruses at and around their s2' site do not offer an obvious explanation for these different entry requirements. though there is one sequence difference occurring immediately upstream of the fp, where fipv has a g instead of the r 963 that occurs in the fecv s sequence, this difference is not consistent when considering other available fipv sequences. besides in strain df2, which is nearly identical to fipv 79-1146 and which is perhaps just another descendant of the same viral isolate from which fipv 79-1146 originated, all other fipvs have an r at this position (fig. 1c) . we can only speculate about the reasons for the different macrophage entry requirements of fecv 79-1683 and fipv 79-1146, such as long-range structural effects on the exposure of the s2' loop that might be caused by other sequence differences in the s2 subunit. with regard to the r/g difference observed at the s2' site of fcov strains 79-1683 and 79-1146 it is interesting to mention an r to g mutation at exactly the same s2' position that occurred at least twice independently during adaptation of pedv field viruses to vero cells wicht et al., 2014) . whereas infection of cultured cells with pedv requires trypsin to activate the spike protein for membrane fusion, this particular mutation appeared to render pedv infection trypsin-independent (wicht et al., 2014) . of the sequence differences between the fcov strains 79-1683 and 79-1146 s 2 proteins those at fecv positions 974, 1096 and 1407 generally contributed most strongly to the macrophage entry phenotype. of these, residue a 974 occurs in the putative fp. the mechanistic consequences of a change to v as it occurs in fipv s2 are hard to imagine. this holds as well for a similar change in hydrophobic residues -i 1407 to v -occurring in the tm domain. residue k 1096 is located in the hr1 domain. its positive charge is likely to be involved in stabilizing the prefusion spike structure and in facilitating the proper assembly of the 6-helix bundle during the membrane fusion reaction. a change into the polar residue q is likely to affect these processes though it remains hard to understand why this is beneficial for infection of macrophages. the other two unique differences -p 1239 vs s and m 1436 vs iexhibited their effects only in combination with the others. though the p to s change certainly has structural consequences, the effects of either of these changes cannot be easily appreciated without more knowledge about the fcov spike structure as well as about the molecular details of the membrane fusion process. an interesting example of how subtle mutations can alter the entry pathway and thereby also the susceptibility of cells to viral infections was recently reported for another alphacoronavirus, hcov-229e, known to cause common cold. primary isolates of this virus were found to prefer cell entry via the cell surface by using the transmembrane protease serine 2 [tmprss2]) as the cellular protease for cleavage activation of their s protein (shirato et al., 2017) . upon serial passaging in culture cells, the virus appeared to rapidly adapt to preferentially using the endosomal pathway by the acquisition of just a single mutation (s577d) that allows fusion activation of the s protein by cathepsin l. consistently, the highly passaged classical hcov-229e isolate (atcc-vr740) was already shown earlier to use this endosomal enzyme to infect cells (kawase et al., 2009) . in this case, sequence comparison of the s protein of this laboratory strain with s proteins of clinical isolates led to the identification of two other responsible mutations. their substitutions (r642 m and n714 k) in the s protein of the laboratory strain reduced hela cell infection and rendered the virus less sensitive to the action of cathepsin l inhibitor while the converse was observed after introduction of the reciprocal mutations in the s protein of a clinical isolate, suggesting these amino acids to be critical for the cathepsin l recognition of the hcov-229e s protein (shirato et al., 2017) . thus, different amino acid substitutions, all in the s2 part of the viral spike protein, gave rise to the same outcome. notably, none of these residues is part of the protease recognition site itself as deduced from 3d structural homology modeling, suggesting their substitutions induce structural changes that alter the s protein's accessibility to cathepsin l. similar effects might also explain the differential abilities of different feline coronaviruses to infect feline macrophages, not only for the serotype ii strains investigated in the current study but perhaps as well for the two serotype i pathotypes that we showed earlier to generally differ by just one amino acid (m1058 l or s1060 a) at a position just upstream of the fp (chang et al., 2012) . lacking a suitable cell culture system for the more relevant serotype i fcovs, studies into the molecular biology of fcovs have for decades been performed almost exclusively with serotype ii viruses, out of necessity. strains 79-1683 and 79-1146 always served as the pathotypic prototypes, particularly after a comparative evaluation of their pathogenicity in kittens had reproduced the distinctive characteristics of the intestinal and systemic infections, respectively, including the typical replication in mature apical columnar epithelial cells by the enteric virus which was also observed for serotype i fecv strain ucd (pedersen et al., 1984b) . more recently, however, concerns have been raised regarding the precise identities of these serotype ii viruses (pedersen, 2009 (pedersen, , 2014 . fipv 79-1146 was originally reported to be isolated from a 4-day-old kitten . it has about 99% homology to fipv strain df2 (except in its orf3abc region) (balint et al., 2012) , which was reported to originate from the spleen of a cat that died from fip (evermann et al., 1981) . the fecv 79-1683 virus was originally isolated from tissue of an adult cat with fatal peracute hemorrhagic enteritis , yet caused mild to inapparent enteritis in spf cats (pedersen et al., 1984b) . all these viruses were isolated around the same time and in the same laboratory, leading to speculations about their possible relationships. thus, it was suggested that the three virulent viruses might share a common ancestor (balint et al., 2012) and that the avirulent virus might actually represent a tissue culture-attenuated derivative of a virulent precursor (pedersen, 2009 (pedersen, , 2014 . the latter concern stems in particular from the observed occurrence of deletions in fecv 79-1683' s genes 3c and 7b, genes that are normally intact in avirulent field strains. these deletions may have been acquired during in vitro passaging of the virus as these genes are known to be dispensable for viral replication in cultured cells; actually, during propagation in cell culture fcov mutants with deletions in the 7b gene are even selected for, suggesting that functional loss of the gene increases viral fitness in vitro (herrewegh et al., 1995) . of note, avirulent field viruses with (generally small) deletions in gene 7b have been described (desmarets et al., 2016; lin et al., 2009a) . it is also worrisome that strain 79-1683 thus far is the only serotype ii enteric coronavirus isolated. though such viruses have been detected in feces from clinically healthy cats by rt-pcr (lin et al., 2009b) , no successful isolations have been described and, thus, no alternative enteric pathotypes are yet available. in our previous study (rottier et al., 2005) we described that neither the functional deletion of gene 7b nor the restoration of 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determined by mutations in the feline coronavirus spike protein clinical isolates of human coronavirus 229e bypass the endosome for cell entry intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence intriguing interplay between feline infectious peritonitis virus and its receptors during entry in primary feline monocytes genetic drift and genetic shift during feline coronavirus evolution feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses pathogenic characteristics of persistent feline enteric coronavirus infection in cats proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture the authors declare that they have no competing interests. key: cord-272383-pzivb0ro authors: reddy, p.seshidhar; idamakanti, neeraja; zakhartchouk, lexander n; babiuk, lorne a; mehtali, majid; tikoo, suresh k title: optimization of bovine coronavirus hemagglutinin-estrase glycoprotein expression in e3 deleted bovine adenovirus-3 date: 2000-11-08 journal: virus res doi: 10.1016/s0168-1702(00)00209-4 sha: doc_id: 272383 cord_uid: pzivb0ro adenoviral vectors expressing foreign genes have many desirable properties in applications such as vaccination. recently, we have generated replication-competent (e3 deleted) bovine adenovirus-3 (bav-3) recombinants expressing significant amounts of glycoprotein d (gd) of bovine herpesvirus-1 (a dna virus). however, attempts to express the rna virus genes using the same strategy were not successful. in an effort to optimize the expression, we have constructed several bav-3 recombinants carrying the hemagglutinin esterase (he) gene of bovine coronavirus (bcv) in the e3 region with or without exogenous transcription control elements. the expression studies suggest that the introduction of a 137 bp chimeric intron upstream of the he cdna is able to increase the level of he gene expression. the introduction of a sv40 early promoter or human cytomegalovirus (hcmv) immediate early (ie) promoter into the expression cassette changed the kinetics of the he expression. however, the recombinant bav-3 containing he under the hcmv ie promoter replicated less efficiently than the wild-type bav-3. these studies should prove useful in expression of other rna viral genes in the e3 region of bav-3 expression system. bovine coronavirus (bcv) contains a positive single stranded rna genome of about 30 kb in length. the bcv genome encodes three membrane glycoproteins, the integral membrane protein (m), the spike protein (s), and the hemagglutinin-esterase (he) (king and brian, 1982) . the s and he proteins are major membrane associated glycoproteins and induce virus neutralizing antibodies . the monoclonal antibodies raised against the he protein neutralize the infectivity of bcv under cell culture conditions and protected the intestinal epithelia of cattle from the virus infection (deregt and babiuk, 1987) . in addition, the immunogenicity of a recombinant human adenovirus-5 (hav-5) expressing the he gene was tested in cotton rats (baca-estrada et al., 1995) . although hav-5 based vectors (expressing vaccine antigens from different bovine pathogens) have been used to test the efficacy and immunogenicity of protective antigens in experimental animals (baca-estrada et al., 1995; breker-klassen et al., 1995; papp et al., 1997) , their use as recombinant vaccines in cattle is limited due to their broad host range, which creates regulatory concerns. in recent years, the idea of using bovine adenovirus-3 (bav-3) as a vector (baxi et al., 1999; reddy et al., 1999; zakhartchouk et al., 1998) for delivery of protective antigen from pathogens of cattle has received considerable attention. deletion of the non-essential e3 region has facilitated construction of replication-competent bav-3 vectors expressing authentic and truncated forms of glycoprotein d (gd) of bovine herpes virus-1 . during infection of bovine cell lines, such recombinant bavs produced large amounts of glycoprotein gd (a dna virus gene), which has been shown to undergo proper post-translational modifications . in addition, intranasal inoculation of calves with these recombinants has been shown to be safe and efficacious, making these vectors particularly attractive for vaccination of cattle (zakhartchouk et al., 1999) . however, similar attempts to obtain high levels of expression of he gene of bovine corona virus in a replication-competent bav-3 vector without any flanking upstream or downstream sequences have been unsuccessful. since the level of expression of an antigen may determine the quality of immune response in animals immunized orally or intranasally with recombinant adenoviruses, it is necessary to maximize the expression of a vaccine antigen. in addition, as the development of a quality immune response would correlate with levels of antigen produced by the recombinant adenovirus vectors, we investigated ways of increasing he gene expression in the bav-3 expression system. the main reason to choose bcv he gene was that it has been difficult to over express he using a variety of heterologous expression systems (unpublished data). in addition, bcv causes neonatal diarrhea in calves resulting in significant economic losses due to mortality and decreased productivity of the survivors. since currently available vaccines against bcv are not effective (waltner-toews et al., 1995) , better vaccines are needed to reduce the economic losses. in the present study, we examined the effect of exogenous transcription control elements on the expression levels of the he gene in e3 deleted bav-3 vector. wild-type and recombinant bav-3s were cultivated in madin darby bovine kidney (mdbk) and vido r2 cells (transformed fetal bovine retina cells; reddy et al., 1999) . the cells were grown in eagle's minimum essential medium (mem) supplemented with 5% fetal bovine serum. the viral dna was extracted from virus infected cell monolayers by the method of hirt (1967) . the original e3 transfer vector, pbav-300 has the genomic dna sequences between nts 24465 and 28593 (nt numbers are based on bav-3 genome sequence; genbank accession no. af030154) with a deletion of 1245 bp of the e3 region from nt 26458 to 27703, cloned into a bacterial plasmid . this transfer vector has an overlap of 1992 bp on the left side and 889 bp on the right side of the e3 region for homologous recombination in e. coli bj 5183 with the e3 deleted full-length clone pfbav-302 . to increase the overlap, initially the kpni-sspi fragment representing the right side of the bav-3 genome between nt 24464 and 34060 was introduced into kpni and blunt-ended noti sites of ppolyii sn 14 to generate a plasmid, pbav-299. the region spanning the kpni and xbai sites of pbav-299 was replaced with that of pbav-300 to generate pbav-301. the plasmid pbav-301 can be digested with kpni (nt 24464) and spei (nt 31570) enzymes and the gel purified fragment can be used for homologous recombination in e. coli bj 5183. this new transfer vector has two unique restriction enzyme sites (srfi and sali) for cloning of foreign genes and an overlap of 1992 bp on the left side and 3866 bp on the right side of the e3 region for efficient homologous recombination with plasmid pfbav-302 , which dramatically increased the frequency of recombination in bj 5183 cells. plasmid pbav301b was constructed by cloning a 137 bp long chimeric intron amplified by pcr from pci-neo (promega) into the srfi site of pbav-301. the intron is composed of the 5%donor site from the first intron of the human beta-globulin gene and the branch, and 3%-acceptor site from the intron of an immunoglobulin gene heavy chain variable region (senapathy et al., 1990) . the transgenes are introduced downstream of the intron in order to prevent utilization of possible cryptic 5%-donor splice sites within the bcv he cdna sequence. the 1.3 kb bamhi fragment of a plasmid pcve3 (parker et al., 1989) containing the complete coding sequence of bcv he gene was treated with t4 dna polymerase and ligated to blunt end repaired srfi digested plasmid pbav301 to create plasmid pbav301.he and blunt end repaired sali digested plasmid pbav301b to create plasmid pbav301b.he. the recombinant bav-3 genomes containing the gene encoding he were generated by homologous recombination in e. coli bj5183 between srfi lin-earized pfbav302 and the 7.2 kb kpni -spei fragment of pbav301.he creating plasmid pf-bav303, and between srfi linerarized pfbav302 and the 7.3 kb kpni -spei fragment of pbav301b.he creating plasmid pfbav332. plasmid psvpia containing a unique sali cloning site was constructed by ligating a 209 bp sv40 promoter (isolated from pcat-promoter plasmid; promega), 137 bp chimeric intron and 240 bp sv40 late poly(a) signal (isolated from pci-neo; promega) to plasmid ppolyiisn. plasmid pcmvpia is similar to plasmid psvpia except that sv40 promoter is replaced by a 510 bp hcmv promoter (isolated from pcmvi; clonetech). the 1.3 kb blunt end repaired bamhi fragment containing the he gene (parker et al., 1989) was ligated, to blunt end repaired sali digested plasmid psvpia to create plasmid psvpia.he, and to blunt end repaired sali digested plasmid pcmvia to create plasmid pcmvia.he. a 1.9 kb fragment of plasmid psvpia.he and a 2.0 kb fragment of plasmid pcmvpia.he containing the he gene under appropriate transcriptional elements were isolated and ligated individually to blunt end repaired srfi digested plasmid pbav301 to create plasmids pbav301.hesv and pbav301.hecmv respectively. finally, the recombinant bav-3 genomes were isolated by homologous recombination in e. coli bj5183 between srfi linearized plasmid pf-bav302 and the 7.8 kb kpni -spei fragment of pbav301.hesv creating plasmid pfbav333, and between srfi linearized plasmid pfbav302 and the 8.1 kb kpni -spei fragment of pbav301.hecmv creating plasmid pfbav334. vido r2 cell monolayers in 60 mm dishes were transfected with 5-10 mg of pac-i digested pfbav303, pfbav332, pfbav333 and pf-bav334 recombinant plasmid dnas using lipofectin reagent. after incubation at 37°c, the transfected cells showing cytopathic effects were collected, freeze thawed two times and the recom-binant viruses were plaque purified on mdbk cells. dna fragments obtained after restriction enzyme digestion of virion dna were transferred from agarose gels to nytran membranes (schleicher and schuell) as described (reddy et al., 1993) . the gene coding for he of bcv was labeled with 32 p dctp by the random primer labeling technique. hybridizations were carried out at 42°c in the presence of 50% formamide. prehybridization, hybridizations and washing of membranes were carried out as described elsewhere (reddy et al., 1993 ). mdbk cells grown in petri dishes were infected with 5 pfu per cell of recombinant bav-3s. total rna was extracted from mock infected or recombinant bav-3 infected cells with acid guanidinium thiocyanate-phenol-chloroform mixture as described by chomczynski and sacchi (1987) . rna (10 ug) was separated on 1% agarose -formaldehyde gels and transferred to nytran membranes (sambrook et al., 1989) . the blots were baked, prehybridized, hybridized and washed as described (sambrook et al., 1989) . the gene coding for he of bcv, labeled with h-32 p dctp by the random primer labeling technique was used as a probe. confluent monolayers of mdbk cells in six well dishes were infected with the virus at a multiplicity of infection of five. the cells were preincubated for 2 h in mem deficient of methionine and cysteine prior to labeling with 50 mci of 35 s methionine (trans ( 35 s) label [1000 ci/ mmol] icn radiochemicals inc., irvine, ca) for 4 h. the cells were washed once with pbs, harvested by scraping and then lysed with ice-cold modified radioimmunoprecipitation assay buffer. the radiolabeled proteins were immunoprecipitated with polyclonal anti-bcv rabbit antibodies (deregt and babiuk, 1987) and analyzed by sds-polyacrylamide gel electrophoresis (page) under reducing conditions. the gels were dried and protein bands were visualized by autoradiography. our initial attempts to insert the bcv he gene in the e3 region of plasmid pfbav302 (e3 deleted full length bav-3 genomic clone; zakhartchouk et al., 1998) by homologous recombination in e. coli bj5183 between the kpni -xbai fragment (with an overlap of 1992 bp on left side and 889 bp on the right side of e3 region) of plasmid pbav300 containing the bcv he gene and srfi linearized plasmid pfbav302 were unsuccessful. in order to increase the efficiency of inserting foreign genes in the e3 region of plasmid pfbav302 by homologous recombination in e coli bj5183, we first constructed a modified transfer plasmid pbav301. this plasmid contains an overlap of 1992 bp on the left side and 3866 bp on the right side of the e3 region with plasmid pfbav302. use of this plasmid dramatically increased the frequency of recombination in e. coli bj5183 and helped to successfully clone bcv he. next, we constructed recombinant bav-3s expressing the bcv glycoprotein he. the full length he gene alone or with different exogenous transcriptional elements was inserted individually into the e3 region of plasmid pfbav302 in the same transcriptional orientation of e3 using homologous recombination in e. coli (chariter et al., 1996) . the pac-i digested pfbav303, pf-bav332, pfbav333 or pfbav334 plasmid dna was transfected into vido r2 cells (transformed fetal bovine retina cells). the infected monolayers showing 50% cytopathic effects were collected, freeze thawed and recombinant viruses were plaque purified and propagated in mdbk cells. the recombinant viruses were named bav303 (he without exogenous elements), bav332 (he with chimeric intron), bav333 (he with sv40 promoter, chimeric intron and sv40 polya) and bav334 (he with cmv promoter, chimeric intron, sv40 polya) (fig. 1) . the viral dna was extracted from infected cells by the hirt method (hirt, 1967) and analysed by agarose gel electrophoresis after digestion with bamhi restriction enzyme. the digestion of wild type bav-3 viral dna with bamhi yields five fragments and the fragment d (3.019 kb) contains the e3 region ( fig. 2a, lane 1) . the difference in the size of modified bamhi 'd' fragment of bav3.e3d (fig. 2a , lane 2) bav303 ( fig. 2a , lane 3), bav332 (fig. 2a, lane 4) , bav333 ( fig. 2a , lane 5) and bav334 (fig. 2a, lane 6) was as expected. this was confirmed by southern blot analysis of bamhi digested genomic dna of wild type and recombinant bav3s. as seen in the fig. 2b , the same modified bamhi 'd' fragment (s) from the recombinant viruses hybridized to h-32 p dctp labeled he gene in the southern blot hybridization (fig. 2b, lanes 3,4,5,6 ). this suggested that recombinants bav303, bav332, bav333 and bav334 contained the he gene. to analyze transcription of the he gene, rna was prepared from mock infected or recombinant bav-3 infected cells at 18 and 28 h postinfection. the rna was separated on agarose -formaldehyde gels, transferred to nytran membrane and probed with the h 32 p labeled he gene the probe was expected to detect four l6 mrnas (100 k, 33 k, 23 k and pviii) and the mrnas of he transcribed from the e3 promoter and mlp. unlike in hav-2 (ziff and fraser, 1978) , the transcripts from the l6 region in bav-3 are polyadenylated at the poly(a) site of the e3 region . thus, all the transcripts of l6 region originating from the major late promoter (mlp) form a nested set of overlapping molecules with common 3' ends. each mrna contains all the sequences in the next smaller mrna plus one additional orf at the 5' end. only the orf at the 5' end of the mrna is translated. when rna was analyzed, several abundant mrnas were identified that had he sequences in them particularly in rna that was extracted at 28 h postinfection (fig. 3) . at 28 h postinfection, the larger transcripts were the dominant species of rna extracted from bav303 (lane 2) and bav333 (lane 6) infected cells. during early stages of hav-5 infection, the e3 promoter is used to express mrnas from the e3 region and during late stages, transcription from the e3 promoter is reduced and some mrnas are made from the mlp (tollefson et al., 1992) . the major late e3 mrnas containing tripartite leader sequences are also produced in bav-3 . since the sizes of the transcripts were considerably larger than the genomic distance between the e3 promoter and poly(a) site of the e3 region, they must have been generated by splicing of the primary transcripts produced from the mlp. (parker et al., 1989 ) as a probe. numbers on the right denote the estimated sizes of rnas in kilobases. proteins from cell lysates, collected at different times post infection of mdbk cells with recombinant bav-3, were analyzed by immunoprecipitation assays using bcv specific polyclonal antiserum. electrophoretic analysis of metabolically radiolabeled immunoprecipitates from bcv (fig. 4abcd, lane 3) infected cell lysates detected a protein of 65 kda. no such protein was detected from mock (fig. 4abcd, lane 1) or bav-3 (fig. 4abcd, lane 2) infected cell lysates. the recombinant bav303 contains the he cdna sequence substituting for bav-3 e3 in the parallel orientation so as to allow expression from endogenous promoters. immunoprecipitation analysis of bav303 infected cell lysates showed little or no he expression (fig. 4a, lanes 4, 5, 6) . the recombinant bav332 has the he sequence in the e3, downstream of an exogenous chimeric intron and upstream of sv40 late poly(a) signal. immunoprecipitation analysis of bav332 infected cell lysates detected a specific band of 65 kda at 36 h post infection (fig. 4b, lane 6) . the recombinant bav333 and recombinant bav334 are similar to bav332 except that they have either sv40 or cmv immediate early promoters upstream of chimeric introns, respectively. immunoprecipitation analysis of bav333 infected cells detected a specific band of 65 kda at 24 (fig. 4c , lane 5) and 36 h (fig. 4c, lane 6) post infection. similarly, immunoprecipitation analysis of bav334 infected cells also detected a specific band of 65 kda at 24 (fig. 4d , lane 5) and 36 h (fig. 4d , lane 6) post infection. to determine whether insertion of exogenous transcription control elements into the e3 had any noticeable effect on the ability of these recombinant to replicate in mdbk cells, virus titer were determined. deletion of the e3 region had no detectable effect on the virus yield. bav303, bav332, and bav333 grew to similar titers as bav3.e3d (e3 deleted) virus. however, bav334 grew to a final titer that was 1.0 log 10 lower than the bav3.e3d (e3 region deleted virus; zakhartchouk et al., 1998) . the e3 region of bav-3 located between nts 26185 and 27776 is 1591 bp in length. the promoter of the e3 is located within the coding sequence of the pviii and shares its poly(a) site with the l6 . recently, we have developed a replication-competent bav-3 vector with a 1.245 kb deletion in the e3 region, removing all e3 open reading frames, leaving the e3 promoter, 5% intiation site, and the poly(a) site intact . recombinant bav-3 expressed high levels of full length or truncated forms of glycoprotein d (gd) of bhv-1 when the genes coding for these proteins were introduced into the e3 region in the same orientation as the e3 transcription unit without any exogenous transcription control elements . however, when the cd-nas representing the protective antigen from rna viruses including bcv were inserted into the e3 region of bav-3, no expression of the proteins was obtained (unpublished observation). in an attempt to optimize the expression of rna virus genes in the bav-3 expression system, we have constructed and characterized four recombinant viruses carrying the he gene of bcv (with or without exogenous transcription control elements). adenoviruses replicate in the nucleus, whereas all except one rna virus replicate in the cytoplasm. if a rna virus gene is to be expressed through a recombinant adenoviral vector system, its mrna must be free of any cryptic splice sites or else the message could be broken into pieces by the splicing machinery present in the cell nucleus. this can be prevented either by swapping codons to remove internal splice sites (expensive) or by inserting an intron upstream of the cdna. since transfection studies have demonstrated that an intron flanking the cdna insert frequently increases the level of cdna expression (huang and gorman, 1990) , it was hypothesized that introduction of an intron upstream of a cdna could prevent utilization of possible cryptic 5%-donor splice sites within the cdna sequence (huang and gorman, 1990) . addition of poly(a) + [e.g. sv40 late poly(a) signal] has also been shown to enhance rna stability and translation (carswell and alwine, 1989; jackson and standart, 1990 ). however, the size of the poly(a) and the intron should be minimal as its inclusion will reduce the ultimate capacity of the adenovirus vector. the intron a, which is most widely used to express cdnas is about 800 bp in length and will reduce the size limit of foreign dna that can be put into bav-3 vectors. keeping this in mind, the he gene was flanked with a chimeric intron of 147 bp in length (senapathy et al., 1990) and the sv40 late poly(a) + signal at the 5% end and 3% end respectively. addition of a chimeric intron and sv40 poly(a) + signal dramatically increased the levels of he gene expression. from the size of majority of mrnas, it appears that the he transcripts must have initiated from the major late promoter (mlp). it seems likely that the chimeric intron provided the strong splice acceptor site. consis-tent with this speculation is the fact that synthesis of he was observed late (36 h) after infection, when much of the transcription occurs from mlp. introduction of a sv40 (bav333) or hcmv ie (bav334) promoter into the expression cassette modified the kinetics of he expression as he glycoprotein could be detected as early as 24 h post infection. it seems likely that early he transcripts must have been initiated from the bav-3 e3 promoter or sv40/hcmv promoter. support for this hypothesis comes from the fact that (a) he specific mrnas of smaller size are abundant in bav333 and bav334 infected cells; (b) bav-3 e3 284r protein expressed from the e3 promoter can be detected at 24 h but not at 12 h post infection . however, it is not clear how many of these transcripts arise from either the e3 promoter or sv40 promoter, and are translated to give rise to the he polypeptide expressed early in recombinant infected cells. the bcv he gene under the control of a sv40 promoter was cloned and expressed using hav-5 (yoo et al., 1992) . expression of the he was seen as early as 6 h postinfection and produced throughout the infection. however, bav-3 differs from hav-5 with respect to replication kinetics. in bav-3 infected cells, viral dna replication begins at about 24 h postinfection and reaches a peak after 40 h, whereas viral dna replication in hav-5 infected cells occurs as early as 12 h postinfection (niiyama et al., 1975) . the green fluorescent protein (gfp) expression was noticed at 12 h postinfection when the gene for gfp was placed under the control of cmv immediate early promoter in the e3 region of bav-3 . this suggests that kinetics of foreign gene expression from the e3 region of bav-3 may be influenced not only by exogenous transcriptional elements but also by the nature of the foreign gene. in conclusion, we have shown that 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bovine adenovirus type 3 as an expression vector nucleotide sequence, genome organization, and transcription map of bovine adenovirus type 3 restriction endonuclease analysis and molecular cloning of porcine adenovirus type 3 molecular cloning: a laboratory manual splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project the 11 600-m w protein encoded by region e3 of adenovirus is expressed early but is greatly amplified at late stages of infection a field trial to evaluate the efficacy of a combined rotavirus-coronavirus/e. coli vaccine in dairy cattle synthesis and processing of haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the e3 region of adenovirus mucosal immunization of calves with recombinant bovine adenovirus-3: induction of protective immunity to bovine herpesvirus-1 construction and characterization of e3 deleted bovine adenovirus type 3 expressing full-length and truncated form of bovine herpesvirus type 1 glycoprotein gd adenovirus type 2 late mrna, structural evidence for 3%-coterminal species we thank stu skinner for his help in the construction of psvpia and pcmvpia, and caron pyne for southern blot analysis. this work was supported by grants from national science and engineering research council of canada, saskatchewan agriculture development fund, saskatchewan beef development fund, and alberta agriculture research institute. p.s.r. was recipient of a postdoctoral research fellowship from the health services utilization and research commission, saskatoon, saskatchewan, canada. this paper is published with the permission of the director of vido as journal series no. 259. key: cord-288253-wqrhiq08 authors: park, jung-eun; park, eui-soon; yu, jung-eun; rho, jaerang; paudel, sarita; hyun, bang-hun; yang, dong-kun; shin, hyun-jin title: development of transgenic mouse model expressing porcine aminopeptidase n and its susceptibility to porcine epidemic diarrhea virus date: 2015-02-02 journal: virus res doi: 10.1016/j.virusres.2014.12.024 sha: doc_id: 288253 cord_uid: wqrhiq08 porcine coronavirus infections have known as they are specific to pigs with predominantly enteric or respiratory diseases. no laboratory animal model is yet been developed in porcine coronaviruses study. here, we report that development of a transgenic mouse model expressing porcine apn which is susceptible to porcine coronavirus infection. the porcine apn transgene was constructed by fusing with mouse proximal apn promoter at 5′ terminus and bovine growth hormone polyadenylation site at its 3′ terminus. after screen on pubs from the microinjected mice, we confirmed two transgenic lines expressing porcine apn in various organs. we confirmed the susceptibility to porcine epidemic diarrhea virus, one of the porcine coronaviruses. these transgenic mice will be an important tool for research into the porcine coronaviruses. the coronaviruses belong to the family coronaviridae within the order nidovirales. they are classified into four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, based on genetic similarities (adams et al., 2014) . the primary replication of the coronaviruses is often confined to respiratory-or gastrointestinal-tract epithelial cells, so they usually induce respiratory or enteric diseases, but also hepatic, renal and neuronal infections (lednicky et al., 2013; masters, 2006; weiss and navas-martin, 2005) . the pathogenesis of several porcine coronaviruses, including transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcov), has been broadly studied (enjuanes et al., 1995; saif, 2004a,b; saif, 2004a,b; weiss and navas-martin, 2005) . tgev is a major cause of viral enteritis and fetal diarrhea in swine, most severely in neonates and with a high mortality rate, which causes significant economic losses worldwide (enjuanes et al., 1995) . prcov is reported to be an attenuated variant of tgev. prcov infects lung epithelial cells, and prcov antigen has been found in type i and type ii pneumocytes and alveolar macrophages, and infection is followed by interstitial pneumonia (halbur et al., 1993; saif, 2004a,b) . porcine epidemic diarrhea virus (pedv) is the causative agent of porcine epidemic diarrhea, which is characterized by watery diarrhea, as is tgev infection (chasey and cartwright, 1978; pensaert and de bouck, 1978; turgeon et al., 1980) . pedv-infected piglets usually show typical enteric signs, including profuse watery diarrhea, weight loss, and loss of milk uptake, entailing high mortality. pedv has been reported in europe and asia, and also recently in the united states (hess et al., 1980; huang et al., 2013; pan et al., 2012; park et al., 2013; wang et al., 2014) . all these porcine coronaviruses belong to the genus alphacoronavirus. a hemagglutinating enteric coronavirus, a member of the genus betacoronavirus, is antigenically unrelated to the other porcine coronaviruses and uses a 5-nacetyl-9-o-acetylneuraminic-acid-containing moiety as its cellular receptor. coronavirus infections are mediated by the spike (s) glycoprotein, a large surface glycoprotein on the viral envelope (delmas and laude, 1990; masters, 2006) . coronavirus s glycoproteins recognize cellular receptors and mediate virus-cell fusion (masters, 2006; peng et al., 2011) . most alphacoronavirus, including tgev, pedv, and prcov, use aminopeptidase n (apn/cd13) as their cellular receptor (delmas et al., 1992; li et al., 2007; ren et al., 2010; tresnan and holmes, 1998; yeager et al., 1992) . apn/cd13 is a 150-kda, zinc-dependent metalloprotease consisting of 967 amino acids (rawlings and barrett, 1995) . mammalian apn is ubiquitously expressed as a glycosylated homodimer on the surfaces of epithelial cells in the liver, intestine, kidney, and respiratory tract, and in fibroblasts and leukocytes, and plays multiple roles in many physiological processes, including coronavirus entry (barnes et al., 1994; luan and xu, 2007; mina-osorio et al., 2008; miura et al., 1983) . the natural hosts of porcine coronaviruses are young piglets, and clinical illness has only been observed in sucking piglets. however, in vivo studies using suckling piglets have many disadvantages, including their high cost, difficulty in handling the piglets, limited reagents, etc. because the use of laboratory animal models (e.g., mouse models) can circumvent these limitations, several transgenic animal models have been generated to study porcine viruses (benbacer et al., 1998; ono et al., 2006) . here, we generated transgenic mice expressing porcine apn under the control of the mouse proximal apn promoter. we tested their susceptibility to pedv with reverse transcription-polymerase chain reaction (rt-pcr) and immunochemical analyses, and found that the porcine apn transgenic mice were susceptible to pedv infection. vero monkey kidney cells were maintained in minimal essential medium containing 10% fetal bovine serum (fbs), 100 u/ml penicillin g, 100 g/ml streptomycin, and 250 g/ml amphotericin b in a 5% co 2 atmosphere at 37 • c. 293t human embryonic kidney cells were maintained in dulbecco's modified eagle's medium containing 10% fbs, 100 u/ml penicillin g, 100 g/ml streptomycin, and 250 g/ml amphotericin b. all tissue culture reagents were purchased from gibco (carlsbad, ca, usa). kpedv-9, a cell-adapted vaccine strain of pedv, was grown and titrated in the vero cells, as described previously, and stored at −80 • c until use (cruz and shin, 2007; hofmann and wyler, 1988) . the polyclonal antibodies specific for pedv and porcine apn were generated in balb/c mice immunized with kpedv-9 and purified porcine apn, respectively, as described previously (cruz et al., 2008) . the antibody specificities were confirmed with enzyme-linked immunosorbent assays. the anti-flag m2 monoclonal antibody (anti-flag) and anti-flag m2 affinity gels (gel beads) were purchased from sigma-aldrich (st. louis, mo, usa). fluorescein isothiocyanate (fitc)-conjugated anti-mouse igg antibody was purchased from santa cruz biotechnology (santa cruz, ca, usa). the mouse proximal apn promoter region (starting at nucleotide-1044) was amplified from c57bl/6j genomic dna by pcr with primers 5 -cccgcggccgcaagatttgaaacagtgga-3 and 5 -cccaagcttgatgccggtggacaggga-3 , containing flanking noti and hindiii restriction endonuclease sites, respectively. the pcr product was cloned into the pbluescript ks (+) vector and the pgl3-basic vector (promega, madison, wi, usa), which contains a promoterless luciferase reporter gene. the porcine apn gene was amplified from the total rna isolated from porcine enterocytes with rt-pcr using specific primers and cloned into the pbluescript ks (+) vector. a sequence encoding the flag epitope (dykddddk) was fused to the 3 terminus of porcine apn with pcr with primers 5 -cccaagcttaccatggccaagggattctac-3 and 5 -cccctcgagtcacttgtcgtcatcgtctttgtagtcgctgtgctctat-gaacca-3 , which contain flanking hindiii and xhoi restriction endonuclease sites, respectively. the bgh-polya sequence was amplified from the pcdna3.1 vector (invitrogen, carlsbad, ca, usa) with pcr using primers 5 -cccctcgagcgactgtgccttctagtt-3 and 5 -cccggtaccccatagagcccaccgcat-3 , which contain flanking xhoi and kpni restriction endonuclease sites, respectively. the pcr product was cloned into the pbluescript ks (+) vector. all pcr products were confirmed with automated sequencing. to generate the porcine apn transgene, porcine apn-flag and the mouse proximal apn promoter were ligated into pbluescript ks-bgh-polya after they were digested with hindiii/xhoi and noti/hindiii, respectively. 293t cells (2.5 × 10 5 cells/ml) were plated in 24-well plates. after 24 h, the cells were transfected with pgl3-basic-mouse proximal apn promoter (mapn-luc) (0.2 g or 1 g). the cells were transfected in triplicate using lipofectamine 2000 (invitrogen), according to the manufacturer's instructions. after 24 h, the cells were lysed for assay with the luciferase assay system (promega), according to the manufacture's protocol. all luciferase activities were normalized to ␤-galactosidase activity. the porcine apn transgene was linearized by restriction with noti and purified with gel extraction (qiagen, valencia, ca, usa). gain-of-function gene transfer was performed by microinjecting the purified dna into the pronuclei of icr mouse zygotes, which were then transferred into the oviducts of female recipient mice. the transgenic mice were identified with pcr analysis of tail genomic dna with primers: pcr1-f: 5 -cccaagcttaccatggccaagggattctac-3 and pcr1-r: 5 -gaagttggagagcatcct-3 ; and pcr2-f: 5 -ggcgtcctacttgcatgc-3 and pcr2-r: 5 -cccctc-gagtcacttgtcgtcatcgtctttgtagtcgctgtgctctatgaacca-3 . the founder mice were backcrossed to the c57bl/6j background for five generations. all the mice used in this study were maintained in a specific-pathogen-free facility at the biomedical research center at the korea advanced institute of science and technology, daejeon, korea. all animals were cared for and the experiments were performed at the animal facility at chungnam national university (cnu), korea, with the permission of and according to protocols approved by the institutional animal care and ethics committee of cnu (permission number 20110825). the porcine apn transgenic and nontransgenic wild-type mice were orally inoculated with 5 × 10 6 tcid 50 of kpedv-9 or phosphate-buffered saline (pbs, ph 7.2) as the negative control. their clinical signs were monitored and their feces collected for 5 days. two mice from each group were killed on the indicated days after viral infection. the tissues were aseptically collected and prepared for rt-pcr and immunohistopathological analysis. total rnas were extracted from the feces and tissue samples using trizol ® reagent (invitrogen) and transcribed to cdna using power cdna synthesis kit (intron biotechnology, korea), according to the manufacturer's protocol. the porcine apn-flag cdna was pcr amplified with the specific primers used for the genotyping pcr. as a control, specific primers were used to amplify ␤-actin (mouse ␤-actin-f: 5 -cggttccgatgccctgaggctctt-3 and mouse ␤-actin-r: 5 -cgtcacacttcatgatggaattga-3 ). the cycling parameters were initial denaturation at 94 • c for 2 min, followed by 30 cycles of denaturation at 94 • c for 15 s, annealing at 60 • c for 40 s, and extension at 72 • c for 90 s, with a final extension step at 72 • c for 5 min. to detect the viral genome, viral rna was extracted from tissue homogenates using the viral gene-spin viral dna/rna extraction kit (intron biotechnology), according to the manufacturer's instructions. m-mlv reverse transcriptase (intron biotechnology) was used for first-strand cdna synthesis, together with 10 l of extracted viral rna in a 50 l randomly primed reaction. the specific primers used for the detection of the n gene were pedv-n forward (5 -ggtaccatggcatctgtcagcttt-3 ) and pedv-n reverse (5 -ggatccttaatttcctgt tcgaa-3 ). rt-pcr was performed at 94 • c for 2 min, followed by 30 cycles of 94 • c for 20 s, 54 • c for 10 s, and 72 • c for 2 min, with a final extension at 72 • c for 10 min. the pcr products were analyzed by gel electrophoresis and visualized with ethidium bromide staining and uv transillumination. 293t cells were lysed with cell lysis buffer (25 mm tris-cl [ph 7.5], 150 mm nacl, 1 mm edta, 1 mm naf, 1 mm sodium orthovanadate, 1 mm pmsf, 5% glycerol, 0.5% triton x-100, and protease inhibitors [roche, indianapolis, in, usa]). the small intestines were rinsed with pbs and homogenized in lysis buffer (1% np40, 150 mm tris-cl, 50 mm nacl, 1 mm edta). the cell or tissue lysates were separated with 8-15% gradient sds-page and transferred into pvdf membrane (amersham-pharmacia biotech, piscataway, nj, usa). the proteins were probed with an anti-flag monoclonal antibody diluted 1:4000 and an anti-␤-actin antibody diluted 1:10,000. the immune complexes were detected with peroxidase-conjugated goat anti-mouse igg antibody (santa cruz biotechnology) and visualized with amersham ecl western blotting detection reagent (amersham-pharmacia biotech). 293t cells were transfected with the porcine apn transgene. after 24 h, the cells were rinsed with pbs and detached with non-enzymatic cell dissociation solution (sigma-aldrich) for 2 min. the detached cells were harvested and resuspended in fluorescenceactivated cell sorting (facs) staining buffer (pbs containing 2% fbs). the cells were stained with the anti-flag m2 monoclonal antibody (1:200) for 15 min at 4 • c and then with fitc-conjugated anti-mouse igg secondary antibody (1:500) for 15 min at 4 • c. the antibody-stained cells were analyzed with flow cytometry (fac-scalibur, becton-dickinson biosciences, franklin lakes, nj, usa). kidney and liver tissues collected from mice were rinsed with pbs, and the renal or hepatic cells were isolated from the tissues with a 70 m mesh filter (becton-dickinson biosciences), resuspended in facs staining buffer, and analyzed as described above. small intestine samples were fixed in 10% buffered formalin and embedded in paraffin. serial 5 m sections were mounted on silane-coated slides. the sections were deparaffinized for immunohistochemical analysis, and the endogenous peroxidases were blocked with 0.6% h 2 o 2 in methanol for 30 min. antigens were retrieved with microwave heating in citrate buffer for 10 min, and the slides were incubated with 1.5% horse serum for 30 min at room temperature. polyclonal anti-porcine apn antiserum, monoclonal anti-flag antibody, or polyclonal anti-pedv antiserum were applied overnight at 4 • c. the biotinylated anti-mouse igg secondary antibody was detected with an avidin-biotin-peroxidase kit (vectastain abc kit, vector laboratories, burlingame, ca, usa). antibody binding was detected with the chromogen diaminobenzidine (vector laboratories), and the cells were counterstained with hematoxylin and eosin. 2.11. pedv replication in the small intestines of porcine apn transgenic mice both wild type and porcine apn transgenic mice were infected with pedv orally on day 0. viral titer in inoculum was 5x tcid 50 10 6 . two mice from each group were killed daily up to 5 days, and small intestine samples from all mice were collected in mem and homogenized. samples were centrifuged and supernatants were kept in −80 • c until processed for titration. titration was performed using vero cells. the pedv titer was measured and described in log value. the transcription of mouse apn is regulated by two different promoters, as shown in fig. 1a (bhagwat et al., 2001) . the distal promoter is located 8 kb upstream from the transcription -1 (3 and 4) , and cnupedm-3 mice (5 and 6). porcine apn rnas were amplified using specific primers targeting porcine apn, as described in section 2. ␤-actin was detected as the loading control. (b) expression of porcine apn protein in the small intestines. small intestine samples were collected from cnupedm-1 and cnupedm-3 mice. porcine apn protein was detected in small intestine homogenates with immunoblotting using an anti-flag monoclonal antibody (2 and 4). no recombinant protein was detected in the small intestine samples derived from nontransgenic wild-type mice (1 and 3). ␤-actin was used as the loading control. (c) immunohistochemical analysis of porcine apn transgenic mice. small intestine sections were prepared from porcine apn transgenic mice (a-c: cnupedm-1; d-f: cnupedm-3) and incubated with anti-flag monoclonal antibody (anti-flag) (a and d), mouse anti-porcine apn polyclonal antiserum (anti-papn) (b and e), or normal mouse serum (c and f). the immune complexes were visualized with avidin-biotin-peroxidase and the cells were counterstained with hematoxylin and eosin. magnification, ×40. start site and controls apn expression in myeloid and fibroblast cells, whereas the proximal promoter regulates apn expression in epithelial cells (olsen et al., 1991; shapiro et al., 1991) . following references and preliminary studies, we choose the mouse proximal apn promoter to express porcine apn in mouse epithelial cells. to test the promoter's activity, the sequence encoding the promoter region was amplified and cloned into the pgl3-basic promoterless vector (fig. 1b) , and the promoter activity was measured by luciferase assay. as shown in fig. 1b , the luciferase activity was about eight-fold higher in 293t human embryonic kidney cells transfected with the vector containing the mouse proximal apn promoter (mapn-luc) than in cells transfected with the empty vector. we constructed a vector encoding porcine apn, which was regulated by the mouse proximal apn promoter (porcine apn transgene) (fig. 2a) . the porcine apn cdna was amplified from porcine enterocytes and tagged at the c-terminus with the flag epitope to distinguish exogenously expressed porcine apn from endogenously expressed murine apn. the 2.9-kb flag-epitopetagged porcine apn cdna was cloned into the pbluescript ks (+) vector under the control of a 1.1-kb genomic sequence containing the mouse proximal apn promoter. the splice sites and polyadenylation signal for the cdna were provided by a 0.2-kb bovine growth hormone polyadenylation signal (bgh-polya). the expression of the porcine apn protein was detected with immunoprecipitation and immunoblotting in 293t cells transfected with the porcine apn transgene. the flag-tagged recombinant protein, with a molecularweight of about 150 kda (the expected molecular mass for porcine apn), was confirmed with immunoprecipitation (fig. 2b) . we also measured the surface expression of porcine apn with flow cytometry using an anti-flag antibody, and found that 21.3% of cells were flag-positive (fig. 2c ). our data demonstrate that the mouse proximal apn promoter efficiently induced porcine apn expression from our construct. the porcine apn transgene was linearized with noti restriction and the dna microinjected into mouse zygotes. to screen for porcine apn expression, we designed two genomic pcr primers, as indicated in fig. 3a (arrowheads) . the presence of the porcine apn transgene in mouse litters was monitored with pcr analysis on tail genomic dna (fig. 3b) . microinjection of the porcine apn transgene produced two transgenic founders, designated cnupedm-1 and cnupedm-3. the transgenic mice were healthy and showed no negative effects of transgene expression. because the major pathological changes of the porcine coronaviruses (e.g., tgev and pedv) involves enteric diseases, we measured porcine apn expression in the small intestine by rt-pcr, immunoblotting, and ihc. all the tested litters descended from cnupedm-1 or cnupedm-3 expressed porcine apn mrna in their small intestines (fig. 4a ). the small intestines expressed a recombinant protein with the molecular mass expected for porcine apn (fig. 4b ). an immunohistochemical analysis, with both anti-flag and anti-porcine apn antibodies, clearly confirmed porcine apn expression in the brush borders of the absorptive cells in the small intestines of the mouse model (fig. 4c) . these results demonstrate that the porcine apn transgenic mice expressed porcine apn in their small intestines. we also screened for porcine apn expression in various other tissues with rt-pcr. porcine apn mrna was strongly expressed in the lung, kidney, and the intestine, and was slightly expressed in the liver (fig. 5a) . the extracellular expression of porcine apn was examined in the kidney and liver by facs analysis (fig. 5b) . consistent with the rt-pcr results, the number of flag-positive renal cells was significantly higher (2-4.3-fold) in the porcine apn transgenic mice than in the nontransgenic wild-type mice, whereas the number of flag-positive hepatic cells was also slightly higher (1.6-3-fold) in summary, we confirmed the expression of porcine apn in the small intestines, lungs, livers, and kidneys of the porcine apn transgenic mice. we examined the susceptibility of the porcine apn transgenic mice to pedv, one of the enteropathogenic porcine coronaviruses. porcine apn transgenic or nontransgenic wild-type mice were inoculated orally with kpedv-9, a vero-cell-adapted korean strain of pedv. in infected transgenic mice, other than slightly watery feces observed at 3 days after inoculation (fig. 6a) , whereas no clinical signs (e.g., watery diarrhea, vomiting, fever, weight loss, or death) were observed for 5 days. viral replication was detected in various tissue extracts with rt-pcr. viral rna was confirmed in the small intestines (for at least 5 days), kidneys (for at least 2 days), and spleen (for at least 3 days) only in the porcine apn transgenic mice (table 1) , and not in the nontransgenic mice. an immunohistochemical analysis detected pedv antigen in the brush borders of the small intestines, at the same location at which porcine apn is expressed (fig. 6b) , which has been demonstrated in pedvinfected piglets (debouck and pensaert, 1980; pensaert et al., 1981) . however, no histopathological changes were observed in the small intestines that shown in pedv infected piglets. overall, these data indicate that porcine apn transgenic mice are susceptible to pedv infection, although they showed none of the enteric disease symptoms typical of pedv-infected piglets (fig. 7) . 3.6. pedv replication in the small intestines of porcine apn transgenic mice pedv replication in both wild type and porcine apn transgenic mice were confirmed and compared by viral load in the small intestines. there were no detectable pedv in any of wild type mice samples. in porcine apn transgenic mice, the mean value of pedv was tcid 50-10 3.1 on day 1. it increased to tcid 50-10 3.8 , tcid 50-10 4.8 , tcid 50-10 5.0 , tcid 50-10 5.1 , on day 2, 3, 4, and 5, respectively. there was no big change in titer between 3 and 5 days pi. we could confirm clear pedv replication in the small intestines of porcine apn transgenic mice. laboratory animal models are crucial tools for the study of viral pathogenesis in vivo, especially for highly pathogenic human viruses or viruses with restricted host ranges (calvert et al., 2014; chiu et al., 2014; deruaz and luster, 2013) . unlike in vitro cell culture systems, laboratory animal models offer researchers invaluable opportunities to study the biological, pathological, and histological characteristics of human and animal diseases. for these purposes, many transgenic mouse models have been developed to study viral pathogenesis, immune responses, and vaccines (darling et both wild type and porcine apn transgenic mice were infected with pedv (5x tcid5010 6 ) orally on day 0. two mice from each group were killed daily up to 5 days, and intestine samples were collected and prepared for titration. titration was done using vero cells. y value is pedv titer in tcid50 in log value. reynaud et al., 2014) . in the field of coronavirus research, studies of severe acute respiratory syndrome coronavirus (sars-cov) and human coronavirus 229e (hcov-229e) have been conducted with transgenic mouse models (lassnig et al., 2005; tseng et al., 2007) . the host range of the porcine coronaviruses is strongly limited to pigs, so pigs are the only animals available in which to study viral pathogenesis. however, in vivo experiments using pigs are relatively challenging because they require special treatments, reagents, and facilities. because most porcine coronaviruses use porcine apn as their receptor, we generated porcine apn transgenic mice, which are susceptible to porcine coronaviruses. we constructed the porcine apn transgene by attaching the mouse proximal apn promoter to the 5 terminus of the porcine apn gene and the bgh-polya signal at its 3 terminus. we thus generated porcine apn transgenic mice expressing porcine apn in the brush borders of their small intestines and various tissues (lungs and kidneys). previous attempts to generate apn transgenic mice susceptible to tgev or hcov-229e have been documented (benbacer et al., 1998; lassnig et al., 2005) . benbacer et al. generated an rt-pcrpositive lineage, but failed to confirm either the expression of the porcine apn protein in the intestine or tgev replication in their porcine apn transgenic mouse model (benbacer et al., 1998) . a susceptible mouse model for hcov-229e infection was successfully developed using comprehensive apn regulatory elements to generate human apn +/+ stat1 −/− double transgenic mice, and a virus was adapted to grow in primary embryonic fibroblasts from these mice (lassnig et al., 2005) . a transgenic mouse model of sars was generated by expressing human angiotensin-converting enzyme 2, a functional receptor for the virus, under the regulation of a global promoter (tseng et al., 2007) . the significance of our study is that we successfully generated transgenic mice expressing porcine apn with minimal modifications, by using the mouse apn proximal promoter and the bgh-polya signal to ensure the strong expression and correct splicing of the gene (pfarr et al., 1986) . therefore, we could express porcine apn in the brush borders of the mouse small intestines and on the surfaces of their renal cells (figs. 4 and 5) . among the porcine coronaviruses, pedv is relatively poorly studied, and many questions and controversies remain regarding its outbreaks and pathogenesis. outbreaks of porcine epidemic diarrhea have been limited to few countries in east asia in the cold season, whereas it has spread to most swine farms in the asian region (mole, 2013; pan et al., 2012; park et al., 2013; song and park, 2012; stevenson et al., 2013) . recently, pedv has also spread rapidly among swine farms in the united states, causing high piglet mortality in more than 17 states (huang et al., 2013; mole, 2013; stevenson et al., 2013; wang et al., 2014) . more importantly, although vaccination is practiced on most swine farms in korea, reported outbreaks are still increasing. moreover, a method for isolating wild-type pedv is not well established. for these reasons, we tested the susceptibility of porcine apn transgenic mice to pedv. although significant clinical illness was not observed when the transgenic mice were infected with pedv, their susceptibility to the virus was confirmed by the detection of viral rna in various organs with rt-pcr and viral proteins in the small intestines with ihc. more clearly, pedv replication was confirmed by increase in viral titer up to 5 days pi compared to those in wild type mice. we could found clear increase in viral titer in the small intestines of papn transgenic mice but not from wild type mice. these results also confirmed that porcine apn plays a role as the cellular receptor for pedv. however, similar experiments using other porcine coronaviruses are required. although we have presented evidence of pedv infection in the small intestines of porcine apn transgenic mice, no clinical signs were observed, apart from moderately soft feces. it has previously been shown that pedv can infect cells lacking extracellular trypsin, although trypsin is essential for the generation of a cytopathic effect (cpe) in infected cells (park et al., 2011) . therefore, we assume that cellular cofactors (e.g., trypsin) other than the primary receptor might be involved in and required for pedv pathogenesis in vivo. however, these factors are probably present at insufficient levels in mice. proteases are generally required for efficient viral entry and the cpes of coronaviruses (matsuyama et al., 2005; simmons et al., 2004 simmons et al., , 2013 . although the viral receptors are usually expressed in various organs, the diseases caused by coronavirus infections are limited to the small intestines and/or lungs, which are enriched cellular proteases. this suggests that proteases play a role in the pathogenesis of the coronaviruses in vivo, as is already well established for influenza virus infections (hatesuer et al., 2013; sakai et al., 2014; tarnow et al., 2014) . the porcine apn transgenic mouse model should extend our understanding of the pathogenesis of and other studies to porcine coronavirus infections. this laboratory animal model should also circumvent all the disadvantages and difficulties related to the study of porcine 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work was supported by a grant from animal and plant quarantine agency of korea (grant no. z-ad-14-2009-10-02), and a grant from the technology development program for agriculture and forestry, ministry for food, agriculture, forestry and fisheries, the korean research foundation grants (grants no. 20120008358, 2011grants (grants no. 20120008358, -0023942, 211-2006. key: cord-326320-flfrdrbi authors: choudhary, shalki; silakari, om title: scaffold morphing of arbidol (umifenovir) in search of multi-targeting therapy halting the interaction of sars-cov-2 with ace2 and other proteases involved in covid-19 date: 2020-08-29 journal: virus res doi: 10.1016/j.virusres.2020.198146 sha: doc_id: 326320 cord_uid: flfrdrbi the rapid emergence of a novel coronavirus, sars-coronavirus 2 (sars-cov-2), originated from wuhan, china, imposed a global health emergency. angiotensin-converting enzyme 2 (ace2) receptor serves as an entry point for this deadly virus while the proteases like furin, transmembrane protease serine 2 (tmprss2) and 3 chymotrypsin-like protease (3clpro) are involved in the further processing and replication of sars-cov-2. the interaction of sp with ace2 and these proteases results in the sars-cov-2 invasion and fast epidemic spread. the small molecular inhibitors are reported to limit the interaction of sp with ace2 and other proteases. arbidol, a membrane fusion inhibitor approved for influenza virus is currently undergoing clinical trials against covid-19. in this context, we report some analogues of arbidol designed by scaffold morphing and structure-based designing approaches with a superior therapeutic profile. the representative compounds a_br4, a_br9, a_br18, a_br22 and a_br28 restricted the interaction of sars-cov-2 sp with ace2 and host proteases furin and tmprss2. for 3clpro, compounds a_br5, a_br6, a_br9 and a_br18 exhibited high binding affinity, docking score and key residue interactions. overall, a_br18 and a_br28 demonstrated multi-targeting potential against all the targets. among these top-scoring molecules a_br9, a_br18, a_br22 and a_br28 were predicted to confer favorable adme properties. coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is an ongoing medical health emergency worldwide (sohrabi et al., 2020) . this outbreak was initially originated from wuhan, hubei, china in december 2019 and rapidly expanded to almost 187 countries throughout the globe (wang et al., 2020a) . on march 11, 2020, world health organization (who) declared the covid-19 situation as pandemic as the confirmed positive cases approached 2 million people with an estimated 8000 deaths (bedford et al., 2020) . as of june 24, 2020, a total of 92,78,515 cases with 4,76840 deaths have been recorded globally due to . amid this pandemic, researchers and scientists around the globe are engaged in finding an effective treatment for this deadly virus. currently, there is no effective drug targeting sars-cov-2, the causative agent of covid-19, however, various drugs from different categories are undergoing clinical trials for drug repurposing (ciliberto et al., 2020; lythgoe and middleton, 2020; rosa and santos, 2020) . most of these drugs belong to antiviral (mevada et al., 2020) , antimalarial , and immunomodulatory (zhao, 2020) categories. for searching an effective therapy, one should understand the pathophysiology of sars-cov-2 infection and its transmission from one person to another at the molecular level. here, instead of describing the detailed molecular biology of the virus, we are briefly discussing the key molecular events which we explored in this designing strategy. the entry of coronavirus in the host cell depends on the binding of the viral spike proteins (sp) to cellular receptors and its priming by host cell proteases (hoffmann et al., 2020) . sars-cov-2 uses the ace2 receptor to enter into the host cell by complexing with sp and further, transmembrane proteases furin and tmprss2 to cause the proteolytic activation of sp (zhou et al., 2020) . thus, the invasion of the virus into the host cell mainly explores the ace2 receptor and two more proteolytic enzymes furin and tmprss2 (bestle et al., 2020) . briefly, the binding of the s1 domain of sp to the enzymatic domain of ace2 present on the cell surface results in endocytosis and translocation of both the virus as well as enzyme into endosomes located within the cells (lan et al., 2020) . this entry process also requires priming of sp which is mediated by the host proteases furin and tmprss2. the s1/s2 domain of sp in newly emerged coronavirus (sars-cov-2) harbor potential protease cleavage site (pcs), nsprrar ^ sva (^ is cleavage site), having four distinct amino acids (in bold), which is absent in sars-cov of the same clade and thereby became a keyhole for viral invasion j o u r n a l p r e -p r o o f (wang et al., 2020b) . these mutations have given the ability to a virus to infect a wider variety of tissues in the body. followed by ace2 mediated viral attachment and transmembrane proteases (furin, tmprss2) mediated membrane fusion and endocytosis, this deadly virus is further processed for replication by viral proteases. viral protease 3clpro (m pro ) with the help of papain-like protease (plpro) is mainly involved in proteolysis and plays an important role in processing the polyproteins that are translated from the viral rna . essentially, the interaction of sp with ace2, its priming by host proteases (furin, tmprss2) at pcs and replication by viral protease 3clpro is the primary reason for sars-cov-2 invasion and fast epidemic spread (mckee et al., 2020) . all the molecular processes are displayed in figure 1 . various studies suggested that the compounds restricting the interaction of sp with ace2 and inhibit the key protease enzymes could make a highly effective treatment to prevent covid-19. arbidol (also known as umifenovir), an effective antiviral drug approved for influenza virus is currently ongoing clinical trials against covid-19 (chen et al., 2020; lythgoe and middleton, 2020; wilkinson and dahly, 2020; zhu et al., 2020) . this broad-spectrum antiviral drug has shown promising results in different pre-clinical and clinical trials (blaising et al., 2014; huang et al., 2020; lythgoe and middleton, 2020; pecheur et al., 2007) . arbidol is a non-nucleoside membrane fusion inhibitor that prevents the interaction of the influenza virus to the host cell. as per the recent report, the binding mode of arbidol to sars-cov-2 sp is similar to that of influenza virus haemagglutinin (ha) (vankadari, 2020) . it is evident from the literature reports that various substituents of arbidol play a different role in its antiviral activity. previous studies discussed the structure-activity relationship (sar) of arbidol in broad-spectrum antiviral activity (di mola et al., 2014; wright et al., 2017) . sar profile of arbidol suggests that the indole core and thiophenyl group present on it are crucial for the activity while the presence of bromine on the indole backbone does not have any significant effect on antiviral activity. besides, the replacement of the remaining functionalities may increase or decrease the activity depending upon the type of virus considered. briefly, the indole ring and thiophenyl group of arbidol are buried inside the hydrophobic cavity of influenza virus ha whereas, polar groups such as hydroxyl and bromine are exposed to solvent. this way, through an induced fit mechanism, arbidol causes the conformational changes in the cavity that in turn break the existing salt-bridge between the virus and host membrane and form a new one (kadam and wilson, 2017; pecheur et al., 2007) . within this frame of reference, we report some analogues of arbidol against sars-cov-2, designed by scaffold morphing and structure-based drug designing approaches. scaffold j o u r n a l p r e -p r o o f 6 morphing is a unique medicinal chemistry tool utilized for rational drug designing by a gradual transformation in the parent molecule to develop novel molecules with a potentially improved therapeutic profile. this drug designing strategy takes into consideration the synthetic feasibility of new scaffolds and is essentially a chemistry-driven approach (shandil et al., 2019) . we utilized the scaffold morphing approach in combination with molecular docking and mm/gbsa (molecular mechanics generalized born and surface area) calculation to identify better therapy than arbidol. the multi-targeting potential of generated analogues was explored against various targets involved in the pathogenesis of covid-19 including sars-cov-2 sp, ace2, furin, tmprss2 (in viral attachment) and 3clpro (in viral replication). considering the current public health emergency, this study is aimed to identify the potential analogues of arbidol which can possibly manage the epidemic spread of sars-cov-2. scaffold morphing is a drug designing approach to improve the synthetic feasibility, potency, and drug-likeness of molecules by gradually modifying its structural features. this method provides a new chemical space the lead molecule that may in turn contribute to improving the overall therapeutic profile of that molecule (langdon et al., 2010) . for scaffold morphing, the bio-isosteric replacement method was adopted that involves swapping the functional groups of a molecule with their bio-isosteres and improve the potency as well as the pharmacokinetic profile of that particular molecule (dick and cocklin, 2020) . in this study, the bio-isosteric transformation in arbidol was done using a freely available web server molopt (shan and ji, 2019) . molopt is a recently developed web tool for in-silico drug designing. this web server automatically generates several analogues based on bio-isosteric transformation rules derived from data mining, deep generative models and similarity comparison. in current study, the rule of data mining was utilized for bio-isosteric replacement. the generated set of molecules is then ranked based on their synthetic possibility. adopting this inbuilt protocol of molopt, the six replacement sites of arbidol, as suggested by this web server, were explored. the generated analogues of arbidol corresponding to each replacement site were sorted based on their synthetic possibility. synthetic accessibility score ranges from 1 (very easy) to 10 (very difficult). a cutoff value of 3 was used for the screening of compounds based on synthetic possibility and topranked molecules were submitted to structure-guided drug binding analysis such as molecular docking studies. molecular docking is a structure-based drug designing approach used to find out the best orientation and key interactions between ligand and receptor. molecular docking experiments were performed on maestro molecular modeling interface (schrödinger suite, llc, ny) (release, 2019) . the 3d x-ray crystal structures of sars-cov-2 sp receptor-binding domain in complex with its receptor ace2 (pdb id: 6lzg, resolution 2.5 å), human furin (pdb id: 5mim, resolution 1.9 å) and main protease 3clpro (pdb id: 6lu7, resolution 2.16 å) were retrieved from the protein data bank accessed at the url (http://www.rscb.org/pdb). since the x-ray crystal structure of tmprss2 was not available, a homology model ( the 3d chemical structure of arbidol was extracted from the pubchem database while the smiles notations of arbidol analogues (generated by molopt webserver) were used to build their 3d structures. ligprep module of schrodinger was used to prepare ligands by adding hydrogen, removing salt and ionizing at ph 7 ± 0.5 (choudhary and silakari, 2019) . since arbidol is reported to impair the membrane fusion of viruses under low ph in the endosome, the docking analysis with proteins involved in viral entry (ace2 and spike protein) was performed at ph 5 ± 0.5 (at which arbidol is reported to show better fusion inhibition) (kadam and wilson, 2017; leneva et al., 2009) . geometry optimization and energy minimization were performed under the opls_3 force field to generate low energy conformers using standard energy function of molecular mechanics with rmsd cut off 0.01 ǻ (shah et al., 2020) . the prepared and minimized molecules were then docked into the grid generated from the accurately prepared protein structures. proteins were prepared using the 'protein preparation wizard' tool of maestro interface by following preprocess, review and modify, optimization and finally minimization under opls_3 force field. during protein preparation, hydrogens were added, bond order was assigned and missing loops and side chains were updated using prime. waters molecules were removed within 5å of het groups to avoid unnecessary hindrance during docking. the receptor grids were generated within the 20 å radiuses around the co-crystallized ligands using 'receptor grid generation' option available with glide. the proteins in which the co-crystallized ligand was not available, sitemap module of maestro was used to predict the putative binding sites and grids with a cubic box of 10 ǻ × 10 ǻ × 10 ǻ were generated around the top-ranked sites (schrödinger, 2013) . the van der waals scaling factor of 1.00 and partial charge cutoff value of 0.25 were selected. for ligand atoms, these cutoff values were kept as 0.80 and 0.15 respectively. the docking analysis was performed using extra precision (xp) docking option which predicts the binding modes and their glide xp g-score (pathak et al., 2020) . a total of ten docking poses were generated corresponding to each ligand and the best poses were selected based on good gscores and appropriate binding orientations. the docked poses were analyzed for the molecular interactions and the formation of hydrogen bonds between the ligand and the active site residues present in the hinge region. to better understand the biological process, the ligand should bind to the protein in a specific manner. therefore, the ligand-binding energies were calculated using the prime mm-gbsa j o u r n a l p r e -p r o o f option of the schrodinger software. mm-gbsa is a method to calculate the free binding energy of a ligand to its protein and is calculated in terms of the mm-gbsa score (haider et al., 2011) . the main contributory factors in mm-gbsa calculations are opls molecular mechanics energies (emm), polar solvation through surface generalized born solvation model (gsgb), and a non-polar solvation term composed of the non-polar solvent accessible surface area and van der waals interactions (sun et al., 2014) . for a better representation of the solvent-accessible surface area, this method uses the gaussian surface instead of a van der waals surface and adopts the surface-generalized born model (du et al., 2011) . the mm-gbsa binding energy is calculated in terms of kcal/mol by using the equation: mm-gbsa δgbind = er: el -el -er where er: el, el, and er are the prime energies of the optimized complex, free ligand and free receptor, respectively (singh and silakari, 2018) . to investigate the pharmacokinetic profile of newly generated analogues, their adme (absorption, distribution, metabolism, and excretion) properties were predicted using the qikprop program of the schrödinger software (qikprop, 2015) . this provided an estimate of the physicochemical properties and the bioavailability of the compounds. various parameters such as polar surface area (psa), solvent accessible surface area (sasa), qppcaco (predicted apparent caco-2 cell permeability in nm/s, cns activity (predicted central nervous system activity on a -2 (inactive) to +2 (active) scales). qplogbb (predicted brain/blood partition coefficient), qppmdck (predicted apparent mdck cell permeability in nm/s), qplogs (predicted aqueous solubility), qplogkhsa (prediction of binding to human serum albumin), and percent human oral absorption (predicted human oral absorption on 0-100% scale) were calculated. among these parameters, caco-2 cells are a model for the gut blood barrier and mdck cells are considered to be a good mimic for the blood-brain barrier. the acceptability of the compounds to be orally bioavailable was estimated on the basis of lipinski's rule of five (lipinski, 2004) . j o u r n a l p r e -p r o o f the chemical structure of arbidol was submitted to the molopt webserver to develop different analogues with improved pharmacokinetic and pharmacodynamic profiles. this server suggested a total of six potential bio-isosteric replacement sites. after bio-isosteric replacement at these six sites, a total of 569 molecules were generated corresponding to these sites. these molecules were then ranked on the basis of synthetic feasibility, which led to 36 top-ranked molecules (table 1) . synthetic accessibility score ranges from 1 (very easy) to 10 (very difficult) (nath et al., 2020) . normally, the molecules with synthetic accessibility score of <5 are considered. to be more precise, in the current study, molecules having this score <3 were considered for further investigation. in these 36 molecules, only the core indole moiety of arbidol was kept intact while the remaining structural features were modified gradually. these 36 molecules were then undergone docking based virtual screening. a list of information on the analogues generated complementary to the six sites of arbidol is provided as supplementary data (excel file). to identify the multi-targeting potential of arbidol analogues against various targets of sars-cov-2, an exhaustive docking analysis was performed on 36 top-ranked analogues of arbidol. all these molecules were docked against sars-cov-2 sp-ace2 complex, furin, tmprss2 and main protease (3clpro) and the binding affinity of their docked complexes was also calculated in terms of mm-gbsa score. the results were compared with arbidol and the molecules were ranked on the basis of their docking score, key residue interactions as well as mm-gbsa scores. the top-ranked molecules demonstrated good docking score (g-score), displayed crucial interactions with binding site amino acid residues and shown better binding affinity (mm-gbsa) than arbidol (table 1 and 2). the best analogue concerning each replacement site of arbidol was also identified against all the targets ( figure 2 ). consequently, six analogues including a_br4, a_br9, a_br18, a_br21, a_br28 and a_br32, identified corresponding to each site, may limit the sars-cov-2 sp and ace2 interaction. these molecules manifested hydrogen bonding and hydrophobic interactions with the interface amino acid residues of sp receptor-binding domain (rbd) and ace2, which are involved in their interaction and complex formation. the docking scores and mm-gbsa scores for these molecules lied in range of -6.13 to -4.19 kcal/mol and -50.11 to -40.12 kcal/mol respectively, which were better than the docking scores (-3.63 to -2.42 kcal/mol) and mmgbsa scores (-39.99 to -30.36 kcal/mol) shown by arbidol against ace2 and sp. on the other hand, a_br5, a_br12, a_br18, a_br22, a_br28 and a_br34 were found to be effective against priming protease furin with docking score range of -5.49 to -3.78 kcal/mol and mm-gbsa ranges from -34.86 to -22.92 kcal/mol. for another priming protease tmprss2, analogues a_br2, a_br11, a_br17, a_br22, a_br28 and a_br32 were found to show crucial interactions with docking score range -2.73 to -2.03 kcal/mol and mm-gbsa ranging from -38.36 to -27.02 kcal/mol (table 1 ). parent drug bio-isosteric replacement 1 bio-isosteric replacement 2 bio-isosteric replacement 3 bio-isosteric replacement 4 bio-isosteric replacement 5 bio-isosteric replacement 6 j o u r n a l p r e -p r o o f further, the analogues which were superior to arbidol, with respect to each replacement site, were also identified for main protease 3clpro (figure 3) . although arbidol did not show any significant interaction, docking score (-4.89 kcal/mol) and mm-gbsa score (-12.23 kcal/mol) with 3clpro, surprisingly, its analogues a_br5, a_br9, a_br18, a_br20, a_br27 and a_br32 exhibited way better results than arbidol with docking score range -7.05 to -4.91 kcal/mol and mmgbsa score ranging from -46.94 to -33.30 kcal/mol (table 2 ). it is reported that the molecules that can block the activity of ace2 as a receptor for sars-cov-2 may serve as a potential therapeutic option for covid-19 (abdelli et al., 2020) . the after binding of sp to ace2, two transmembrane proteases including furin and tmprss2 leads to proteolytic cleavage of sp, which facilitates the entry of the virus into the host cell, viral replication and cell-to-cell transmission (hasan et al., 2020; south et al., 2020) . thus, the designed arbidol analogues were docked into the binding sites of these two proteases and their among all the arbidol analogues, submitted to docking based virtual screening, a_br18 and a_br28 displayed good results against sars-cov-2-ace2 and furin, whereas, a_br22 was found effective against furin and tmprss2. therefore, these three molecules can be considered as dual inhibitors. moreover, a_br28 was found effective against all the three targets involved in viral attachment and membrane fusion step. at the same time, analogues a_br5, a_br6, a_br9 and a_br18 demonstrated promising results against main protease (3clpro) that is involved in viral replication. overall, based on in-silico results, a_br18 and a_br28 implied multi-targeting potential against covid-19. the 3d view of the docked complex of these most active molecules with their respective proteins is shown in figure 8 (a_br18), figure 9 (a_br22), figure 10 (a_br34) and figure 11 (a_br28). the main protease (3clpro) of sars-cov-2 is essential for the processing of polyproteins which are translated from viral rna. 3clpro acts on the leu-gln^ser-ala-gly cleavage site of polyproteins . since there is no reported protease in humans with the same cleavage site specificity, inhibiting this enzyme would not show any toxic effect on humans. therefore, the top-ranked arbidol analogues were docked within the catalytic site of 3clpro. it is reported that the catalytic triad (his41, cys145 and ala285) of 3clpro is essential for the enzymatic activity and n-terminal residue glu166 keeps the s1 domain of this enzyme in an active conformation. those inhibitors which show interaction with the catalytic triad and glu166 are considered to be very good inhibitors of 3clpro. from docking results, it was observed that j o u r n a l p r e -p r o o f arbidol and its analogues a_br8, a_br15 and a_br36 did not show any interaction with the key amino acids. however, remaining analogues were found to form h-bond interactions with glu166. the top-ranked analogues interacted with his41, cys145 and glu166 while no interaction was observed with ala285. in most of the molecules, the phenyl ring was found to interact through π-π stacking with his41. the tertiary amine group of a_br5 in this virtual screening process, the adme parameters of 36 novel arbidol analogues were investigated to assess their drug-like properties. the drug-likeness was recommended for the molecules obeyed lipinski's rule of five (mol_mw <500, qplogpo/w <5, donorhb≤5, sasa: total solvent accessible surface area (300-1000), qplogpo/w: predicted octanol/water partition coefficient (-2 to 6.5), qplogs: predicted aqueous solubility (-6.5 to 0.5), cns: predicted central nervous system activity on a -2 (inactive) to +2 (active) scale, qppcaco: caco-2 cell permeability in nm/sec (<25 poor, >500 great), qppmdck: predicted apparent mdck cell permeability in nm/sec (<25poor, >500great), qplogbb: brain/blood partition coefficient (-3 to 1.2), qplogkhsa: binding to human serum albumin (-1.5 to 1.5), percent human-oral absorption: human oral absorption on 0 to 100% scale (>80% high,<25% poor). j o u r n a l p r e -p r o o f 44 a combination of scaffold morphing and a structure-based drug designing approach was successfully utilized to identify putative multi-targeting analogues of arbidol against covid-19. initially, the bio-isosteric replacement was done on six suggested sites of arbidol to generate a library of its analogues. from a library of 569 analogues, 36 were selected based on synthetic possibility and submitted for docking analysis against different targets of sars-cov-2. the binding affinity and adme properties of these molecules were also determined. the in-silico adme prediction conferred the drug-like properties of these analogues. the most active molecules a_br4, a_br9, a_br18, a_br22 and a_br28 suggest plausible binding mode with the interface amino acid residues which are responsible for the interaction of spike protein with ace2 as well as with priming proteases furin and tmprss2. on the other hand, a_br5, a_br6, a_br9 and a_br18 were found effective against the main protease (3clpro). overall, a_br18 and a_br28 displayed multi-targeting potential against maximum targets considered in the study. however, further experimental validation is required to confirm their inhibitory activities against sars-cov-2. on the basis of these results, it can be suggested that a slight structural modification in the arbidol i.e. the replacement of tertiary amine group with primary amine and bromine with methoxy group may improve its therapeutic profile. the protocol adopted in this study may be used as a framework in the future for the development of novel multi-targeting small molecules against the covid-19. huang, j., song, w., huang, h., sun, q., 2020 j o u r n a l p r e -p r o o f predicted octanol/water partition coefficient (-2 to 6.5), qplogs: predicted aqueous solubility (-6.5 to 0.5), cns: predicted central nervous system activity on a -2 (inactive) to +2 (active) scale, qppcaco: caco-2 cell permeability in nm/sec (<25 poor, >500 great), qppmdck: predicted apparent mdck cell permeability in nm/sec (<25poor, >500great), qplogbb: brain/blood partition coefficient (-3 to 1.2), qplogkhsa: binding to human serum albumin ( in silico study the inhibition of angiotensin converting enzyme 2 receptor of covid-19 by ammoides verticillata components harvested from western algeria central nervous system manifestations of covid-19: a systematic review covid-19: towards controlling of a pandemic tmprss2 and furin are both essential for proteolytic activation and spread of sars-cov-2 in human airway epithelial cells and provide promising drug targets arbidol as a broad-spectrum antiviral: an update favipiravir versus arbidol for covid-19: a randomized clinical trial 2019. hces1 and hces2 mediated activation of epalrestat-antioxidant mutual prodrugs: unwinding the hydrolytic mechanism using in silico approaches drug repurposing against covid-19: focus on anticancer agents structureactivity relationship study of arbidol derivatives as inhibitors of chikungunya virus replication bioisosteric replacement as 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virus inhibition by the broad-spectrum antiviral arbidol llc clinical trials on drug repositioning for covid-19 treatment neurological manifestations in covid-19 caused by sars-cov-2 schrödinger release 2013-3: sitemap, version 2.9 in silico studies on therapeutic agents for covid-19: drug repurposing approach molopt: a web server for drug design using bioisosteric transformation scaffold morphing to identify novel dpre1 inhibitors with antimycobacterial activity molecular dynamics guided development of indole based dual inhibitors of egfr (t790m) and c-met world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) controversies of renin-angiotensin system inhibition during the covid-19 pandemic assessing the performance of mm/pbsa and mm/gbsa methods. 5. improved docking performance using high solute dielectric constant mm/gbsa and mm/pbsa rescoring arbidol: a potential antiviral drug for the treatment of sars-cov-2 by blocking the trimerization of viral spike glycoprotein? a review of the 2019 novel coronavirus (covid-19) based on current evidence a unique protease cleavage site predicted in the spike protein of the novel pneumonia coronavirus (2019-ncov) potentially related to viral transmissibility statistical review of favipiravir versus arbidol for covid-19: a randomized clinical trial structure-based optimization and synthesis of antiviral drug arbidol analogues with significantly improved affinity to influenza hemagglutinin analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors cytokine storm and immunomodulatory therapy in covid-19: role of chloroquine and anti-il-6 monoclonal antibodies systemic analysis of tissue cells potentially vulnerable to sars-cov-2 infection by the protein-proofed single-cell rna profiling of ace2, tmprss2 and furin proteases arbidol monotherapy is superior to lopinavir/ritonavir in treating covid-19 the authors thank dr. anshuman dixit, institute of life sciences (ils), bhubaneswar, for help and support in molecular docking analysis. s choudhary would like to acknowledge the indian council of medical research (icmr), new delhi for providing srf under sanction no: isrm/11(61)/2017. the authors have no competing conflict of interest to declare.j o u r n a l p r e -p r o o f 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-345999-iiw4cs8p authors: khare, prashant; sahu, utkarsha; pandey, satish chandra; samant, mukesh title: current approaches for target-specific drug discovery using natural compounds against sars-cov-2 infection date: 2020-09-24 journal: virus res doi: 10.1016/j.virusres.2020.198169 sha: doc_id: 345999 cord_uid: iiw4cs8p the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) recently caused a pandemic outbreak called coronavirus disease 2019 (covid-19). this disease has initially been reported in china and also now it is expeditiously spreading around the globe directly among individuals through coughing and sneezing. since it is a newly emerging viral disease and obviously there is a lack of anti-sars-cov-2 therapeutic agents, it is urgently required to develop an effective anti-sars-cov-2-agent.through recent advancements in computational biology and biological assays, several natural compounds and their derivatives have been reported to confirm their target specific antiviral potential against middle east respiratory syndrome coronavirus (mers-cov) or severe acute respiratory syndrome(sars-cov).these targets including an important host cell receptor, i.e., angiotensin-converting enzyme ace2 and several viral proteins e.g. spike glycoprotein (s) containing s1 and s2 domains, sars cov chymotrypsin-like cysteine protease (3cl(pro)), papain-like cysteine protease (pl(pro)), helicases and rna-dependent rna polymerase (rdrp). due to physical, chemical, and some genetic similarities of sars cov-2 with sars−cov and mers−cov, repurposing various anti-sars−cov or anti-mers−cov natural therapeutic agents could be helpful for the development of anti−covid-19 herbal medicine. here we have summarized various drug targets in sars−cov and mers−cov using several natural products and their derivatives, which could guide researchers to design and develop a safe and cost-effective anti-sars−cov-2 drugs. the outbreaks of coronavirus (cov) infection that have already threatened the world by sars and mers in the first decade of 21 st century have recently come up with a novel strain of lethal coronavirus named as 2019 novel coronavirus (sars-cov-2). in december 2019, the disease was originally started in the local seafood market of wuhan of china (hui et al., 2020; perlman et al., 2020; zhu et al., 2020) . since then this new coronavirus strain has spread across the globe very rapidly with the catastrophic effects. coronaviruses are the non-segmented, enveloped viruses with positive-sense rna as their genetic material belonging to the family coronaviridae. they are pleomorphic and club-shaped spikes are present on their cell surface. the disease is characterized as respiratory disorders with flu-like symptoms such as a sore throat, fever, cold, cough and severe pneumonia is also reported in more critical cases. sars-cov-2 can be transmitted through coughing and sneezing droplets of infected individuals; these virions containing droplets retained on the hard surfaces for a longer time and can spread to a fresh individual by direct inhalation or by touching the infected surfaces. as of 31 st august 2020, the complete number of affirmed covid-19 cases reported globally is more than 25 million and the mortality has crossed more than 850,600. recently many efforts have been made to develop the therapeutic agents to control covid-19, but so far no medicine is significantly effective against sars-cov-2 (tu et al., 2020) , and further supportive care is also needed to the individual for proper breathing. while the development of a vaccine may also take 12-18 months (pandey et al., 2020) , repurposing of the drugs (from ebola to malaria to arthritis) is the only feasible option for treating the patients in this current situation (simsek yavuz and unal, 2020) . progress in drug discovery and development largely depends on the identification of potential drug targets. for the management j o u r n a l p r e -p r o o f of covid-19 infection, various molecular targets playing important role in the sars-cov-2 life cycle including host cell receptor-angiotensin-converting enzyme ace2 (pdb id 3d0g) and viral proteins such as s protein (containing s1 and s2 domains) (pdb id 6xm0); various cysteine proteases such as papain-like cysteine protease (pl pro ) (pdb id 6wx4) or chymotrypsin like nprotease (3cl pro ) (pdb id 1p9u), helicases and rna-dependent rna polymerase (rdrp) (pdb id 6m71) could be evaluated . nature has provided us with an immense supply of natural products. interestingly, the nutraceuticals market hugely depends on the success of natural drugs for the treatment of infectious diseases (williamson et al., 2020) . so these natural products and their derivatives could offer new scope for the control and prevention of various ailments including viral infections (figure-1a-d and table1 a-c) (chen and du, 2020; ganjhu et al., 2015; islam et al., 2020; jo et al., 2020; lin et al., 2014; wang et al., 2014) . this article gathers information on the use of herbal-based drugs and/or their derivatives for target-specific drug discovery against sars cov2 infection (figure-2). initially, cov was known to cause mild disease, but the recent outbreaks (sars-cov outbreak of china and mers-cov outbreak of saudi arabia and now covid-19 originated from wuhan, hubei, china) signifies the importance of understanding the structure, metabolism, and pathophysiology of cov-associated diseases to identify major drug targets (j alsaadi et al., the viral rna codes for some conserved genes: orf1a, orf1b, oef3s, e, m, and n gene. the orf1a/b genes code for viral replicase polyproteins (pps) pp1a and pp1ab. these j o u r n a l p r e -p r o o f pps are further processed to form sixteen mature non-structural proteins (nsps), which play a crucial role in the formation of the replicase transcriptase complex. other structural proteins viz. membrane (m), envelope (e), spike (s), nucleocapsid as well as other accessory proteins are encoded by rest of the genome (mcbride et al., 2014) and the beta-covs also have hemagglutinin esterase (he) glycoprotein (hilgenfeld, 2014) . all these proteins play a significant role in virulence and for viral multiplication. hence these viral proteins could be the potential targets for the treatment of sars cov2 infection. spike proteins are glycoprotein which facilitates the attachment of coronavirus to the target cells via a specific receptor present on the cell surface of host i.e. angiotensin-converting enzyme ace2 receptor in sars-cov(li et al., 2003; zhou et al., 2020) and dipeptidyl peptidase-4 in mers-cov(mubarak et al., 2019) . the coronavirus relies on the association of viral envelope protein with host cell membrane for delivering their nucleocapsid. the spike proteins (s) are responsible for viral entry inside the host cell and are accountable for disease progression in a specific types of host cells. during the fusion of s protein with a specific receptor on the host cell membrane, a crucial conformational change occurs in s glycoprotein (belouzard et al., 2012) . so the s-glycoprotein could be evaluated as a potential drug target. so far various natural compounds and their derivatives have been tested for anti-sars-cov activity against this protein (ho et al., 2007) . several extracts/derivatives from the herbs belonging to family polygonaceae have been reported to inhibit the sars-cov s protein interaction with angiotensin-converting enzyme ace2 receptor. anthraquinone compound namely emodin (1), a plant extract isolated from genus polygonum, and rheum has efficiently impeded the interaction of s protein and angiotensin-converting enzyme ace2 receptor. moreover, it also j o u r n a l p r e -p r o o f hampered s protein-pseudo typed retrovirus infectivity to vero e6 cells. these observations indicated the potential role of emodin as a drug candidate against s protein (ho et al., 2007; yi et al., 2004) . two naturally occurring compounds tetra-o-galloyl-β-d-glucose (tgg) (2) and luteolin (3) derived from galla chinensis were reported to possess anti-sars-cov activities. tgg and luteolin have a high affinity for s2 domain of spike protein. this indicates the anti-sars activity of tgg and luteolin is due to inhibition of virus and host cell fusion however the exact mechanism remains unknown (yi et al., 2004) . these observations indicate that tgg and luteolin could be used for drug development against covid-19 targeting s2 domain helicase also known as ntpase is involved in the replication of viral genomic rna as well as in transcription and translation (frick and lam, 2006) . sars-cov helicase is an enzyme of the sf1 family, which hydrolyzes all ntps and utilizes atp, datp, and dctp as substrates (karpe and lole, 2010). cov helicase nsp13 has been reported to retain dsrna unwinding activity with translocation along the nucleic acid by atp hydrolysis (adedeji et al., 2012) . various natural compounds have also been reported to inhibit helicases of sars-cov-2. the activity of two naturally occurring flavonoids namely myricetin (4) and scutellarein (5) have been shown to inhibit potential against sars cov helicase nsp13. these compounds have been reported to inhibit helicase protein by affecting the atpase activity (yu et al., 2012) .therefore, helicases could be a potential drug target for anti-covid-19 therapy. j o u r n a l p r e -p r o o f angiotensin-converting enzyme ace2 receptor is a human receptor to the sars and sars-cov-2 . angiotensin-converting enzyme ace2 receptor is mostly present as cell surface receptors and rarely circulates in soluble form. these receptors facilitate entry of three cov strains (e.g. nl63, sars-cov, and sars-cov-2), which are present most abundantly in the lungs (predominantly in type 2 pneumocytes and macrophages), testis, brain, heart, blood vessels, and kidney (verdecchia et al., 2020) . the overexpression of ace2 receptor from human, pig, civet in hela cells permitted replication of sars-cov-2, thus proving it to be the principal receptor for cov entry (zhou et al., 2020) . drugs targeting the ace2 receptor could be efficient for anti cov drugs. various natural compounds such as baicalin, (6) scutellarin (7), nicotianamine (8) (docking score -5.1) and glycyrrhizin (9) (docking score -9) (supplementary table 1 ) have been reported to have potential anti-2019-cov effects by preventing the attachment and entry of virus (chen and du, 2020) , particularly baicalin, extracted from plant scutellaria baicalensis georgi demonstrated an excellent antiviral and anti-sars activity . another such compound scutellarin, is reported to reduce ace2 activity in brain tissues (wang et al., 2016) and therefore this compound can also be evaluated as an ace2 receptor inhibitor to block the entry of sarscov2. stilbenoids belonging to other phenolic natural compounds were reported to possess inhibitory activity against ace2 receptor (wahedi et al., 2020) . furthermore, natural extracts isolated from garlic were also observed to have inhibitory effects against ace2 receptor (thuy et al., 2020) . (11) (docking score --9.565), and pectolinarin (12) demonstrated anti-sars-cov 3cl pro activity (jo et al., 2020). 3cl pro has 3 domains at substrate binding site -s1, s2, and s3. s1 represents the polar site of 3cl pro , s2 represents the hydrophobic site, while s3 has no strong tendency. molecular docking showed the binding affinity of three flavonoids with 3 domains of 3cl pro (jo et al., 2020). another flavonoid amentoflavone (13) (docking score −11.42) is the most effective flavonoid inhibiting sars-cov 3cl pro (ryu et al., 2010) (supplementary table 1 (19), isotheaflavin-3-gallate [(tf2b) (20)] and theaflavin-3, 3'-digallate [(tf3) (21)] belonging to polyphenols of tea were reported to exhibit antiviral properties by their inhibitory potential against 3cl pro . triterpenes [betulinic acid (22) and savinin (23)] were reported to possess anti 3clpro activity (wen et al., 2007) . recently, a sum of 28 natural compounds was identified from the shuanghuanglian preparations. out of which two major bioactive compounds baicalin (6) and baicalein, (24) were found to possess significant inhibitory activity against sars-cov 3cl pro by inhibiting the proliferation in vero e6 cells (su et al., 2020) the papain-like cysteine protease (pl pro ) plays an important role in sars-cov viral genomic rna replication. it cleaves the n terminal site of polyproteins (pps) to generate three nonstructural proteins (nsps-1, 2, and 3) (hilgenfeld, 2014; lindner et al., 2005) . pl pro also contains a catalytic core domain and a consensus sequence lxgg which is required for cleaving replicase substrate (barretto et al., 2005) . thus pl pro could be used as a crucial drug target for anti-sars drug development (park et al., 2017) . recently 13 chalcones that includes isobavachalcone (25) (dockind score -8.82) , 4-hydroxyderricin (26) it is a big challenge to develop an effective antiviral therapeutic agent. various inverse agonists are currently being explored against covid-19. the nucleoside inhibitor (gilead's nuc inhibitor) which has shown disappointment in the treatment of ebola is effective in the treatment of a 2019-cov patient in the usa, but the higher rate of mutation in this virus have restricted the use of this drug for treating the n-cov patients (nguyen et al., 2020) . moreover, remdesivir another drug recommended for the treatment of ebola and other rna viruses have also been found useful in some of the patients (gordon et al., 2020; hillaker et al., 2020; shannon et al., 2020) . recently anti-influenza drug favipiravir or avigan was considered as an efficient treatment regimen for covid-19 patients as compared to other antiviral agents (chibber et al., 2020; rosa and santos, 2020; zhu et al., 2020) . likewise, chloroquine and hydroxychloroquine which is effective against malaria, lupus, and rheumatoid arthritis (garcia-cremades et al., 2020; rosa and santos, 2020; zhu et al., 2020) have also been found effective in coronavirus infection . due to the involvement of in silico approaches in pharmaceutical research, now it is quite possible to identify the specific drug targets and understanding the mechanism of action of various natural products and their derivatives (supplementary information). in this review, we have summarized various drug targets for natural drugs and their synthetic compounds, which were used to treat sars cov and mers cov. we have discussed the importance of various herbal-based compounds that can inhibit viral infectivity by blocking the ace2 receptor of host or interrupt the activity of various viral proteins/enzymes such as spike glycoproteins (s protein), 3cl protease, pl pro , helicase, and rna dependent rna polymerase. we have documented the mechanism of action of various herbal-based drugs so; these natural compounds could be important substitutes of synthetic drugs for the treatment of viral infections due to their low cost and safety efficacy. in summary, we have identified and discussed the target-specific antiviral potential of several natural compounds against various strains of cov, which might directly impede the covid-19 pandemics. further pharmaceutical companies should also give more emphasis on natural product research for the development of novel therapeutic agents against various viral infections to achieve sustainable development goals on health. j o u r n a l p r e -p r o o f (yi, 2004) 3. luteolin 10.6μm spike protein (s) (yi, 2004) 4. myricetin 2.5-3.0 μm helicase (yu, 2012) 5. scutellarein 0.4-1.24 μm helicase (yu, 2012) 6. baicalin 2.24mm angiotensin-converting enzyme 2 (ace2) receptor (deng et al., 2012) 7. scutellarin 44-52 μm ace2 receptor (wang et al., 2016) 8. nicotianami ne 84nm ace2 receptor 9. glycyrrhizin na ace2 receptor j o u r n a l p r e -p r o o f chymotrypsin like protease (3cl pro ) (wen, 2007) 23. savinin 25 μm chymotrypsin like protease (3cl pro ) (wen, 2007) 6. baicalin 6.41 ± 0.95 μm chymotrypsin like protease (3cl pro ) (su et al., 2020) 24. baicalein 0.94 ± 0.20 μm chymotrypsin like protease (3cl pro ) (su et al., 2020) j o u r n a l p r e -p r o o f 54. saikosaponin b2 1.7 ± 0.1 μmol/l 55. saikosaponin c 19.9 ± 0.1 μmol/l 56. saikosaponin d 0.02 ± 0.001μmol/l 57. r-halitunal na (koehn et al., 1991b) diterpenes 58. ferruginol 0.40 µg/ml (wen et al., 2007) 59. dehydroabieta-7one 4.00 μm (wen et al., 2007) 60. sugiol na (wen et al., 2007) 61. cryptojaponol >3.3 µg/ml (wen et al., 2007) 62. 8β-hydroxyabieta-9(11), 13-dien-12-0.44 µg/ml (wen et al., 2007) one 63. 7βhydroxydeoxycrypt ojaponol 1.15 μm (wen et al., 2007) 64. 6,7dehydroroyleanone 5.55 μm (wen et al., 2007) 65. 3β, 12diacetoxyabieta-6, 8,11,13-tetraene 0.48 µg/ml (wen et al., 2007) 66. pinusolidic acid 4.71 μm (wen et al., 2007) 67. forskolin 3.1 µg/ml (wen et al., 2007) sesquiterpenes 68. cedrane-3β,12-diol >2.3 µg/ml (wen et al., 2007) 69. cadinol 1.04µg/ml (wen et al., 2007) triterpenes 22. betulinic acid >4.5 µg/ml (wen et al., 2007) 70. betulonic acid 0.29 µg/ml (wen et al., 2007) lignins: 71. hinokinin >10μm (wen et al., 2007) 23. savinin 0.40 µg/ml (wen et al., 2007) 72. 4,4′-obenzoylisolariciresinol na (wen et al., 2007) 73. honokiol 6.5μm (wen et al., 2007) 74. magnolol 3.80μm (wen et al., 2007) 75. curcumin >10μm (wen et al., 2007) 76. niclosamide <0.1μm (wen et al., 2007) 77. valinomycin 1.82 µg/ml (wen et al., 2007) 78 mechanism of nucleic acid unwinding by sars-cov helicase the proximal origin of sars-cov-2 the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity mechanisms of coronavirus cell entry mediated by the viral spike protein toona sinensis roem tender leaf extract inhibits sars coronavirus replication inhibition of sars-cov 3c-like protease activity by theaflavin-3, 3'-digallate (tf3) in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds potential natural compounds for preventing 2019-ncov infection preprint virus 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taxilluschinensis inhibit sars-cov replication trends in use, pharmacology, and clinical applications of emerging herbal nutraceuticals identification of phenanthroindolizines and phenanthroquinolizidines as novel potent anti-coronaviral agents for porcine enteropathogenic coronavirus transmissible gastroenteritis virus and human severe acute respiratory syndrome coronavirus small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells identification of myricetin and scutellarein as novel chemical inhibitors of the sars j o u r n a l p r e -p r o o f coronavirus helicase, nsp13 angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china fangchinoline hcov-oc43 1.01 µm key: cord-259935-xyo2pe4g authors: wang, ching-ying; lu, chien-yi; li, shih-wen; lai, chien-chen; hua, chun-hung; huang, su-hua; lin, ying-ju; hour, mann-jen; lin, cheng-wen title: sars coronavirus papain-like protease up-regulates the collagen expression through non-samd tgf-β1 signaling date: 2017-05-02 journal: virus res doi: 10.1016/j.virusres.2017.04.008 sha: doc_id: 259935 cord_uid: xyo2pe4g sars coronavirus (cov) papain-like protease (plpro) reportedly induced the production of tgf-β1 through p38 mapk/stat3-meidated egr-1-dependent activation (sci. rep. 6, 25754). this study investigated the correlation of plpro-induced tgf-β1 with the expression of type i collagen in human lung epithelial cells and mouse pulmonary tissues. specific inhibitors for tgf-βri, p38 mapk, mek, and stat3 proved that sars-cov plpro induced tgf-β1-dependent up-regulation of type i collagen in vitro and in vivo. subcellular localization analysis of smad3 and smad7 indicated that non-smad pathways in tgf-β1 signaling involved in the production of type i collagen in transfected cells with psars-plpro. comprehensive analysis of ubiquitin-conjugated proteins using immunoprecipitation and nanolc–ms/ms indicated that sars-cov plpro caused the change in the ubiquitination profile of rho gtpase family proteins, in which linked with the increase of rho-like gtpase family proteins. moreover, selective inhibitors tgf-βri and stat6 (as1517499) ascertained that stat6 activation was required for plpro-induced tgf-β1-dependent up-regulation of type i collagen in human lung epithelial cells. the results showed that sars-cov plpro stimulated tgf-β1-dependent expression of type i collagen via activating stat6 pathway. severe acute respiratory syndrome (sars)-associated coronavirus (cov), a member of betacoronoviruses in the coronaviridae family, is identified as the causative agent for the outbreak of sars in asia and other countries in [2002] [2003] . like other human coronaviruses (hcovs) hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1, and mers-cov (li and lin, 2013; huang et al., 2015) , sars-cov genome is an approximately 30 kb positive-strand rna consisting of a 5′ cap, a 3′ poly (a) tract, and 14 open reading frames (orfs). the largest orfs orf1a and orf1ab encode for the polyprotein replicases 1a and 1ab mainly involving in the sars-cov replication, as cleaved in cis and in trans by orf1a-encoded papain-like protease (plpro) and 3c-like protease (3clpro). plpro, a de-ubiquitinating/de-isgylating enzyme (barretto et al., 2005; ratia et al., 2006) , has the antagonistic activities of type i interferon (ifn) via blocking irf-3 and erk1 phosphorylation, preventing the iκbα degradation, and de-ubiquitinating the sting-traf3-tbk1 complex (li et al., 2011; frieman et al., 2009; sun et al., 2012) . recently, plpro shows the inhibitory effect on tolllike receptor 7 (tlr7) mediated cytokine production through removing lys63-linked ubiquitin chains of traf3 and traf6 (li et al., 2016a) . sars-cov up-regulates pro-inflammatory cytokines like ifn-γ, il-18, tgf-β1, tnf-α, il-6, ip-10, mcp-1, mig, and il-8 (huang et al., 2005; he et al., 2006) , in which recruits immune responder cells into the lungs, triggers acute respiratory distress syndrome (ards), and even causes lung fibrosis in the late phase (huang et al., 2005; he et al., 2006) . among sars-cov proteins, the nucleocapsid induces a smad3dependent induction of tgf-β1 expression (zhao et al., 2008) ; spike protein stimulates the il-8 up-regulation in lung cells (chang et al., 2004) ; nsp1 provokes the expression of ccl5, cxvl10, and ccl3 (law http://dx.doi.org/10.1016 (law http://dx.doi.org/10. /j.virusres.2017 received 10 january 2017; received in revised form 3 april 2017; accepted 10 april 2017 et al., 2007) ; plpro elavates the production of tgf-β1 and pro-fibrotic markers via ubiquitin proteasome, p38 mapk, and erk1/2-mediated signaling (li et al., 2012) . recently, sars-cov plpro notably initiates ros/p38 mapk/stat3 pathway to activate egr-1 dependent expression of tsp-1, tgf-β1 and vimentin in vitro and in vivo (li et al., 2016b) . therefore, plpro becomes a virulent factor in sars pathogenesis. tgf-β1 plays a crucial mediator of tissue fibrosis in lung, skin, liver, heart, and kidney through modulating the expression of pro-fibrotic proteins including type i collagen, fibronectin, α-sma, and vimentin (kubiczkova et al., 2012) . in the canonical smad signalling pathway, tgf-β1 interacts with the tgf-β type i receptor, activates receptorregulated smads (smad2 and smad3) complexed with smad4, and then translocate to the nucleus to synthesize tgf-β1-induced transcriptional genes like type i collagen (leask and abraham, 2004; kubiczkova et al., 2012) . in non-canonical tgf-β signaling pathways, tgf-β1 activates mapks (erk, jnk and p38 mapk), rho-like gtpases (rhoa, rac and cdc42), pi3k/akt, wnt/β-catenin, or ca 2+ signaling cascades in tgf-β1-induced transcriptional response (zhang, 2009 ). the activation of p38 mapk is responsible for tgf-β-induced epithelial-tomesenchymal transition of mouse mammary gland epithelial cells under a receptor independent of receptor-mediated smad activation (yu et al., 2002) . the activation of rhoa in tgf-β-induced emt response is also independent of smad2 and/or smad3 (bhowmick et al., 2001) . our prior study demonstrated sars-cov plpro triggering the tgf-β1 production in vitro and in vivo that linked with up-regulating the expression of pro-fibrotic proteins (vimentin and glial fibrillary acidic protein) (li et al., 2016b) . this study assesses possible effects and mechanisms of sars-cov plpro-induced tgf-β1 upregulation on the expression of type i collagen. the induction ability of sars-cov plpro on in vitro and in vivo expression of type i collagen was characterized. in addition, subcellular localization of smad3, smad7, and stat6 was performed to elucidate the key factors involved in the induction of type i collagen by plpro. the relationship between tgf-β upregulation and the mechanism of type i collagen induction by plpro was validated by the specific inhibitors of tgf-β receptor kinase, p38 mapk and stat6. human alveolar basal epithelial a549 cells grew in dulbecco's modified eagle's medium (hyclone laboratories) and were transfected with control vector pcdna3.1/his c (invitrogen), or psars-plpro containing sars-cov plpro gene, as described in our prior reports (li et al., 2011 (li et al., , 2012 (li et al., , 2016a (li et al., , 2016b . in addition, psars-plpro (h273a) that had the alanine substitution for histidine at position 273 by ala within plpro gene was constructed using pcr-based sitedirected mutagenesis with a mutated primer pair (5′-ggtaact atc-agtgtggtgcttacactcatataactgctaag-3′ and 5′-cttagcagt tatatgagtgtaagcaccacactgatagttacc-3′). a549 cells transiently expressing recombinant plpro 2 days post transfection was analyzed using western blotting, real-time rt-pcr, sirius staining, and immunofluorescent staining assays. to detect the mrna expression of type i collagen, total rnas extracted from transfected cells treated with or without a tgf-β1 inhibitor (sb-431542) were further examined the mrna levels of type i collagen using quantitative real-time pcr. the relative fold mrna levels were normalized by gapdh mrna, presented as the relative ratio (b). for verifying the protein levels of type i collagen, transfected cells with serial doses of indicated plasmids were stained using sirius red staining kit (c). ** p value < 0.01 by student's t-test. the lysate of transfected cells was performed by western blotting with primary antibodies including rabbit anti-tgf-β1 (cell signaling), anti-e. coli synthesized plpro mouse serum, anti-phospho stat6 (tyr641) (cell signaling), and anti-β-actin mab (abcam), and hrpconjugated secondary antibodies like goat anti-mouse or anti-rabbit igg. immune complexes were detected using enhanced chemiluminescent hrp substrate (millipore). to measure the expression of type i collagen, tgf-β1, and vimentin in transfected cells, total rnas extracted from transfected cells 2 days post transfection and mouse lung tissues were analyzed using two-step real time rt-pcr with sybr green i, as described in our prior reports (li et al., 2016b) . primer pairs included (1) 5′-gttcgtgaccgtgac-ctcg-3′ and 5′-tcttgtccttggggttcttgc-3′ for human type i collagen, (2) 5′-gagcggagagtactggatcg-3′ and 5′-tactcgaac-gggaatccatc-3′ for mouse type i collagen, (3) 5′-ggcctttcctgc-ttctcatgg-3′ and 5′-ccttgctgtactgcgtgtcc-3′ for human tgf-β1, (4) 5′-tctctgaggctgccaaccg-3′ and 5′-cgaaggtgacgagc-catttcc-3′ for human vimentin, (5) 5′-cagaacagcctcccgaatg-3′ and 5′-tgctacgctcactccattac-3′ for human rac1, (6) 5′-agcc-acatcgctcagacac-3′ and 5′-gcccaatacgaccaa atcc-3′ for human gapdh, and (7) 5′-tgaggccggtgctgagtatgtcg-3′ and 5′-ccacagtcttctgggtggcagtg-3′ for mouse gapdh. specific pcr product was quantified using the abi prism 7900ht sequence detection system (pe applied biosystems). relative mrna levels of indicated genes were normalized relative to gapdh mrna. for the detection of collagen expression, the tissue sections were stained with sirius red solution for 2 h, and then rinsed 10 times with 0.5% glacial acetic acid in pbs. after dehydrating with ethanol, stained sections were mounted on the glass slides, and then examined using light microscopy (olympus, bx50). the mouse mode with a direct chest injection was performed as described in our prior report (li et al., 2016b) . empty vector pcdna3.1 or recombinant plasmin psars-plpro (50 μg/100 μl) in 3% sucrose/ pbs was injected into the right chest of 5 eight-weeks-old balb/c male mice using a 1-ml syringe with a 28-gage needle every 2 days. after 15 injections, the mice were sacrificed; the lung tissues were fixed, dehydrated, embedded in paraffin, and cut at 4-5 μm thickness using a rotary microtome. for immunohistochemistry (ihc) staining, mouse lung tissues were performed with anti-e. coli synthesized plpro serum, as descried in our previous report (li et al., 2016b) . for h & e staining, sections were stained with hematoxylin for 3 min, eosin for 3 min, dehydrated in ethanol, and then mounted as slides that were examined and photographed using light microscopy (olympus, bx50). sirius staining and sybr green real time rt-pcr assays were mentioned above. for determining the effects of sars-cov plpro on the nuclear translocalization of smad3, smad7, and stat6, a549 cells grew on the glass coverslip in 6-wellt were transfected with psars-cov plpro or pcdna3.1, and treated with or without 1 μm kartogenin (sigma). for testing the role of rac1 in stat6 signal, the rac1 mutant plasmid, pmx-ig-rac1 t17n provided by dr. takehito uruno (kyushu university, japan), was co-transfected into cells. after 2-day incubation, cells were fixed with 3.7% formaldehyde in pbs for 1 h, blocked with 1% bovine serum albumin in pbs for 1 h, and then incubated with specific primary antibodies against smad3, smad7, and stat6 at 4°c to detect the mrna expression of type i collagen, total rnas extracted from lung tissues were examined the mrna levels of type i collagen using quantitative real-time pcr. the relative fold mrna levels were normalized by gapdh mrna, presented as the relative ratio (b). ** p value < 0.01 by student's t-test. overnight. subsequently, cells were reacted with fitc-or af546conjugated secondary antibodies in a dark box for 2 h, finally, cells were stained with 4′,6-diamidino-2-phenylindole (dapi) for 10 min. after washing with pbs, stained cells were photographed using the immunofluorescence microscopy (olympus, bx50). the lysates from plpro-expressing and empty vector cells were reacted with anti-ubiquitin antibodies for 4 h at 4°c, and then incubated with protein a-sepharose beads. the ubiquitin-conjugated proteins were collected after centrifugation, washed four times with net buffer, embedded in sds-page gel, and then digested in gel. the peptides of ubiquitin-conjugated proteins were recovered for nanolc-ms/ms spectra. proteins were identified according to mass spectra obtained were compared to swissport database (release 51.0) via mascot algorithm (version 2.2.07), as described in our prior reports (li et al., 2012) . peptides were identified if mascot individual ion scores exceeded 30. all data were collected from 3 independent experiments and analyzed using student's t-test or χ 2 test. statistical significance was considered at p < 0.05. to examine the association of sars-cov plpro-induced tgf-β1 production with the collagen up-regulation, a549 lung epithelial cells transiently transfected with pcdna3.1 and psars-plpro were analyzed the production of tgf-β1 and type i collagen using western blot, realtime rt-pcr and sirius red staining assays (fig. 1) . transfected cells with psars-plpro, but not pcdna3.1, secreted the active form of tgf-β1, and significantly increased the mrna and protein expression of type i collagen. importantly, sb-431542 (a selective tgf-βri inhibitor) treatment at 100 nm caused the 4-fold reduction of type i collagen mrna in transfected cells with psars-plpro (fig. 1b) . in a mouse model, the expression of type i collagen in lung tissues from chest injection with pcdna3.1 or psars-plpro was examined using sirius staining and quantitative rt-pcr (fig. 2) . the expression of sars-plpro in lung tissues of mice was determined using ihc staining with anti-e. coli synthesized plpro serum, and ihc positivity for plpro expression within lung tissues was observed in the group infected with psars-plpro ( fig. 2a) . h & e and sirius staining assays indicated that pulmonary inflammation with the infiltration of immune cells and the increase of type i collagen was identified in the psars-plpro group, but not vector control and solvent groups ( fig. 2a) . real-time pcr confirmed that plpro triggered the mrna expression of type i collagen fig. 3 . analysis of tgf-β1 and type i collagen levels in transiently transfected cells in response to the inhibitors for p38 mapk and stat3. to detect the mrna expression of tgf-β1 or type i collagen, total rnas extracted from transfected cells treated with or without the rac1 mutant plasmid (a), an inhibitor for p38 mapk (sb203580) (c) or stat3 (stattic) (d) were examined the mrna levels of type i collagen using quantitative real-time pcr. the relative fold mrna levels were normalized by gapdh mrna, presented as the relative ratio (a, b). for verifying the protein levels of type i collagen, transfected cells treated with or without an inhibitor for p38 mapk (sb203580) or mek (u0126) were stained using sirius red staining kit (c). ** p value < 0.01 by student's t-test. c.-y. wang et al. virus research 235 (2017) [58] [59] [60] [61] [62] [63] [64] [65] [66] in mouse lung tissues in comparison with vector control and solvent groups (fig. 2b) . to examine the proteolytic enzymatic activity of plpro on the production of tgf-β1 and type i collagen, the catalytic mutant of plpro (h273a) was constructed, and then used to investigate whether plpro (h273a) up-regulated tgf-β1 and type i collagen in vitro (fig. 3a) . importantly, plpro(h273a) with the catalytic mutation lose the ability to induce the expression of tgf-β1 and type i collagen in vitro. since sars-plpro had been demonstrated to stimulate p38 mapk/stat3mediated activation of tgf-β1 production (li et al., 2016b) , sb203580 (a specific p38-mapks inhibitor), u0126 (a mek1/2 inhibitor), and stattic (a small-molecule inhibitor of stat3 activation) were used to further confirm the correlation between the tgf-β1 production and type i collagen up-regulation in transfected cells with psars-plpro and pcdna3.1 (fig. 3b-d) . sb203580 significantly reduced the mrna expression of tgf-β1, in which was linked with down-regulation of type i collagen in transfected cells with psars-plpro in presence of sb203580 or u0126 ( fig. 3b and c) . stattic also suppressed the mrna expression of type i collagen in transfected cells with psars-plpro ( fig. 3d) . overall, results of the in vitro and in vivo data demonstrated that the proteolytic enzymatic activity was required for sars-cov plpro-dependent tgf-β1-mediated up-regulation of pro-fibrotic gene type i collagen. to examine whether smad-dependent pathways involve in tgf-β1mediated up-regulation of type i collagen in response sars-cov plpro, subcellular localization of receptor-regulated smad3 and inhibitory smad7 in transfected cells were detected using the immunofluorescent and dapi staining (fig. 4) . imaging analysis of transfected cells indicated that smad3 localized in the nucleus of pcdna3.1-transfected cells, but not psars-plpro-transfected cells (fig. 4a) . moreover, smad7 was detected in the nucleus of both transfected cells (fig. 4b) . interestingly, kartogenin, a stimulator for tgf-β1/smad3 signal pathway , was used to verify the inhibitory effect of plpro on tgf-β1/smad3 signal in transfected cells (fig. 4c) . after 24 h treatment with kartogenin, samd3 nuclear translocation was spotted within the nucleus in vector control cells, but not in plproexpressing cells. most samd3 was in the cytoplasm of psars-plpro transfected cells treated with kartogenin. the result indicated that sars-cov plpro inactivated smad-dependent pathways, implying that non-smad pathways in tgf-β1signaling for the production of type i collagen would be initiated by sars-cov plpro. non-smad pathways in tgf-β1 signaling include map kinase, rholike gtpase, and phosphatidylinositol-3-kinase/akt pathways (zhang, 2009) . to examine the possible pathways involved in tgf-β1-dependent up-regulation of type i collagen by sars-cov plpro, the profiles of ubiquitin-conjugated proteins in transfected cells with vector control and psars-plpro were determined using immune-precipitation and nanolc-ms/ms. interestingly, several proteins of the ras gtpase family were identified (table 1 ), in which indicated the ubiquitination of these ras family gtpase proteins was influenced by sars-cov plpro. fig. 5a represented the mass spectrum of rac1 identified by lc-ms/ms. rac1, a rho gtpase, had an increase of the ubiquitination in plpro-expressing cells compared to vector control cells. real-time rt-pcr and western blotting assays revealed the elevation of rac1 mrna and protein expression in transfected cells with psars-plpro compared to vector control ( fig. 5b and c) . rho gtpases were identified in the regulation of g protein-coupled receptor signaling through activation of jak/stat and modulated stat-dependent gene expression (pelletier et al., 2003) . in addition, stat-6-dependent collagen production has been demonstrated in human skin fibroblasts and mouse airway fibroblasts in response to bovine milk and platelet-derived growth factor, respectively (kippenberger et al., 2015; lu et al., 2014) . for analyzing the correlation between plpro-induced rac1 upregulation and stat6 activation, co-transfection of pcdna3.1 or psars-plpro plus the rac1 mutant plasmid pmx-ig-rac1 t17n was performed, and the stat6 activation in co-transfected cells was measured using immunofluorescence staining with anti-stat6 and fitc-conjugated secondary antibodies. plpro expression caused the stat6 expression and induced the translation of stat6 in the nucleus. remarkably, the rac1 mutant slightly affected the stat6 up-regulation by plpro, but significantly reduced plpro-induced stat6 nuclear translocation (fig. 5d) . later, the functional activity of stat6 in tgf-β1-dependent collagen up-regulation was further characterized in vector control and plpro-expressing cells (figs. 6 and 7) . nuclear localization and phosphorylation of stat6 was observed in transfected cells with psars-plpro, but not vector control cells (figs. 6 and 7 a) . importantly, sb-431542, a selective tgf-βr inhibitor, significantly reduced the entry of stat6 in the nucleus of plpro-expressing cells (fig. 6) . the result indicated that rho gtpases/stat6 was responsible for one of non-smad pathways in sars plpro-induced tgf-β1 signals. furthermore, a stat6 inhibitor as1517499 markedly suppressed tgf-β1-dependent collagen expression in plpro-expressing cells (fig. 7) . the results demonstrated that stat6 activation was required for sars plpro-induced tgf-β1-dependent production of type i collagen. plpro has been demonstrated to trigger the tgf-β1 production in human promonocytes, human lung epithelial cells, and pulmonary tissues in mouse models (li et al., 2012 (li et al., , 2016b . sars-cov plpro induced the ros-mediated p38 mapk and stat3 activation of egr-1 expression, in which egr-1 specifically bound to the tgf-β1 promoter region between −175 to −60, resulting in the increase of tgf-β1 production. this study indicated that sars-cov plpro stimulated the production of type i collagen in vitro and in vivo ( fig. 1-3) . sars-cov plpro-induced collagen deposition in pulmonary tissues was associated with lung inflammation and pulmonary fibrosis in mice injected with psars-plpro (fig. 3) . the specific inhibitor for tgf-β receptor, sb-431542, blocked the up-regulation of type i collagen in transfected cells with psars-plpro. moreover, the inhibitors for p38 mapk and stat3 that involved in egr-1-dependent tgf-β1 production significantly reduced the expression of tgf-β1 and type i collagen. overall, the results revealed that sars-cov plpro elicited tgf-β1dependent up-regulation of type i collagen. tgf-β1-dependent profibrotic response was considered as the crucial character in sars-cov plpro-induced pathogenesis. an elevated level of pro-inflammatory cytokines including tgf-β1 was detected in sars-cov-infected cells in autopsy tissues from died sars patients (he et al., 2006) , in which implied the association of pro-inflammatory cytokines with the severity and mortality of sars. the increase of serum tgf-β1 concentration during the early phase of sars could be associated with lung infiltration (beijing group of national research project for sars, 2003) . meanwhile, the decrease of serum tgf-β1 concentration markedly lowered in the severity of sars patients (zhang et al., 2004) . in canonical pathway, tgf-β1interacts with type i and ii tgf-β receptors and causes the activation of a serine/threonine kinase domain in tgf-β receptors and the recruitment and phosphorylation of smad2 and smad3. the phosphorylated smad2/3 complexed with smad4 was trans-localized into nucleus to regulate the target gene expression (kubiczkova et al., 2012) . subcellular localization analysis demonstrated that smad3 was predominant in cytoplasmic, but not in the nucleus in transfected cells with psars-plpro compared to vector control (fig. 4) , revealing that canonical smad-dependent signaling pathway was not involved in plpro-induced tgf-β1-dependent upregulation of type i collagen. tgf-β also activates non-canonical non-smad pathways, including mapks (erk1/erk2, jnk and p38), pi3k kinases, akt/pkb, mtor, and rho-like gtpase family proteins (ras, rhoa, rac1, and cdc42) (zhang, 2009; kubiczkova et al., 2012) . particularly, activation of rhoa pathway was required for tgf-βmediated process of epithelial-to-mesenchymal trans-differentiaion in a dominant-negative smad3 cells (bhowmick et al., 2001) . in addition, rac1 activation promoted tgf-β-dependent collagen expression in mesangial cells (hubchak et al., 2009) . lc-ms/ms analysis demonstrated that sars-cov plpro influenced the ubiquitination status of ras-related c3 botulinum toxin substrate 1 (rac1), ras gtpaseactivating-like protein iqgap1, ras-related protein rab (rab5c), putative ras-related protein rab (rab1c), and ras gtpase-activating protein-binding protein (g3bp1) ( table 1) . real-time rt-pcr and western blotting assays indicated the increased expression of rac1 in transfected cells with psars-plpro compared to vector control (fig. 5) . since activation of jak/stat pathway in a rac-dependent manner was identified in response to the agonist of protein-coupled receptors (pelletier et al., 2003) , stat-dependent signals were investigated in this study. the tgf-βri inhibitor sb-431542 significantly attenuated the phosphorylation and nuclear localization of stat6 in transfected cells with psars-plpro (fig. 6) . meanwhile, the stat6 inhibitor as1517499 meaningfully reduced tgf-β1-dependent up-regulation of type i collagen in plpro-expressing cells (fig. 7) . the results indicated fig. 6 . nuclear localization of stat6 in transfected lung epithelial cells in response to sb-431542. for analyzing the effect of sb-431542 on nuclear localization of stat6, transfected cells were treated with or without sb-431542 for 2 days, washed, fixed, and reacted with primary antibodies against phospho-stat6, followed by fitc-conjugated secondary antibodies. after staining with dapi for 10 min, imaging was analyzed by immunofluorescent microscopy. that stat6 activation was required for plpro-induced tgf-β1-dependent expression of type i collagen in human lung epithelial cells and mouse lung tissues. the result was accordant with the previous reports in that stat6-dependent collagen production has been demonstrated in human skin fibroblasts and mouse airway fibroblasts (kippenberger et al., 2015; lu et al., 2014) . sars-cov plpro affected on the ubiquitination and expression profile of rho-like gtpase family proteins that could link with the activation of stat6 signaling in tgf-β1dependent collagen expression, in which suggested that a new gtpase proteins/stat6 pathway might play a critical role in tgf-β1-dependent collagen production in plpro-expressing cells. in summary, sars-cov plpro induced p38 mapk/stat3-mediated tgf-β1-dependent up-regulation of type i collagen, causing pulmonary pro-fibrotic responses. plpro diminished the nuclear localization of smad3, changed the expression profiling of rho-like gtpase family proteins, and activated stat6-mediated tgf-β1-dependent production of type i collagen. the results let us conclude that sars-cov plpro induced non-smad signals including stat6 activation in tgf-β1dependent pulmonary pro-fibrotic responses. fig. 7 . analysis of stat6-mediated plpro-induced production of type i collagen. to determine the phosphorylation of stat6, transfected cells with 0.5, 1, 2, 5 or 10 μg of pcdna3.1 or pplpro were harvested 2 days post transfection; the cell lysates were examined using western blotting with anti-phospho-stat6 (tyr641) or anti-β actin antibodies (a). for analyzing mrna levels of type i collagen (b) and vimentin (c), transfected cells were treated with or without as1517499 for 4 h, and then their mrna levels of type i collagen and vimentin were measured by quantitative pcr. relative mrna levels were normalized by gapdh mrna, presented as relative ratio. ** p value < 0.01 by student's t-test. the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity dynamic changes in blood cytokine levels as clinical indicators in severe acute respiratory syndrome transforming growth factor-beta1 mediates epithelial to mesenchymal transdifferentiation through a rhoa-dependent mechanism 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 severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace2+ cells in sars patients: relation to the acute lung injury and pathogenesis of sars an interferon-gamma-related cytokine storm in sars patients epidemiology of human coronavirus nl 63 infection among hospitalized patients with pneumonia in taiwan rac1 promotes tgfbeta-stimulated mesangial cell type i collagen expression through a pi3k/aktdependent mechanism stat6-dependent collagen synthesis in human fibroblasts is induced by bovine milk tgf-β-an excellent servant but a bad master role for nonstructural protein 1 of severe acute respiratory syndrome coronavirus in chemokine dysregulation tgf-beta signaling and the fibrotic response human coronaviruses: clinical features and phylogenetic analysis severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferon-induced responses through downregulation of extracellular signalregulated kinase 1-mediated signalling pathways correlation between tgf-β1 expression and proteomic profiling induced by severe acute respiratory syndrome coronavirus papain-like protease sars coronavirus papain-like protease inhibits the tlr7 signaling pathway through removing lys 63-linked polyubiquitination of traf3 and traf6 sars coronavirus papain-like protease induces egr-1-dependent upregulation of tgf-β1 via ros/p38 mapk/stat3 pathway platelet-derived growth factor mediates interleukin-13-induced collagen i production in mouse airway fibroblasts rho family gtpases are required for activation of jak/stat signaling by g protein-coupled receptors severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling a heterocyclic molecule kartogenin induces collagen synthesis of human dermal fibroblasts by activating the smad4/ smad5 pathway tgf-β receptor-activated p38 map kinase mediates smad-independent tgf-β responses analysis of serum cytokines in patients with severe acute respiratory syndrome non-smad pathways in tgf-beta signaling severe acute respiratory syndrome-associated coronavirus nucleocapsid protein interacts with smad3 and modulates transforming growth factor-beta signaling this study was supported by china medical university under the aim for top university plan of the ministry of education, taiwan (chm106-6-2). this project was also funded by grants from the ministry of science and technology, taiwan (most102-2628-b-039-044-my3, most105-2320-b-039-053-my3) and china medical university (cmu105-s-20, cmu103-asia-07, and cmu105-asia-10). key: cord-332075-gxmae2rs authors: wang, jianzhong; cong, yanlong; yin, renfu; feng, na; yang, songtao; xia, xianzhu; xiao, yueqiang; wang, wenxiu; liu, xiufan; hu, shunlin; ding, chan; yu, shengqing; wang, chunfeng; ding, zhuang title: generation and evaluation of a recombinant genotype vii newcastle disease virus expressing vp3 protein of goose parvovirus as a bivalent vaccine in goslings date: 2015-05-04 journal: virus res doi: 10.1016/j.virusres.2015.04.006 sha: doc_id: 332075 cord_uid: gxmae2rs newcastle disease virus (ndv) and goose parvovirus (gpv) are considered to be two of the most important and widespread viruses infecting geese. in this study, we generated a recombinant rmna-vp3, expressing gpv vp3 using a modified goose-origin ndv na-1 by changing the multi-basic cleavage site motif rrqkr↓f of the f protein to the dibasic motif grqgr↓l as that of the avirulent strain lasota as a vaccine vector. expression of the vp3 protein in rmna-vp3 infected cells was detected by immunofluorescence and western blot assay. the genetic stability was examined by serially passaging 10 times in 10-day-old embryonated spf chicken eggs. goslings were inoculated with rmna-vp3 showed no apparent signs of disease and developed a strong gpv and ndv neutralizing antibodies response. this is the first study demonstrating that recombinant ndv has the potential to serve as bivalent live vaccine against goose parvovirus and newcastle disease virus infection in birds. goose parvovirus (gpv) infection also known as derzsy's disease, goose hepatitis, or gosling plague is an acute, contagious, and fatal disease to domestic goslings and muscovy ducklings (derzsy, 1967; gough et al., 2005; schettler, 1971 ). this is a gastrointestinal disease characterized mainly by diarrhea and fibrous hemorrhagic necrotic enteritis. with high mortality and morbidity, gpv has caused substantial economic losses to goose farming countries of europe and asia (jansson et al., 2007; takehara et al., 1995) . to control the disease caused by gpv, live attenuated vaccines are widely used in breeder geese or goslings in addition to flock management (gough and spackman, 1982; kisary, 1977) . usually, the attenuated viruses for vaccines were obtained by serial passages in cultured goose-embryo fibroblasts (gef) or embryonated goose eggs to reduce the pathogenicity . however, the potential risk of reversion to virulence of attenuated live vaccines is always a concern. additionally, the current available vaccines are prepared with chorioallantoic fluid collected from infected embryos, which is costly and inefficient due to the limited availability of spf (specific pathogen free) goose and duck embryos (ju et al., 2011; lee et al., 2010) . therefore, the development of alternative strategies for producing safe and effective vaccines may be of great significance. newcastle disease, caused by newcastle disease virus (ndv), is one of the most serious infectious diseases of many avian species and has caused substantial losses in the poultry industry worldwide (aldous and alexander, 2001; alexander, 2000 the basis of their pathogenicity for birds, ndv has been classified into three different pathotypes: velogenic (highly virulent), mesogenic (moderately virulent) or lentogenic (low virulence) viruses. the molecular basis for the pathogenicity of ndv is mainly determined by the amino acid sequence of the protease cleavage site located in f protein. mesogenic and velogenic viruses have a polybasic amino acid motif of 112 r/kr-q-k/r-r-f 117 , while viruses of low virulence have a monobasic amino acid sequence of 112 g/e-k/r-q-g/e-r-l 117 at the cleavage site (choi et al., 2010; panda et al., 2004; peeters et al., 1999) . lentogenic and, in some cases, mesogenic strains of ndv are widely used as live attenuated vaccines in poultry. in recent years, they also have been developed as a vector to express foreign protective antigens based on reverse genetic techniques. the use of such live vector vaccines for immunization not only protect birds against ndv and another poultry pathogens, such as highly pathogenic avian influenza virus (hpaiv) (ge et al., 2007) and infectious bursal disease virus (ibdv) (huang et al., 2004b) , but also has the potential to serve as effective vaccines for human use against sars-cov or other emerging viruses (dinapoli et al., 2007a (dinapoli et al., ,b, 2010 . in this study, we generated a recombinant ndv expressing the vp3 protein of gpv using a modified avirulent virus vector derived from ndv na-1 strain isolated from goose flocks. the safety, stability, and feasibility of this recombinant rmna-vp3 to serve as a bivalent live vaccine in goslings were evaluated. bhk-21 (atcc, ccl-10) and df-1 (atcc, crl-12203) cell lines were cultured in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs) and maintained at 37 • c with 5% co 2 . the gpv vaccine strain, gd-01, was obtained from the china veterinary culture collection. the viral stock was inoculated into the chorioallantoic cavity of 12-day-old embryonated goose eggs. ndv strain na-1 was a virulent strain isolated by our laboratory from geese stocks in nong'an county of jilin province, china in 1999 (genbank access no. dq659677) and recovered from the full-length antigenomic cdna using a reverse genetics system as describe previously (wang et al., 2015) . to attenuate the ndv na-1 strain the sequence encoding the protease cleavage site of the f protein was modified by means of pcr mutagenesis. thus, a fragment between pmei and sacii restriction sites (nt 3165-4900) was amplified from the full-length cdna clone of na-1, and subcloned into pbluescript ii sk+ (stratagene) at the ecorv site. then, the fragment pmei-sacii of pci-na-1 was pcr amplified using the primers fmut-pf (5 -gcttgtttaaacaa-aacaacagccctctctcaccc-3 , with pmei site underlined) and fmut-pr1 (5 -ccaagagctacactgccaataacggcacctataa-ggcgcccctgtctccctcctccagacgtggacacagac-3 , with modified sequence bolded). the pcr products were subjected to a second round pcr with the primes fmut-pf and fmut-pr2 (5 -gggccgcggctgctgttatctgtgccgctgttgcaaccccaagagct-acactgccaataacggc-3 , with sacii site underlined) resulting in a new fragment encoding an avirulent protease cleavage site grqgr↓l of lasota mutated from rrqkr↓f of na-1. finally, the new fragment was excised from pbluescript ii sk+ using the restriction enzymes pmei and sacii and was used to replace the corresponding fragment in the full-length cdna clone of the pci-na-1. the resultant plasmid was designated as pci-mna-1. the open reading frame (orf) of vp3 gene of gpv strain gd-01 was amplified from the genome dna by using the primer pair of 5 -gcgcgtttaaacttaagaaaaaatacgggtagaagccgccaccat-ggcagagggaggaggcgg-3 and 5 -gcgcgtttaaacttacagat-tttgagttagatatc-3 , in which the gene end and gene start sequences of ndv (bold), the optimal kozak sequence (italic), and the pmei restriction sites (underlined) were included. the amplified product was ligated into ecorv-cut pbluescript ii sk+ vector, sequenced, and inserted into the ndv full-length cdna clone contained in pci-mna-1 through the introduced pmei site in the p-m noncoding region at nucleotide position 3165 of the ndv genome, as described previously (wang et al., 2015) . the new full-length plasmid was designated as pci-na-vp3. rescue of the infectious viruses were performed by transfecting the full-length cdna clone and supporting plasmids into bhk-21 cells as described previously (wang et al., 2015) .the rescued viruses were named rmna-1 and rmna-vp3, respectively. a 2% solution of phosphotungstic acid (ph 6.8) was added to the virus samples for negative staining. then the mixtures were transferred to carbon-coated grids, and the specimens were photographed with an electron microscope h-7650 (hitachi ltd., hitachi, japan). for immunofluorescence analysis, bhk-21 cells were plated on coverslips in 35-mm-diameter dishes and infected with ndv rmna-1 or rmna-vp3. after 24 h, the cells were fixed in ice-cold 3% paraformaldehyde in phosphate buffered saline (pbs) for 15 min at room temperature and then washed with pbs 3 times. cells were blocked in pbs containing 1% (w/v) bovine serum albumin (bsa) at 4 • c for 1 h. cells were then incubated with chicken serum anti-ndv or horse serum against gpv for 1 h at room temperature and were washed 3 times with pbs containing 0.05% tween 20. then, the cells were stained with fitc (fluorescein isothiocyanate)conjugated goat anti-chicken antibody (sigma) or an alexa fluor 555-conjugated rabbit anti-horse igg (bioss) for 30 min. finally, cells were washed 3 times with pbs, and dapi (4 ,6 -diamidino-2-phenylindole) was used to stain the cell nucleus. cells were analyzed with a confocal laser microscope (olympus corp, tokyo, japan). df1 cells were infected with rescued virus rmna-1 or rmna-vp3 at a multiplicity of infection (moi) of 5 and incubated for 48 h. the expression of cell-associated proteins was analyzed by western blotting. proteins from the lysates of infected cells were separated using sds-page under denaturing conditions and analyzed with chicken serum anti-ndv or rabbit anti-vp3 polyclonal antibody (bioss), or mono-clonal antibody against ␤-actin (cell signaling). immunostained proteins were visualized using a 3,3diaminobenzidine (dab) reagent. mock-infected df1 cells were used as the negative control. to evaluate the genetic stability of the recombinant viruses, rmna-1 or rmna-vp3 was serially passaged by 10 times in 10day-old embryonated eggs, respectively. the viral genome rna was isolated from allantoic fluids of each passage and subjected to rt-pcr to check the modified region in the f gene cleavage site of rmna-1 or rmna-vp3 and confirm the presence of the vp3 gene in the genome of rmna-vp3 by dna sequencing. the pathogenicity of the modified virus was evaluated by the mean death time (mdt) and the intracerebral pathogenicity index (icpi) according to the standard pathogenicity assay (alexander, 2008) . ten-day-old embryonated chicken eggs were inoculated with rmna-1, rmna-vp3 and rna-1, at 100tcid 50 per egg. viral growth was analyzed at different time points after inoculation. a 50% tissue culture infective dose (tcid 50 ) of each virus was determined by immunofluorescence assay. ninety-six well plates of 80%-confluent df1 cells were infected with serial 10-fold dilutions of virus (four wells per dilution). cells were incubated for 3 days and fixed with 80% cold acetone. viral antigens were detected with chicken serum anti-ndv and fitc-conjugated goat anti-chicken antibody. goslings at 4 days of age were randomized into three groups of 10 birds each. group 1 and group 2 were injected with 10 6 eid 50 of the rescued rmna-1 or rmna-vp3 in a 0.2-ml volume by subcutaneous administration and a booster injection was performed 2 weeks later. the third group of goslings was injected with pbs used as negative controls. all animals were housed in a specific pathogen-free facility with free access to water and food. blood samples were collected at 3, 6, 9, and 12 weeks post-immunization and the sera were separated by low speed centrifugation and stored at −20 • c. the neutralizing antibodies (nas) of individual goose serum samples against gpv and ndv were tested in the primary goose embryo fibroblasts (gefs) and df1 cells respectively. for the gpv nas assay, gefs were prepared from 13-day-old goose embryos and double dilutions of serum samples were pre-incubated with 100 tcid 50 gpv gd-01 at 37 • c for 1 h and then added to 10 5 gef cells in 96-well plates. for the ndv nas assay, 100 tcid 50 ndv na-1 was incubated for 1 h with serial dilutions of the test sera and then were added to df1 cells in 96-well plates. cells were observed microscopically for cytopathic effects (cpe) starting at 48 h post-infection and the titers were calculated by using the method detailed by reed and muench (1938) . statistical analysis was performed using of the analysis of variance (anova) method and considered significant when p ≤ 0.05. data are shown as means ± standard deviation (sd). to generate an attenuated ndv na-1 strain, modification were made to the full-length cdna clone of na-1 by changing the amino acid sequence of the f protein cleavage site from the naturally occurring rrqkr↓f to an avirulent motif grqgr↓l, resulting in a new cdna clone pci-mna-1 (fig. 1a) . the recovery of the modified virus rmna-1 was confirmed by rt-pcr and sequence analysis of the f gene from the ha-positive allantoic fluid (fig. 1b) . pathogenicity of the recombinant viruses was assessed by performing mdt and icpi tests. the results showed that the mdt of rmna-1 was 132 h, while that of rna-1 was 59 h. additionally, the icpi of rmna-1 was 0, while that of rna-1 was 1.9. these results suggest that rmna-1 is highly attenuated. to investigate the capability of cpe and the trypsin-dependent infectivity in cell culture, df1 cells were infected with rna-1, rmna-1, or lasota viruses at a moi of 0.01 in the presence or absence of 10% normal allantoic fluid as exogenous protease supplementation (fig. 1c) . the results showed that syncytium formation could be observed in cells infected by parental virus rna-1 with or without exogenous protease. lasota only induced the formation of syncytia in the presence of added protease. in contrast, rmna-1, which has exactly the same f protein cleavage site as lasota, did not cause any apparent syncytia even in the presence of exogenous protease. the genetic stability of the modified sites of rmna-1 was examined by serially passaging them 10 times in 10-day-old embryonated chicken eggs and sequencing the f gene from each passage. the results indicate the cleavage sites were preserved and stably maintained after 10 passages in chicken embryos (data not shown). to obtain the cdna clone pci-mna-vp3, an artificial transcription cassette coding for the vp3 protein of gpv was inserted into pci-mna-1 between the p and m genes of the genome (fig. 2a) . the resultant recombinant virus, rmna-vp3, was recovered entirely from this cdna by using our established reverse genetics procedures (wang et al., 2015) . the presence of the vp3 gene in the genome was confirmed by rt-pcr (data not show). the virions of rmna-vp3 were examined by observation with tem after negative staining, and compared with rna-1 and rmna-1, the vp3 gene inserted into the genome did not affect viral morphology (fig. 2b) . expression of the vp3 protein by rmna-vp3 was examined by indirect confocal immunofluorescence staining infected bhk-21 cells. as expected, cells infected with rmna-1 were not stained by horse serum against gpv, but they were positive for immunostaining using chicken serum against ndv (fig. 3a ). cells infected with rmna-vp3 were stained by horse serum against gpv as well as chicken serum against ndv (fig. 3a) . the vp3 expression by the recombinant virus was further confirmed by western blotting analysis of infected df1 cell lysates with rabbit anti-vp3 polyclonal antibody (fig. 3b) , whereas no band was detected in purified rmna-vp3 virus (data not shown). these results suggested that the recombinant rmna-vp3 stably expressed thevp3 protein and that the vp3 protein was probably not incorporated into the virions. the biological properties of the recombinant viruses were compared to parental rna-1 by determination of growth characteristics in tissue culture and in embryonated chicken eggs. as the cleavage site of f protein was modified to a monobasic motif from the dibasic amino acid motif, both the rmna-1 and rmna-vp3 cannot be propagated as well as rna-1 in tissue culture (data not shown). the growth kinetics of the recombinant viruses were determined and compared in embryonated chicken eggs at different time points after inoculation. a 50% tissue culture infective dose (tcid 50 ) of each virus was determined in df-1 cells by immunofluorescence assay. the results showed that both rmna-vp3 and rmna-1 have similar growth patterns compared to parental rna-1. at 60 h postinoculation, rna-1 and rmna-1 reached the maximum titers of 10 8.25 tcid 50 /ml and 10 8 tcid 50 /ml respectively, whereas the maximal viral titer of rmna-vp3 achieved at 72 h post-inoculation and was approximately 20-fold lower (fig. 4a) . the genetic stability of rmna-vp3 was also examined by serial passage 10 times in 10-day-old embryonated spf chicken eggs as described previously. rt-pcr confirmed the presence of the vp3 gene in each passage, and sequence analysis showed that the vp3 gene remains unchanged during 10 passages. also the mutant sequence at the cleavage sites in the f gene were proved to be preserved and stably maintained after serial passage through chicken embryos by dna sequencing (data not shown). the pathogenicity of recombinant ndv expressing the vp3 protein was evaluated by the mdt and icpi assays (fig. 4b) . the value of mdt exceeds 168 h and the icpi was 0. these results show that rmna-vp3 continues to keep the characteristics of the vector virus rmna-1. fig. 3 . analysis of expression of the vp3 protein by rmna-vp3. (a) immunofluorescence analysis of vp3 protein expression. bhk-21 cells were infected with rmna-1 or rmna-vp3 at an moi of 0.01. the infected cells were fixed and probed with chicken serum against ndv and horse serum against gpv and then incubated with a fitcconjugated goat anti-chicken antibody and an alexa fluor 555-conjugated rabbit anti-horse igg. dapi (sigma) was used for cell nucleus staining. cells were analyzed by using a confocal laser microscope. (b) western blot analyses of vp3 expression. lysates of df1 cells infected with rmna-vp3 or rmna-1 were incubated with chicken serum anti-ndv, rabbit anti-vp3 polyclonal antibody or mono-clonal antibody against ␤-actin. binding was visualized with 3, 3-diaminobenzidine reagent after incubation with peroxidase-conjugated secondary antibodies. the locations of marker proteins are indicated on the left and the antiserum or antibody used is indicated on the right. to investigate the immunogenicity of the recombinant rmna-vp3, groups of 10 4-day-old goslings were inoculated with rmna-1, rmna-vp3, or pbs. during the experimental period, the birds inoculated with the recombinant virus appeared healthy without any signs of vaccine side-effects. serum samples of the birds were collected before and after vaccination. the antibody titres against gpv were determined by virus neutralization test. at 3 weeks after the first dose, the mean titer of gpv na in the blood of goslings inoculated with rmna-vp3 was 3.25log 2 (fig. 5a) . at 3 weeks after the second dose, the mean titer reached up to 6log 2 . then the mean titer of 5.25log 2 was detected at 9 weeks after first immunization and still maintained a titer of 5.25log 2 until 12 weeks. no gpv-specific antibodies were detected from goslings in the rmna-1 or pbs vaccine control group after either initial or boost vaccination. meanwhile, ndv nas were detected in all serum samples (fig. 5b) . the two groups of goslings inoculated with rmna-1 or rmna-vp3 displayed no significant differences in ndv vnas antibody titers (p > 0.05). the mean titers of 4.5log 2 in rmna-vp3 group and 4.75log 2 in rmna-1 group were detected at 3 weeks after the first dose. three weeks after the second dose, the mean titer for the two groups reached 10.5log 2 and 10log 2 , respectively, showing substantial boost responses. after 9 weeks, the antibody titers are maintained at 7.25log 2 in rmna-vp3 group and at 7.75log 2 in rmna-1 group, and at 12 weeks after vaccination the mean titers is still 6.25log 2 and 6.75log 2 respectively. fig. 5 . immunogenicity evaluation in goslings. groups of 10 goslings were inoculated subcutaneously with 10 6 eid50 of rmna-1 or rmna-vp3 or with pbs as a control in a 0.2-ml volume respectively. at 3 weeks after initial vaccination, the goslings received a second dose. the serum samples from goslings were collected prior to vaccination and at different times after vaccination and the vna titers against gpv (a) and ndv (b) were detected. in this study, we generated a recombinant rmna-vp3, expressing gpv vp3 using a modified goose-origin ndv na-1 as an avirulent, safe, and effective vaccine vector, and evaluated its potential as a bivalent vaccine against gosling plague and nd in goslings. the results of the gosling immunizing experiment show that rmna-vp3 could induce strong na responses to gpv and ndv simultaneously. this is the first study to demonstrate that a highly virulent ndv could serve as vaccine vector expressing exogenous protein after modification by changing the multi-basic cleavage sites of the f protein to the dibasic sequence of avirulent strain lasota. the recombinant rmna-vp3 has the potential to serve as a bivalent live vaccine against gosling plague and nd. gpv is a pathogen of major economic importance to the goose industry worldwide. currently, prophylactic vaccination is used as protection against gpv infection. two types of immunization strategies were adopted in field: vaccination of breeder geese before laying, by which the offspring was protected depending on maternally derived antibodies in the egg yolks, and vaccination of susceptible goslings, where the virulence of the vaccine strains needs to be much lower than that of strains used to immunize parent flocks. preparation of vaccines with varying levels of virulence makes the production very costly and labor-effective. therefore, the development of a safe, effective and low-cost vaccine is necessary. among the possible strategies, one of the most promising is the live viral vectored vaccines. the genomes of gpv encode non-structural and structural proteins, including three capsid proteins of vp1, vp2, and vp3. the vp3 protein is the most abundant of the three core proteins in purified virions and can induce neutralizing antibodies with high immunogenicity (le gall-reculé et al., 1996; le gall-recule and jestin, 1994) . therefore, vp3 may be a suitable candidate antigen for developing a vaccine against gpv. as a viral vector, ndv has outstanding advantages. firstly, ndv grows to very high titers in many cell lines and embryonated eggs, which allows cost-effective and easy manufacture of the vaccine. secondly, ndv has a simple genome encoding only a few proteins than other viruses such as fowl pox virus and adenovirus. thirdly, the live ndv vaccines can induce not only humoral immunity and cellular immunity simultaneously, but also a strong mucosal immunity, which makes this expression vector more attractive (huang et al., 2003) . ndv lentogenic vaccine strains such as lasota can be developed in vectors directly; while mesogenic strains should be modified by changing the multi-basic cleavage site sequence of the f protein to the dibasic sequence of avirulent strains because these strains might cause disease in poultry and most strains are classified as select agents (kim et al., 2014) . recently, it has been demonstrated that the virulent isolates also can be attenuated by modification in the same way to serve as a live-attenuated vaccine candidate (hu et al., 2009 (hu et al., , 2011 xiao et al., 2012) . ndv has a single serotype, but many genotypes. to date, ndv had caused four major nd panzootics and different genotypes contributed to each panzootic. epidemiological studies revealed that genotype vii ndv strains were circulating predominantly worldwide after the fourth panzootic (huang et al., 2004a; miller et al., 2009 miller et al., , 2010 and found to be responsible for the nd outbreaks in asia, africa, middle east, and south america (munir et al., 2012; perozo et al., 2012; wang et al., 2006) . these strains also caused many outbreaks of nd in domestic geese with high mortality and morbidity in southern and eastern china since the late 1990s, which was referred as goose paramyxovirus (gpmv) in earlier reports (cai et al., 2011; jinding et al., 2005) . however, the genotype ii vaccine strains such as lasota or hitcher b1 remain in current and widespread use, and the differences in their antigenicity and genotypes between the presently circulating strains and commercial vaccine strains are considered the main reasons for the nd outbreaks in vaccinated poultry flocks (liu et al., 2003; miller et al., 2007) . recent work has demonstrated that genotype-matched vaccine strains provide better protection than conventional vaccines in terms of reducing virus shedding and transmission following challenge with ndv virulent virus (hu et al., 2009 (hu et al., , 2011 xiao et al., 2012) . in 1999, a highly virulent genotype vii ndv strain na-1 was isolated from outbreaks in goose flocks in our lab (xu et al., 2008) . to evaluate whether this genotype vii isolate could be used as a vaccine vector for geese we generated a recombinant virus rmna-vp3 expressing vp3 protein of gpv after modifying the polybasic f cleavage site of na-1 to the dibasic motif of lasota. it is interesting that the modified na-1 which has exactly the same f protein cleavage site as avirulent lasota did not form syncytium in virus-infected cells as lasota did. even in the presence of exogenous protease no syncytium was observed (fig. 1c) . these results are repeatedly confirmed and consistent with previous research on the mutation of a highly virulent indonesian strain (xiao et al., 2012) . it is demonstrated that the syncytium formation is not determined by the motif of the f protein cleavage site alone; some other factors may also be involved. anyway, the inability to induce the formation of syncytia, may be indicative of a highly attenuated of the modified viruses comparing with lasota. as a feasibility study, one concern that requires investigation is that whether the mutated f protein cleavage site of vector virus might revert to a wt or wt-like sequence after serial passage. to evaluate its heredity stability, the recombinant virus rmna-1 and rmna-vp3 were passaged continuously in chicken embryos and the sequence of f protein cleavage site was determined every generation. the results demonstrated that the mutated f protein cleavage site always remained stable after 10 passages in chicken embryos. the safety issue of the recombinant viruses should be further evaluated through its serial passage in geese prior to its use in field. immunogenicity of the recombinant virus rmna-vp3 was evaluated in goslings. a detectable immune response against gpv was evidenced after the first immunization dose, and after the second immunization administered 3 weeks later, significant booster responses to vna of gpv were induced. moreover, the antibody level did not drop markedly during at least the first 12 weeks. meanwhile, the neutralizing antibodies against ndv vaccine vector were also detected. the mean titer induced by rmna-vp3 was comparable to that induced by rmna-1, indicating that this recombinant virus holds its characteristic in stimulating effective immune response against ndv infection. in summary, as a new type of engineered vaccine the recombinant virus rmna-vp3 has several advantages over the existing gpv vaccines. firstly, the recombinant rmna-vp3 will be highly economical for the poultry industry, because rmna-vp3 can be cultured in many cell lines and embryonated eggs. secondly, the recombinant virus can potentially overcome maternal antibody interference. therefore, it can be used for vaccination with or without maternal antibody. even in the presence of the maternal antibody, it can be used to enhance protection in the early danger period of gosling life. finally, this recombinant virus would be developed an in-ovo vaccine against gpv after further modified as describe in in-ovo vaccination against ibdv and ndv elsewhere (ge et al., 2014) . (i) all the authors have agreed to its submission and are responsible for its contents; (ii) all the authors have agreed that zhuang ding may act on their behalf regarding any subsequent processing of the paper; (iii) the authors have declared that no competing interests exist. detection and differentiation of newcastle disease virus (avian paramyxovirus type 1). avian pathol newcastle disease and other avian paramyxoviruses newcastle disease, other avian paramyxoviruses, and pneumovirus infections genetic characterization and evolutionary analysis of 4 newcastle disease virus isolate full genomes from waterbirds in south china during antigenic and immunogenic investigation of the virulence motif of the newcastle disease virus fusion protein a viral disease of goslings. i. epidemiological, clinical, pathological and aetiological studies newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of h5n1 highly pathogenic avian influenza virus protect against virus challenge in monkeys immunization of primates with a newcastle disease virus-vectored vaccine via the respiratory tract induces a high titer of serum neutralizing antibodies against highly pathogenic avian influenza virus newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous h5n1 avian influenza viruses novel in-ovo chimeric recombinant newcastle disease vaccine protects against both newcastle disease and infectious bursal disease isolation and identification of goose parvovirus in the uk studies with a duck embryo adapted goose parvovirus vaccine a vaccine candidate of attenuated genotype vii newcastle disease virus generated by reverse genetics generation of a genotype vii newcastle disease virus vaccine candidate with high yield in embryonated chicken eggs genomic sequence of an isolate of newcastle disease virus isolated from an outbreak in geese: a novel six nucleotide insertion in the non-coding region of the nucleoprotein gene recombinant newcastle disease virus as a vaccine vector a recombinant newcastle disease virus (ndv) expressing vp2 protein of infectious bursal disease virus (ibdv) protects against ndv and ibdv epidemiologic investigation of an outbreak of goose parvovirus infection in sweden a goose-sourced paramyxovirus isolated from southern china goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose modified newcastle disease virus vectors expressing the h5 hemagglutinin induce enhanced protection against highly pathogenic h5n1 avian influenza virus in chickens immunological aspects of derzsy's disease in goslings expression of muscovy duck parvovirus capsid proteins (vp2 and vp3) in a baculovirus expression 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protein is a major determinant for virulence biological and phylogenetic characterization of a genotype vii newcastle disease virus from venezuela: efficacy of field vaccination a simple method of estimating fifty per cent endpoints virus hepatitis of geese. ii. host range of goose hepatitis virus an outbreak of goose parvovirus infection in japan cloning of the genome of a goose parvovirus vaccine strain syg61v and rescue of infectious virions from recombinant plasmid in embryonated goose eggs development of a reverse genetics system based on rna polymerase ii for newcastle disease virus genotype vii genotyping of newcastle disease viruses isolated from 2002 to 2004 in china generation by reverse genetics of an effective, stable, live-attenuated newcastle disease virus vaccine based on a currently circulating, highly virulent indonesian strain genomic analysis of newcastle disease virus strain na-1 isolated from geese in china the research was supported by the national natural scientific fund (31272561, 31472195, and 31402195), and chinese special fund for agro-scientific research in the public interest (201303033). key: cord-311628-ep795pil authors: fu, yu; li, jinming title: a novel delivery platform based on bacteriophage ms2 virus-like particles date: 2016-01-04 journal: virus res doi: 10.1016/j.virusres.2015.08.022 sha: doc_id: 311628 cord_uid: ep795pil our objective here is to review the novel delivery platform based on bacteriophage ms2 virus-like particles (vlps), including introduction to their structure, their potential as a delivery platform, and their expected use in medicine and other fields. bacteriophage ms2 vlps are nanoparticles devoid of viral genetic material and can self-assemble from the coat protein into an icosahedral capsid. as a novel delivery platform, they possess numerous features that make them suitable and attractive for targeted delivery of rnas or dnas, epitope peptides, and drugs within the protein capsid. in short, as a novel delivery platform, ms2 vlps are suitable for delivery of targeted agents and hold promise for use in diagnostics, vaccines, and therapeutic modalities. virus-like particles (vlps)-nanoparticles devoid of viral genetic material that are usually formed by one or several structural proteins-have external structure and antigenicity similar to those of native viruses. recently, considerable efforts have been devoted to construction of vlps, making them attractive as possible http://dx.doi.org/10.1016/j.virusres.2015.08.022 0168-1702/© 2015 elsevier b.v. all rights reserved. nanocarriers (carrico et al., 2008; kovacs et al., 2007; wu et al., 2005) . combining a good safety profile with strong immunogenicity, vlps are expected to gain widespread use in numerous fields, such as in vitro diagnostics, vaccines, and therapeutic modalities. before 2013, more than 110 vlps had been constructed from viruses belonging to 35 families (zeltins, 2013) , among which ms2 vlps-an icosahedral capsid self-assembled from 180 copies of a single coat protein (cp) and measuring 22-29 nm in diameter-represent a novel delivery platform and became a hot research area within several years because of their attractive features. first, ms2 vlps can offer effective, convenient ways to package and deliver rnas or dnas, epitope peptides, and drugs within bacteriophage capsids wei et al., 2009; wu et al., 2005; zhang et al., 2015a,b) . second, they can have excellent adjuvant properties and thus induce innate and cognate immune responses. additionally, they are safer and more effective than traditional vaccines derived from attenuated or inactivated infectious viral strains. furthermore, they are capable of tissue-specific targeting after modification with a ligand, and this feature can ensure that the targeted agents carried by ms2 vlps are more effective. clinical applications of ms2 vlps are perhaps their most exciting feature. their main applications belong to the field of vaccine development. a series of research findings showed that an ms2 vlp-based vaccine can effectively induce innate and cognate immune responses and can be used as a specific preventive intervention in some diseases, such as foot-and-mouth disease (bittle et al., 1982; dong et al., 2015; van lierop et al., 1992; wong et al., 2000) , prostate cancer (li et al., 2014) , and illnesses caused by human papilloma virus (hpv) (tumban et al., 2012) . another important application of ms2 vlps is therapeutic modalities because these particles can deliver drugs or biologics to a specific tissue (ashley et al., 2011; pan et al., 2012a; yao et al., 2015) . moreover, by packaging specific rna molecules into the recombinant ms2vlps, researchers can construct so-called armored rnas. because their structure is similar to that of the native virus and their concentration is already calibrated, the armored rnas can be used as "standards" or "calibrators" for detection of the corresponding native viruses and for validation of an experiment with the help of a "control" (das et al., 2006; zhan et al., 2009) . our objective here is to review the novel delivery platform based on bacteriophage ms2 vlps, including introduction to their structure, their potential as a delivery platform, and their expected use in medicine and other fields. ms2 is an icosahedral rna bacteriophage with the triangulation number t = 3, and its crystallographic structure was solved at 2.8 å resolution (fig. 1a ) (borodavka et al., 2012; golmohammadi et al., 1993; valegård et al., 1990 valegård et al., , 1991 . the genome of bacteriophage ms2 is a positive-sense single-stranded rna molecule of 3569 nucleotides and encodes four proteins: the major coat protein (cp), the maturation protein (a-protein), the replicase (an rna polymerase necessary for genome multiplication), and the lysis protein (fig. 1b) . cp makes up the bulk of the bacteriophage, assembling into an icosahedral structure ∼26 nm in diameter. cp molecules first form a dimer. the dimer can bind to an rna hairpin and then undergoes an allosteric conformational change in the fg loop region (bleckley and schroeder, 2012) , forming an asymmetric a/b dimer rather than a symmetric c/c dimer (borodavka et al., 2012; , 2007) . the fg loops of the b conformation of cp surround the fivefold vertices of the icosahedron, and the a and c conformations interdigitate at the threefold vertices of the icosahedron, thus forming an icosahedron of 60 a/b dimers and 30 c/c dimers valegård et al., 1990) . eventually, bacteriophage ms2 can form a simple ribonucleoprotein structure composed of 180 cp molecules. the three quasiequivalent conformers of cp differ primarily in the conformation of the fg loop. the loops of the a subunit are similar to those of subunit c because they have extended conformations and interact with each other at the capsid's threefold axes, but in the b subunit, the loop is bent back toward the globular core of the subunit, interacting with the other b loops at the particle's fivefold axes (fig. 1c ) (valegård et al., 1990) . both in vitro (mastico et al., 1993; stockley et al., 1993) and in vivo (knolle and hohn, 1975) , cp molecules are capable of selfassembling into capsids at t = 3. in very rare cases, aberrant forms, such as capsids at t = 1, have also been documented (knolle and hohn, 1975) . 2.2. self-assembly of bacteriophage ms2 as discussed above, we know that bacteriophage ms2 can assemble from 180 copies of cp into a monodisperse, icosahedral capsid with a diameter of 22-29 nm (mastico et al., 1993; stockley et al., 1993) . generally, the final fully formed capsids represent the state of lowest free energy of the component proteins and nucleic acids . therefore, after its assembly, the final fully formed ms2 vlp has a stable structure, and this feature forms the basis for delivery of all kinds of targeted agents by ms2vlps. regarding the self-assembly of ms2vlps, stockley et al. (1994) reported that the assembly of this bacteriophage can be achieved via interaction between the bacteriophage cp and a 19-nucleotide sequence-specific ms2cistron, the so-called pac site. that is to say, the ms2 rna genome has a special functional structure when encapsidated inside the virus particle. borodavka et al. (2012) and rolfsson et al. (2010) showed that cps and rna act as mutual chaperones by altering each other's conformation because of formation of the complex in a context-specific way, as the capsid shell grows from an initiation complex. toropova et al. (2008 toropova et al. ( , 2011 reported that 90% of the encapsidated rna is well ordered and forms two layers inside the icosahedral viral particle. dykeman et al. (2011) stated that the symmetry of the icosahedron and the locations of the rna hairpins in the a/b dimers point to possible assembly mechanisms for aseries of hairpins in the ms2 genome. thus, according to the above findings, the cp-binding rna hairpins may play an important role in the assembly of the virus particle with the correct size and symmetry (basnak et al., 2010; koning et al., 2003; morton et al., 2010; stockley et al., 2007) . nonetheless, many viral cps can assemble in the absence of rna, i.e., around nonviral rna or polyanions (cadena-nava et al., 2011; hu et al., 2008) and even nanoparticles (chen et al., 2006) . the accepted mechanisms underlying the assembly of ms2 vlps mostly represent protein-centric models. in other words, viral proteins are the key component of the process of assembly of ms2 vlps (zlotnick and mukhopadhyay, 2011). b acteriophage ms2 is an excellent model for studies on the factors that can affect capsid stability . several factors, including physicochemical ones, such as temperature, ph, and ionic strength or structural ones, such as mutationsin cp, can affect ms2 vlp stability to some extent. in one experiment with temperature stability, plevka et al. (2009) found that the melting temperature of the ms2 phage and wild-type vlps is 66 • c. stonehouse and stockley (1993) reported that these particles can withstand temperatures up to 68 • c. no capsids are detectable, however, if the temperature is increased by just l • c. in order to determine the role of ms2 rna in capsid stability, the experiment was repeated with wild-type phage particles, prepared for electron microscopy in the same way as the empty capsids. a similar result was obtained for the denaturation temperature, showing that rna cannot exerta substantial influence on thermal stability of the capsid. the influence of salts and natural organic matter on the stability of bacteriophage ms2 was studied by mylon et al. (2010) . they found that aggregation of ms2 cannot be induced within a reasonable kinetic time frame in the presence of monovalent electrolytes, such as licl, nacl, and kcl, and this situation is observed even at salt concentrations greater than 1.0 m. on the other hand, ms2 can aggregate in a solution containing divalent electrolytes (e.g., ca 2+ ); this finding points to transition from repulsive to attractive interactions among ms2 virus particles as monovalentions are replaced by divalent ones. in addition to thermal and salt-related characteristics of ms2vlps, ph may have a strong physicochemical influence on stability of ms2vlps. some studies showed that bacteriophage ms2 can stay stable at ph 7.0 but becomes less stable as ph decreases (da poian et al., 1993; lago et al., 2001; sugiyama et al., 1967) . additionally, early in 1970, dubovi and akers (1970) found a decrease in the recovery of phage ms2 after prehumidification of anaerosol sample before collection, if no peptone was added to the spraymedium. stonehouse and stockley (1993) indicated that a wide range of amino acid substitutions can be tolerated by ms2vlps, even at intermolecular interfaces, with only modest effects on overall stability. some other substitutions, however, could result in problems with cp folding or stability. thus, a change in the amino acid sequence of this protein may lead to a functional change. as to the effects of site-directed mutagenesis of cp on the assembly of ms2 vlps, a study conducted by stonehouse and stockley (1993) showed that three mutants, l86s, g74a, and c46r, have thermal stability identical to that of wild-type cp. another group of mutants, t59s, y129c, and c46w, showed a small but significant 2-4 • c increase in the denaturation temperature. the third group, p78n and e76d, showed a 2 • c decrease in the denaturation temperature. in another experiment, peabody (2003) found that only the t15c mutant among five selected amino acid substitutions (g13c, g14c, t15c, d114c, and g115c) can produce significant quantities of soluble cp that assembles into particles with the same electrophoretic mobility as those of the wild-type virus. additionally, in comparison with the wildtype, the capsid stability decreases when mutations of cp are located in the fg loop (axblom et al., 1998; peabody and ely, 1992; stonehouse et al., 1996) . by means of genetic engineering, two or more different polypeptides can be recombined easily. early in 1996, peabody and lim (1996) fused two copies of the cp gene by removing the stop codon from the first gene and the second residue (serine) from the second copy. the final covalent cp dimer (ccpd) gene encoded 257 amino acid residues. the ccpd molecules were then found to assemble into vlps. these particles are indistinguishable from the vlps assembled from the wild-type cp at the resolution of an electron microscope. another study showed that both wild-type and the ccpd particles are more stable than the dimers of the wild-type subunits (peabody, 1997; peabody and lim, 1996) , even though they appear to have a similar diameter: 280 ± 20 å (plevka et al., 2008) . after the initial reports about ms2vlps as a form of a nonviral delivery system wei et al., 2009; wu et al., 2005) that can be targeted to specific cell types via chemical or covalent conjugation with ligands (ashley et al., 2011; brown et al., 2002) , bacteriophage ms2 vlps have been shown to have many features as a delivery platform. on the one hand, these particles can be prepared easily by means of the recombinant-protein technology ). on the other hand, the ms2 capsid, which interacts with the pac site of rna or dna, can package the target rna or dna located at the 5 terminus of the pac site and thus protect the target rna or dna from degradation by nucleases zhan et al., 2009; zhang et al., 2015a,b) . capable of offering effective, convenient ways of packaging all kinds of agents and ways of delivery to target cells, ms2 vlps hold great promise as a novel delivery platform for diagnostic, vaccine-related, and therapeutic purposes (fig. 2) . as early as 1990, studies on mrna vaccines became a hot area of research when direct injection of naked synthetic mrna had proven to be feasible and could induce an antigen-specific immune response in clinical settings. therefore, vaccine-based mrna opens up a new avenue for the development of nextgeneration vaccines (rittig et al., 2011) . in addition, mrna vaccines have also been considered as an efficient alternative to protein and dna vaccines not only because of the good antigenic potenms2vlps possess several features that make them attractive as potential nanocarriers for passive and targeted drug delivery and can be effective against certain diseases (in animal models). adapted from pan et al. (2012a,b) . (e) ms2 vlp-based rnas and dnas (armored rna or dna). owing to a structure similar to that of the native rna virus and good biosafety, recombinant ms2 vlps are widely used as "control", "standard", and "calibrator" in assays for viruses. tial and immunostimulatory properties (scheel et al., 2004) but also owing to safety, low cost, and high purity (van lint et al., 2011) . it is known that naked mrnas are easily degraded by rnases, and this instability limits their practical applications (ulmer et al., 2012) . several approaches, such as liposome-entrapped mrna (martinon et al., 1993) , self-replicative mrna (ying et al., 1999) , mrna-coated gold particles (qiu et al., 1996) , and in vitro mrnatransfected dendritic cells (dcs) (gardner et al., 2012; perche et al., 2011) have been developed to improve intracellular stability and translational efficacy of mrna. nevertheless, these approaches do not offer a perfect carrier for delivery. first, the greatest challenge is still the relative instability of mrna owing to degradation by prevalent rnases. besides, the high costs of gold particles and the gene gun with inconvenient settings for administration to experimental animals hinder the development of ballistic delivery of mrna for vaccination. furthermore, in vitro mrna-transfected dcs have been used for cancer treatment in the clinic and turned out to be expensive (van lint et al., 2013) , let alone the scarcity of the source of autologous dcs. all of these disadvantages hamper practical applications and development of mrna vaccines. thus, there is a high demand for a simple, safe, efficient, stable, and inexpensive method for the delivery of mrna. an ms2 vlp-based rna approach can be utilized for development of an mrna vaccine resistant to rapid extracellular degradation ( fig. 2a) . because of the highly specific interaction between cp and the pac site, the target mrna can be packaged into cp of bacteriophage ms2 and kept stable within 18 h (uhlenbeck, 1998) . therefore, mrna delivery in vivo, which does not require complicated procedures and can directly target antigen-presenting cells, may be preferable. several studies have shown that an mrna vaccine is effective at protecting from prostate cancer when the ms2 delivery platform is used. johnson et al. (2006) demonstrated that prostatic acid phosphatase (pap) mrna vaccine could induce a pap-specific antibody and t-cell immune response, whereas a pap dna vaccine could not do the job without an adjuvant: granulocyte-macrophage colonystimulating factor (gm-csf). mockey et al. (2007) reported that an ms2vlp-based hpap-gm-csf mrna vaccine-which first showed its prophylactic potential in protection of mice from prostate cancer-proved to have a better preventive effect than a liposomeencapsulated mrna vaccine (mockey et al., 2007; zhou et al., 1999) and is as effective as a naked mrna vaccine (kreiter et al., 2010) . nevertheless, the encapsulated mrna is more stable than naked mrna under the protection of cp. a study by li et al. (2014) indicated that an ms2 vlp-based hpap-gm-csf mrna vaccine could suppress prostatic-tumor growth in c57bl/6 mice, suggesting that this vaccine may evoke an efficient immune response within a short period and is a promising treatment for prostate tumors. in the above studies, the ms2vlp-based hpap mrna vaccine was inferior to the hpap-gm-csf mrna vaccine; this observation means that gm-csf has good adjuvanticity and is effective at activating the immune system. in immunology, an epitope, also known as an antigenic determinant, is the part of an antigen that is recognized by the immune system, in particular, by antibodies, b cells, or t cells, and then elicits an adaptive immune response. as a nonviral delivery system, ms2 vlps can be designed to deliver an epitope peptide for clinical purposes. first, after insertion of a dna oligonucleotide into a unique site of the ms2cp gene, ms2 vlps can present the epitope peptide to the immune system (mastico et al., 1993; peabody et al., 2008; wei et al., 2008) . it is, of course, not difficult to elicit a desired antibody response by a specific epitope peptide (meloen et al., 2000; partidos and steward, 2002) . furthermore, vlps can present viral antigens as dense repetitive arrays, which are known to be highly stimulatory to immunocytes (bachmann et al., 1993; milich et al., 1997) . additionally, compared to other vlp platforms ms2 vlps are advantageous because of their good stability and proper size for effective presentation of viral antigens (bundy et al., 2008) . when ms2 vlps are used as a platform for the presentation of epitopes, in the majority of cases, this approach is implemented through modification of the vlp gene sequence, so that fusion proteins of vlp components with the foreign epitope are assembled into vlps during the expression of these proteins. this feature of presenting targeted epitopes to the immune system (fig. 2b ) may lead to widespread use of ms2vlps in clinical practice. one known clinical application of a vlp vaccine is protection from foot-and-mouth disease virus (fmdv). fmdv's genome is a single-stranded positive sense rna that contains a unique open reading frame encoding four structural proteins (vp1-vp4) (acharya et al., 1989) . studies confirmed that the epitope peptide vp1 141-160 (also known as ep141-160) can induce not only neutralizing antibodies but also fmdv-specific t cells (bittle et al., 1982; dong et al., 2015; van lierop et al., 1992; wong et al., 2000) . the above studies mean that ep141-160 vlps may offer protection from fmdv. an ms2vlp vaccine can also be used for prevention of infection with hpv. the current hpv vaccines are derived from vlps consisting of the hpv major capsid protein l1. to develop secondgeneration hpv vaccines based on the hpv minor capsid protein l2, tumban et al. (2012) inserted hpv l2 peptides into the n terminus of cp of bacteriophage ms2 via genetic recombination and found that the vlp-based vaccine targeting hpv l2 can elicit broadly cross-reactive and cross-protective antibodies to heterologous hpvs. thus, with the ms2 vlp platform, the recombinant l2-vlps could serve as the basis for a broadly protective secondgeneration hpv vaccine. mirna is a type of 20-to 24-nucleotide noncoding rna that can bind to the 3 untranslated region of a targeted mrna, causing its degradation or translational suppression. the powerful generegulatory role of mirnas is well recognized at present, and some mirna-based therapeutic strategies have shown effectiveness in experimental tumor models. this type of treatment may be more effective because the regulatory mechanism of a single mirna affects hundreds of targets in multiple pathways rather than one or two transcripts (selbach et al., 2008) . owing to many limitations, however, such as rapid extracellular degradation and the necessity of a reliable delivery system capable of transferring mirnas into the intracellular space without cytotoxicity (davidson and mccray, 2011; wang et al., 2010) , the development of an efficient mirna delivery system has been a challenge. a number of approaches to mirna delivery involving the use of viral or nonviral vector systems have also been explored (davidson and mccray, 2011) . nonetheless, the typically low transduction efficiency, possible cytotoxicity, and integration-induced tumorigenesis are always concerns when plasmid or viral vectors are used for mirna expression (davidson and mccray, 2011) . therefore, as a novel attractive delivery system, ms2vlps recently became a focus of research attention in the field of mirna delivery. after delivery by ms2 vlps, mirnas can be used for gene therapy (fig. 2c ). studies showed that several known mirnas, such as mir-21, mir-125, and mir-146a, have potent capacity for gene regulation (ceribelli et al., 2011; dai and ahmed, 2011) . among these mirnas, mir-146a-an mirna important for negative regulation of acute responses during activation of innate immunity-acts as an effective inhibitor of autoimmunity, myeloproliferation, and cancer (boldin et al., 2011; li et al., 2010; tang et al., 2009) . overexpression of mir-146a can effectively suppress the expression of its target genes and proteins (pan et al., 2012a) . tang et al. (2009) reported that expression of mir-146a is reduced in peripheral blood mononuclear cells (pbmcs) of patients with systemic lupus erythematosus (sle). boldin et al. (2011) found that ablation of the mir-146a gene in mice results in several adverse immune-system-related phenotypes leading to premature death. in this study they also demonstrated that mir-146a levels in mice, after delivery via ms2vlps, first significantly increase, and then this mirna exerts its therapeutic action on lupus-prone mice via inhibition of production of a pathogenic autoantibody. in other words, this treatment with ms2-mir146a vlp can have profound effects on lupus-prone bxsb mice by upregulating mature mir-146a, which causes significant downregulation of autoantibodies, of total igg, and of proinflammatory cytokines, including interferon␣, il-1␤, and il-6. pan et al. (2012b) confirmed that the induction of dysregulated mirnas by an ms2 vlp-based delivery system may lead to novel therapeutic interventions for sle. moreover, a study by yao et al. (2015) demonstrated that osteoclastogenesis can be inhibited in cell precursors by means of an ms2 system that transports mir-146a into human pbmcs. as a result, the number of osteoclasts decreased markedly, and a bone resorption assay demonstrated inhibition of their activity. thus, this mir-146a delivery system can be used to induce overexpression of mir-146a and to inhibit the differentiation and function of osteoclasts. ms2 vlps possess several features that make them attractive as potential nanocarriers for passive and targeted drug delivery (fig. 2d ). during the self-assembly of ms2cp, ms2 vlps can be loaded with drugs. even for the drugs with a greater-than-average molecular weight, ms2vlps have sufficient volume of the interior cavity and can deliver such a drug to specific target cells. in the study by ashley et al. (2011) , macromolecular drug candidates, such as doxorubicin, cisplatin, and 5-fluorouracil were encapsidated specifically and efficiently inside a protective cp shell by first covalently linking them to the rna operator that triggers in vitro capsid assembly from disassembled cp dimers. these drug packages can then be directed toward hepatocellular carcinoma by covalent modificationof the outside surface with ligands for receptor-mediated endocytosis and eventually can selectively kill the hepatocellular carcinoma cells. in another experiment, brown et al. (2002) tested cytotoxicity of 5fdu/anti-df3-anucleotide analog combined with the anti-df3 monoclonal antibody packaged in synthetic virions-against zr-75-1 breast carcinoma cells, which over express the df3 antigen. the results showed that 5fdu/anti-df3 is slightly more toxic than free 5fdu at a lower concentration, suggesting that the synthetic virions enhance the toxicity of the oligonucleotide by protecting it from degradation by nucleases in the serum or by enhancing its uptake by zr-75-1 cells. in some diagnostic methods, an absolute concentration of a specific rna has to be detected with high sensitivity. a specific type of rna, calibrated to known concentrations, plays a core role in the reliable detection of rna. this kind of rna, on the one hand, may serve as a "control" for validation of the experiment; on the other hand, it can serve as a quantitative "standard" or "calibrator" against which the samples are measured. on the other hand, this kind of rna is easily degraded by rnases in vitro, but it is possible to place rna in long-term storage to prevent the degradation. in recent years, recombinant ms2 vlps that are packaged with rna, the so-called armored rnas, were developed for use as "controls", "standards", and "calibrators" (stevenson et al., 2008) (fig. 2e ) in diagnostic procedures for the following reasons. first, possessing structure similar to that of the native rna virus, ms2vlps can serve as a substitute for the native virus during the extraction procedureand thus dramatically reduce the error rate of the detection of the virus. additionally, the rnas in the particles are rnase resistant by virtue of their encapsulation within ms2cp. moreover, they are not pathogenic during the experiment because these particles cannot replicate by themselves. armored rnas based on ms2 vlps are now used for the detection of various viruses, such as human immunodeficiency virus (pasloske et al., 1998) , severe acute respiratory syndrome coronavirus (drosten et al., 2001) , enteroviruses (beld et al., 2004; donia et al., 2005) , avian influenza virus (das et al., 2006) , measles virus (zhang et al., 2015a,b) , and ebola virus . although armored rna is a good candidate for an rnaseresistant positive "control" or "standard", its application is limited by the maximal length of the exogenous rna that can be packaged into vlps by means of the existing armored-rna technology. nevertheless, zhan et al. (2009) constructed a long armored rna containing a 3034-base exogenous rna, which can be used for detection of hiv-1 in a one-plasmid double-expression system. it is completely resistant to rnases, remains stable in normal human edta-preserved plasma at 4 • c for at least 6 months, and yields reproducible linear results in the versant hiv-1 rna 3.0 assay. additionally, in a recent study, zhang et al. (2015a,b) produced armored hepatitis b virus and hpv dna for use in nucleic-acid assays; these constructs were confirmed to be stable, homogeneous, noninfectious, nuclease resistant, and safe for shipping. in that study, double-stranded dna 1.3, 3.0, 3.5, and 6.5 kb long were successfully packaged into ms2viral capsids with high reassembly efficiency. this is the first successful use of bacteriophage ms2 as a platform for encapsulation of double-stranded dna. this armored dna may become an ideal "control" and "standard" for use in clinical laboratory tests and diagnostics, holding promise for varied applications, especially as a new platform for production of assay standards for dna viruses. according to the above properties of ms2 vlps, these particles seem to be ideal carriers for delivery of a variety of agents. it should be noted that when a large protein domain serves as an antigen, it may pose difficulty for display on ms2vlps because such an insertion may disrupt the vlp assembly. in that case, chemical conjugation may be a solution. another problem with the delivery of rna within ms2 vlps is that these vlps are immunogenic, and antibody responses to the particles may interfere with the delivery to the intended destination. bacteriophage ms2 vlps can self-assemble from its cp into an icosahedral capsid. these particles offer an effective and convenient way to package rnas, dnas, epitope peptides, and drugs into bacteriophage capsids, forming different kinds of vlps. ms2 vlps can not only deliver various kinds of agents with a good safety profile and strong immunogenicity but also ensure tissue-specific targeting, which is determined by the species of the virus. thus, ms2 vlps possess extensive prospects for practical applications. the most promising applications of ms2vlps are vaccine development and therapeutic modalities. besides, ms2 vlps can be used as "control" or "calibrator" in assays for native viruses. the three-dimensional structure of foot-and-mouth disease virus at 2.9 a resolution cell-specific delivery of diverse cargos by bacteriophage ms2 virus-like particles structure of phage fr capsids with a deletion in the fg loop: implications for viral assembly the influence of antigen organization on b cell responsiveness viral genomic single-stranded rna directs the pathway toward a t = 3 capsid highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-pcr with an armored rna internal control protection against foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence incorporating global features of rna motifs in predictions for an ensemble of secondary structures for encapsidated ms2 bacteriophage mir-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice evidence that viral rnas have evolved for efficient, two-stage packaging rna bacteriophage capsid-mediated drug delivery and epitope presentation escherichia coli-based cell-free synthesis of virus-like particles exploiting fluorescent polymers to probe the self-assembly of virus-like particles thermal stability of rna phage virus-like particles displaying foreign peptides oxidative coupling of peptides to a virus capsid containing unnatural amino acids micrornas in systemic rheumatic diseases nanoparticle-templated assembly of viral protein cages microrna, a new paradigm for understanding immunoregulation, inflammation, and autoimmune diseases reversible pressure dissociation of r17 bacteriophage: the physical individuality of virus particles development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse transcription-pcr with lyophilized reagents current prospects for rna interference-based therapies promising ms2 mediated virus-like particle vaccine against foot-and-mouth disease use of armored rna as a standard to construct a calibration curve for real-time rt-pcr taqman 5 -nuclease human immunodeficiency virus type 1 pcr assay with phage-packaged competitive internal control for high-throughput blood donor screening airborne stability of tailless bacterial viruses s-13 and ms-2 simple rules for efficient assembly predict the layout of a packaged viral rna dynamic allostery controls coat protein conformer switching during ms2 phage assembly all-atom normal-mode analysis reveals an rna-induced allostery in a bacteriophage coat protein sipuleucel-t (provenge) autologous vaccine approved for treatment of men with asymptomatic or minimally symptomatic castrate-resistant metastatic prostate cancer the refined structure of bacteriophage ms2 at 28 a resolution packaging of a polymer by a viral capsid: the interplay between polymer length and capsid size safety and immunological efficacy of a prostate cancer plasmid dna vaccine encoding prostatic acid phosphatase (pap) cold spring harbor visualization by cryo-electron microscopy of genomic rna that binds to the protein capsid inside bacteriophage ms2 dual-surface-modified bacteriophage ms2 as an ideal scaffold for a viral capsid-based drug delivery system intranodal vaccination with naked antigen-encoding rna elicits potent prophylactic and therapeutic antitumoral immunity probing the kinetics of formation of the bacteriophage ms2 translational operator complex: identification of a protein conformer unable to bind rna messenger rna vaccine based on recombinant ms2 virus-like particles against prostate cancer microrna-146a and human disease induction of virus-specific cytotoxic t lymphocytes in vivo by liposome-entrapped mrna multiple presentation of foreign peptides on the surface of an rna-free spherical bacteriophage capsid mimotopes: realization of an unlikely concept role of b cells in antigen presentation of the hepatitis b core influence of salts and natural organic matter on the stability of bacteriophage ms2 mrna-based cancer vaccine: prevention of b16 melanoma progression and metastasis by systemic injection of mart1 mrna histidylated lipopolyplexes the impact of viral rna on assembly pathway selection ms2 vlp-based delivery of microrna-146a inhibits autoantibody production in lupus-prone mice development of a microrna delivery system based on bacteriophage ms2 virus-like particles mimotopes of viral antigens and biologically important molecules as candidate vaccines and potential immunotherapeutics armored rna technology for production of ribonuclease-resistant viral rna controls and standards control of translational repression by protein-protein interactions complementation of rna binding site mutations in ms2 coat protein heterodimers immunogenic display of diverse peptides on virus-like particles of rna phage ms2 a viral platform for chemical modification and multivalent display subunit fusion confers tolerance to peptide insertions in a virus coat protein enhancement of dendritic cells transfection in vivo and of vaccination against b16f10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger rna crystal packing of a bacteriophage ms2 coat protein mutant corresponds to octahedral particles structure and stability of icosahedral particles of a covalent coat protein dimer of bacteriophage ms2 gene gun delivery of mrna in situ results in efficient transgene expression and genetic immunization intradermal vaccinations with rna coding for taa generate cd8+ and cd4+ immune responses and induce clinical benefit in vaccinated patients mutually-induced conformational switching of rna and coat protein underpins efficient assembly of a viral capsid immunostimulating capacities of stabilized rna molecules widespread changes in protein synthesis induced by micrornas the use of armored rna as a multi-purpose internal control for rt-pcr a simple, rna-mediated allosteric switch controls the pathway to formation of a t = 3 viral capsid molecular mechanism of rna phage morphogenesis molecular mechanism of rna-phage morphogenesis effects of amino acid substitution on the thermal stability of ms2 capsids lacking genomic rna crystal structures of ms2 capsids with mutations in the subunit fg loop ribonucleoprotein complexes formed between bacteriophages ms2 rna and ms2 protein in vitro a new rna vaccine platform based on ms2 virus-like particles produced in saccharomyces cerevisiae microrna-146a contributes to abnormal activation of the type i interferon pathway in human lupus by targeting the key signaling proteins the three-dimensional structure of genomic rna in bacteriophage ms2: implications for assembly visualising a viral rna genome poised for release from its receptor complex vlps displaying a single l2 epitope induce broadly cross-neutralizing antibodies against human papilloma virus a coat for all sequences rna-based vaccines structure determination of the bacteriophage ms2 the three-dimensional structure of the bacterial virus ms2 proliferative lymphocyte responses to foot-and-mouth disease virus and three fmdv peptides after vaccination or immunization with these peptides in cattle mrna: from a chemical blueprint for protein production to an off-the-shelf therapeutic mrna: delivering an antitumor message external quality assessment of molecular detection of ebola virus in china delivery of sirna therapeutics: barriers and carriers development of an antisense rna delivery system using conjugates of the ms2 bacteriophage capsids and hiv-1 tat cell-penetrating peptide rnase-resistant virus-like particles containing long chimeric rna sequences produced by two-plasmid coexpression system plasmids encoding foot-and-mouth disease virus vp1 epitopes elicited immune responses in mice and swine and protected swine against viral infection delivery of antisense oligonucleotides to leukemia cells by rna bacteriophage capsids using a novel microrna delivery system to inhibit osteoclastogenesis cancer therapy using a self-replicating rna vaccine construction and characterization of virus-like particles: a review armored long rna controls or standards for branched dna assay for detection of human immunodeficiency virus type 1 external quality assessment for the detection of measles virus by reverse transcription-pcr using armored rna a novel method to produce armored double-stranded dna by encapsulation of ms2 viral capsids rna melanoma vaccine: induction of antitumor immunity by human glycoprotein 100 mrna immunization virus assembly, allostery and antivirals this work was supported by the grant from the national natural science foundation of china (no. 81171981). key: cord-300883-rws11uel authors: padhi, abinash; verghese, bindhu title: positive natural selection in the evolution of human metapneumovirus attachment glycoprotein date: 2007-10-10 journal: virus res doi: 10.1016/j.virusres.2007.08.014 sha: doc_id: 300883 cord_uid: rws11uel human metapneumovirus (hmpv), a newly discovered virus of the family paramyxoviridae, has been associated with upper and lower respiratory tract infections in different age groups in many countries. the putative attachment (g) glycoprotein of this virus was previously reported to have shown more extensive nucleotide and deduced amino acid sequence polymorphism than any other genomic regions of this virus, leading to four sub-lineages. using a maximum likelihood-based codon substitution model of sequence evolution, here we report that sequences of extracellular domain of 8 amino acid sites in lineage 1a, and 3 amino acid sites each in lineage 1b, 2a, and 2b have a higher rate of nonsynonymous substitutions (d(n)) than the synonymous substitutions (d(s)) with a posterior probability above 0.95, thus suggesting the evidence of adaptive evolution driven by darwinian selection. although it is unclear whether these amino acid adaptations are driven by differential immune pressure or some other factors, identification of these positively selected amino acid sites would help in better screening using epitope mapping technology to identify and localize the sites that can be recognized by the immune system. we also observed surprisingly higher nucleotide substitution rates per site, per year for each lineage of hmpv than the rates that were previously reported for the human respiratory syncytial virus, suggesting rapid evolutionary dynamics of hmpv. human metapneumovirus (hmpv) of the family paramyxoviridae and subfamily pneumoviridae was first discovered in the netherlands from infants and children suffering from acute respiratory tract disease (van den hoogen et al., 2001) . since then considerable progress has been made in identification and characterization (cote et al., 2003; mackay et al., 2003; ebihara et al., 2004; maertzdorf et al., 2004; skiadopoulos et al., 2004; hamelin and boivin, 2005; leung et al., 2005; gray et al., 2006a,b; ulbrand et al., 2006; van den hoogen, 2007) as well as in understanding its genetic diversity (bastien et al., 2003 (bastien et al., , 2004 biacchesi et al., 2003; ishiguro et al., 2004; peret et al., 2004; carr et al., 2005; ludewick et al., 2005; galiano et al., 2006; boivin et al., 2007) . to date this virus has been identified in many countries from different age groups and reported to cause upper respiratory tract infections, flu-like infections, and has also been associated with lower respiratory tract infections * corresponding author. tel.: +1 918 631 3062; fax: +1 918 631 2762. e-mail address: abinash-padhi@utulsa.edu (a. padhi). (van den hoogen et al., 2001; stockton et al., 2002; biacchesi et al., 2003; bastien et al., 2003 bastien et al., , 2004 ishiguro et al., 2004; peret et al., 2004; carr et al., 2005; ludewick et al., 2005; fouchier et al., 2005; regev et al., 2006; galiano et al., 2006; kahn, 2006; gray et al., 2006a,b) , a pattern similar to that reported for human respiratory syncytial virus (hrsv). although comparative genome mapping analyses suggested that this virus has structural and functional similarities with hrsv (kahn, 2006) , recent studies reported that the attachment (g) glycoprotein of these paramimyxoviruses exhibit extensive nucleotide and amino acid variation, with most differences located in the extracellular domain (peret et al., 2004; kahn, 2006) . therefore, g-protein has been widely used to infer evolutionary relationships among the isolates from different geographic regions (e.g., peret et al., 2004; ishiguro et al., 2004) . although phylogenetic analyses of hmpvs from the complete nucleotide coding sequences revealed the existence of two major lineages of hmpvs , recent analyses based on g-protein phylogeny revealed the existence of two minor subgroups within each major lineage (peret et al., 2004) . despite the knowledge of identification and characterization of hmpvs, the possible mechanism by which hmpv g-proteins have evolved is poorly understood. earlier studies on the molecular evolution of hrsv gprotein reported that certain amino acid sites that correspond to sites of o-glycosylation, or amino acid sites that were previously described as monoclonal antibody-induced in vitro escape mutants, are under positive selection and thus showed strong association between these positively selected sites and the mapped neutralizing epitopes (zlateva et al., 2004) . recently, zhang et al. (2006) also reported that certain amino acid sites in severe acute respiratory syndrome (sars) coronavirus (cov) are evolved by positive darwinian selection. these lines of evidence suggest an interesting evolutionary pattern of the respiratory viruses. at the genomic level, whether a gene, or a particular amino acid within a gene, is under relaxed selection or remains functionally constrained throughout evolution can be detected by comparing the rate of nonsynonymous nucleotide substitutions per nonsynonymous site (d n ) with that of synonymous substitutions per synonymous site (d s ) (hughes and nei, 1989) . if d n /d s (hereafter referred as ω) is greater than one, then positive selection is said to be operating. alternatively, if ω < 1, the gene is under strong purifying selection and presumed to be functionally constrained. identifying genes that have evolved by adaptation is central to understanding molecular evolution. however, not all amino acid differences observed among the closely related sequences from ecologically/geographically isolated strains are adaptive (e.g., zlateva et al., 2004) . therefore, analyzing patterns of amino acid substitutions would provide insight into understanding protein adaptation by identifying candidate codon sites on which positive selection has been operating. identifying the positively selected amino acid sites would also help in further immunization studies. maximum likelihood (ml)-based codon substitution models, which account for variable ω ratios among codon sites and detect codon sites that are subjected to positive selection (yang et al., 2000) , have been widely used in detecting positive selection in a number of respiratory viral groups (e.g., zlateva et al., 2004; zhang et al., 2006) . here we used yang et al's (2000) ml codon substitution models to test whether there was evidence at the nucleotide sequence level that a subset of amino acid sites in g-protein of hmpv sequences that represent each subgroup has been under positive selection. in addition, we used a bayesian mcmc approach implemented in beast version 1.4.4 ) that utilize the number and temporal distribution of genetic differences among viruses sampled at different times (drummond et al., 2002 to estimate the evolutionary change for each lineage. a total of 144 published unique nucleotide coding sequences of g-protein representing four sub-lineages (1a = 46, 1b = 40, 2a = 38, 2b = 20) were retrieved from genbank (table 1 ) . sequences were aligned using mesquite version 1.2 (maddison and maddison, 2006) , dambe version 4.5.2 (xia, 2000; xia and xie, 2001) , and bioedit version 7.0.5.3 (hall, 1999) software packages. to infer phylogenetic relationship among these strains of hmpvs, we reconstructed a neighbor joining tree from their predicted amino acid sequence data with p-distance implemented in mega version 3.1 (kumar et al., 2004) . using the same program, nodal supports were estimated with 10,000 nonparametric bootstrap replicates. for selection analyses, we reconstructed unrooted ml trees for each lineage from their respective nucleotide sequence data using the appropriate nucleotide substitution model identified by the hierarchical likelihood ratio test implemented in modeltest version 3.5 (posada and crandall, 1998) . phyml version 2.4.4 (guindon and gascuel, 2003) was used to conduct ml analyses. overall substitution rate (nucleotide substitutions per site per year) of each lineage was estimated using the bayesian skyline model, with both relaxed (variable) molecular clock (with uncorrelated lognormal model) and strict clock implemented in the beast version 1.4.4 . this model employs a bayesian mcmc approach and utilize the number and temporal distribution of genetic differences among viruses sampled at different times (drummond et al., 2002 . bayesian skyline plots with 10 grouped intervals were reconstructed to infer demographic history (drummond et al., 2005) . phylogenies were evaluated using a chain length of 30 million states under the hky85 + 4 substitution model and with uncertainty in the data reflected in the 95% high-probability density (hpd) intervals. convergence of trees was checked using tracer version 1.3 . to determine the synonymous and nonsynonymous sequence divergence distribution pattern across the entire coding region of each lineage (fig. 1) , we used a sliding window approach (window size = 6, step = 1) implemented in dnasp version 4.0 (rozas et al., 2003) . to assess whether positive selection is operating in any codon sites, we used the alignment and ml trees of respective lineages as input for the codeml program of paml version 3.15 (yang, 1997) . the paml program incorporates six different codon substitution models that account for variable ω for each codon site. the six codon substitution models are: m0 (one-ratio), m1a (nearly neutral), m2a (positive selection), m7 (β distribution; 0 ≤ ω ≤ 1), m8 (β + ω > 1: continuous) (yang et al., 2000) , and m8a (β + ω = 1) (swanson et al., 2003) . the m0 model estimates overall ω for the data. the m1a model estimates a single parameter, p 0 , with ω 0 = 0, and the remaining sites with frequency p 1 (p 1 = 1 − p 0 ) assuming ω 1 = 1. the m2a model adds a class of positively selected sites with frequency p 2 (where p 2 = 1 − p 1 − p 0 ) with ω 2 estimated from the data. in the m7 model, ω follows a beta distribution and is allowed to vary between 0 and 1, and two parameters (p and q) of the beta distribution are estimated from the data. in the m8 model, a proportion, p 0 , of sites have ω drawn from the beta distribution and the remaining sites with proportion p 1 are positively selected (ω 1 > 1). the lrts between nested models were conducted by comparing twice the difference in log-likelihood values (2ln l) against a χ 2 -distribution with degrees of freedom equal to the difference in the number of parameters between models (yang, 1997) . three lrts were conducted. the first comparison was made between m1a, which allows for two site classes (0 < ω < 1, ω = 1), and m2a, which allows three site classes (0 < ω < 1, ω = 1 or ω > 1). the second comparison was between m7 and m8, and the last comparison was between m8 and m8a, in which ω for hmpv13 m8a was constrained to 1. in all lrts good evidence for positive selection is found if the lrt indicates that models that allow for selection (i.e. m2a and m8; alternative models) are significantly better than their respective null models (m1a, m7 and m8a) (yang, 1997) . posterior probabilities of the inferred positively selected sites were estimated by the bayes empirical bayes (beb) approach that takes sampling errors into account (yang et al., 2005) . consistent with earlier studies (peret et al., 2004) , g-protein based phylogeny in the present study has also revealed the existence of multiple lineages of this virus (fig. 1) . all four lineages showed some degree of spatial structure; however, few strains in each lineage did not show any spatial structure, indicating extensive viral gene flow across the regions in a given epidemic season. relatively weak temporal structure across the regions further suggested that either certain strains can remain stable for more than one epidemic season (e.g., hrsv, zlateva et al., 2004 zlateva et al., , 2005 , or mutations might not have occurred in a linear fashion with the preservation of changes in the circulating viral strains. thus, virus genotypes would frequently appear and disappear along with new mutations in the populations. however, hrsv (zlateva et al., 2004 (zlateva et al., , 2005 showed a strong correlation between the accumulation of genetic divergence and the isolation date of the sequences. based on the relaxed clock assumption, the evolutionary rate of each major lineage of hmpvs (1 and 2; table 2 ) are 5.18 × 10 −3 and 6.49 × 10 −3 substitutions/site/year, respectively. although these rates are compatible with the substitution rates reported for influenza viruses (chen and holmes, 2006) , these rates are higher than the estimates of hrsv ( matheson et al., 2006) and other paramyxoviruses (e.g., measles: woelk et al., 2002) . these discrepancies in the evolutionary rates could be associated with the differential selective pressures targeting different genomic regions. for example, the presence of a greater number of adaptively evolved amino acid sites in the gene can cause an accelerated rate of evolution. as a consequence, the overall evolutionary rate is expected to be higher . both major lineages of hmpvs showed interesting population dynamics (fig. 2) . the times to the most recent common ancestor for lineage 1 and 2 are 49.452 (29.08-70.8) and 26.091 (21-36.651) years, respectively. while the population size of lineage 1 recently declined, the lineage 2 population size did not show any declining trend. this contrast in the population size could be associated with fitness of the virus. despite the weak temporal and spatial structure, viral strains belonging to lineage 1a (australia, canada, the netherlands, south africa, usa, argentina, and japan) and 1b (canada, the netherlands, south africa, usa, japan, china, and argentina) have a wider geographic spread than the strains belonging to lineage 2a (canada, uk, the netherlands, usa, china, and south africa) and 2b (the netherlands, canada, usa, peru, and japan), indicating that fitness of the viral strains might have estimates with relaxed clock are better fit to the data. played a crucial role in the uneven distribution across the wide geographic regions. the extensive polymorphisms of the hmpv g-gene may have resulted from mutations occurring during virus propagation in cell culture; however, peret et al. (2004) reported identical sequences of the same viral strain after multiple passages, and thus, the observed variation in the g-gene of hmpvs due to multiple passages is more unlikely. however, it is unclear whether the hmpv g-gene experienced differential selection pressures, or all the deduced amino acid sites evolved due to stochastic mutational processes? sliding window analyses of each lineage revealed that in the majority of regions synonymous divergence exceeds the corresponding nonsynonymous divergence, thus suggesting that the g-gene of hmpv is influenced by purifying selection (fig. 1) . however, a few coding regions in all the lineages showed relatively higher nonsynonymous divergence than synonymous divergence, therefore indicating the pervasive role of positive selection in certain amino acid sites. to identify the codon sites that are positively selected, we performed ml-based codon substitution analyses. consistent with sliding window results, the m0 model revealed that the average ω for each lineage is less than one (table 3 ) , thus suggesting each lineage experienced purifying selection. however, comparison of the models that assume positive selection (m2a, m8) with the models (m1a, m7, and m8a) that assume no positive selection, detected approximately 6%, 1.3%, 7.3%, and 3% positively selected codons in lineage 1a, 1b, 2a, and 2b, respectively (table 3 ). there are eight positively selected sites (site 93, 105, 106, 154, 158, 171, 173, and 188) with posterior probability ≥0.95 within lineage 1a, whereas lineage 1b (site 146, 183, and 196 ) and lineage 2a (85, 232, and 239) each have three positively selected sites with posterior probability ≥0.95. lineage 2b has only two positively selected sites (site 100 and 105) with posterior probability ≥0.95. except site 105, which is positively selected in lineage 1a and 2b, none of the positively selected sites are overlapping among the lineages. it is unclear whether these positively selected sites are associated with the fitness of this virus. research with monoclonal antibodies has shown that the hmpv f-protein carries neutralizing epitopes (skiadopoulos et al., 2004; ulbrand et al., 2006) ; therefore, antigenic variation due to immune selection in the hmpv f-protein is more likely. although, the overall excess of synonymous substitutions at the hmpv g-protein indicates that host immune selection might not be the dominant selective force, the findings of several hotspots of nonsynonymous substitutions in this protein suggests that host immune selection might also play a role in maintaining diversity. recent study has shown that a majority of the neutralizing epitopes in the hrsv g-gene is strongly associated with positively selected sites, and some of the positively selected sites correspond to the sites of o-glycosylation (zlateva et al., 2004) . like hrsv, although all the positively selected codons of hmpv g-gene are located in the extracellular domain and some of them correspond to sites of o-glycosylation, the putative role is still unclear for these positively selected sites, as is whether some of these positively selected sites are associated with the region of antigenic determinants. we intended to map these positively selected sites with the hrsv g-protein to see whether the same sites were also positively selected in hrsv (zlateva et al., 2004 (zlateva et al., , 2005 ; however, the predicted g-gene amino acid sequences of the two viruses could not be aligned (van den hoogen et al., 2002; kahn, 2006) . although a vast majority of codon sites (>95% in most cases) are shown to have been under purifying selection, significantly higher ω values (>1) at certain codon sites (table 3) indicate the hmpv g-gene is under positive selection. identification of these positively selected amino acid sites would help in better screening using epitope mapping technology to identify and localize the sites that can be recognized by the immune system. knowledge of 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polymorphism analyses by the coalescent and other methods the two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (f) protein is a major contributor to this antigenic relatedness human metapneumovirus as a cause of community-acquired respiratory illness pervasive adaptive evolution in mammalian fertilization proteins natural history of human respiratory syncytial virus inferred from phylogenetic analysis of the attachment (g) glycoprotein with a 60-nucleotide duplication isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in in vitro and in vivo a newly discovered human pneumovirus isolated from young children with respiratory tract disease increased positive selection pressure in persistent (sspe) versus acute measles virus infections data analysis in molecular biology and evolution dambe: data analysis in molecular biology and evolution paml: a program package for phylogenetic analysis by maximum likelihood codon-substitution models for heterogeneous selection pressures at amino acid sites bayes empirical bayes inference of amino acid sites under positive selection adaptive evolution of the spike gene of sars coronavirus: changes in positively selected sites in different epidemic groups genetic variability and molecular evolution of the human respiratory syncytial virus subgroup b attachment g protein molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment g glycoprotein we thank the university of tulsa for providing facilities to carry out this work and dr. peggy s.m. hill for editing and comments for improving the manuscript. we thank two anonymous reviewers for the helpful comments on the earlier version of the manuscript. key: cord-293562-69nnyq8p authors: imran, mudassar; usman, muhammad; malik, tufail; ansari, ali r. title: mathematical analysis of the role of hospitalization/isolation in controlling the spread of zika fever date: 2018-08-15 journal: virus res doi: 10.1016/j.virusres.2018.07.002 sha: doc_id: 293562 cord_uid: 69nnyq8p the zika virus is transmitted to humans primarily through aedes mosquitoes and through sexual contact. it is documented that the virus can be transmitted to newborn babies from their mothers. we consider a deterministic model for the transmission dynamics of the zika virus infectious disease that spreads in, both humans and vectors, through horizontal and vertical transmission. the total populations of both humans and mosquitoes are assumed to be constant. our models consist of a system of eight differential equations describing the human and vector populations during the different stages of the disease. we have included the hospitalization/isolation class in our model to see the effect of the controlling strategy. we determine the expression for the basic reproductive number r(0) in terms of horizontal as well as vertical disease transmission rates. an in-depth stability analysis of the model is performed, and it is consequently shown, that the model has a globally asymptotically stable disease-free equilibrium when the basic reproduction number r(0) < 1. it is also shown that when r(0) > 1, there exists a unique endemic equilibrium. we showed that the endemic equilibrium point is globally asymptotically stable when it exists. we were able to prove this result in a reduced model. furthermore, we conducted an uncertainty and sensitivity analysis to recognize the impact of crucial model parameters on r(0). the uncertainty analysis yields an estimated value of the basic reproductive number r(0) = 1.54. assuming infection prevalence in the population under constant control, optimal control theory is used to devise an optimal hospitalization/isolation control strategy for the model. the impact of isolation on the number of infected individuals and the accumulated cost is assessed and compared with the constant control case. the zika virus is transmitted to humans primarily through aedes mosquitoes and through sexual contact. it is documented that the virus can be transmitted to newborn babies from their mothers. we consider a deterministic model for the transmission dynamics of the zika virus infectious disease that spreads in, both humans and vectors, through horizontal and vertical transmission. the total populations of both humans and mosquitoes are assumed to be constant. our models consist of a system of eight differential equations describing the human and vector populations during the different stages of the disease. we have included the hospitalization/isolation class in our model to see the effect of the controlling strategy. we determine the expression for the basic reproductive number r 0 in terms of horizontal as well as vertical disease transmission rates. an in-depth stability analysis of the model is performed, and it is consequently shown, that the model has a globally asymptotically stable disease-free equilibrium when the basic reproduction number r 0 < 1. it is also shown that when r 0 > 1, there exists a unique endemic equilibrium. we showed that the endemic equilibrium point is globally asymptotically stable when it exists. we were able to prove this result in a reduced model. furthermore, we conducted an uncertainty and sensitivity analysis to recognize the impact of crucial model parameters on r 0 . the uncertainty analysis yields an estimated value of the basic reproductive number r 0 = 1.54. assuming infection prevalence in the population under constant control, optimal control theory is used to devise an optimal hospitalization/isolation control strategy for the model. the impact of isolation on the number of infected individuals and the accumulated cost is assessed and compared with the constant control case. the zika virus spreads among humans primarily through an infected mosquito bite, which has been increasing at an alarming incidence rate worldwide over the past few years (dick et al., 1952) . it belongs to the family of flaviviruses which includes more than fifty viruses, such as dengue, yellow fever, and the west nile virus. this virus was first identified in the zika forests of uganda and east africa during the investigations on the ecology of the yellow fever (anderson et al., 2016) . the first isolation was made in april of 1947 from the serum of pyrexia rhesus monkey caged. the second isolation was made in 1947 in the same forest (dick et al., 1952) . just a year later, in 1948, the virus was recovered from mosquito aedes africanus from the zika forest. the first human case of zika fever was reported in uganda in 1952. the first outbreak of zika fever was reported in 2007 on the pacific island of yap, this outbreak caused 108 symptomatic cases. another epidemic outbreak occurred in french polynesia between 2012 and 2014. during this time, it was estimated that about 28,000 people were reported to have zika like symptoms (anderson et al., 2016) . epidemic, reporting the most cases of people infected with the zika virus worldwide. in 2016, the state of rio de janeiro alone reported over 71,000 probable zika virus infections, however, this number dropped to only 2210 cases in 2017. sexual transmission of the zika virus has also been reported from both males and females to their partners (cdc, 2018; ecdc, 20185; hastings and fikrig, 2003; summers and acosta, 2015) . zika virus can be sexually transmitted from a person who is infected by the virus, even while they are not symptomatic. furthermore, it has been suggested that zika virus can be transmitted from a pregnant woman to her fetus during pregnancy. zika virus can be transferred horizontally as well as vertically. during the zika epidemic, brazilian health officials reported an increase in the number of cases of microcephaly disease, a condition in which a baby's head is smaller than normal in zika affected areas. the main symptoms of zika fever include a fever, the maculopapular rash often spreading from the face to the body, joint and muscle pain, vomiting, or bilateral non-purulent conjunctivitis (ecdc). the first well-documented case of zika fever was reported in 1964 and started with a mild headache with later development of a maculopapular rash, fever, and back pain (hayes, 2009) . the symptoms of zika fever are thus quite similar to those of dengue fever and there is a strong possibility of misdiagnosis in regions where dengue virus is common. the incubation period for the zika virus is between 3 and 14 days (krow-lucal et al., 2017) . disease-related symptoms are developed within one week of infection for 50% of the infected individuals and within 2 weeks among 99% (krow-lucal et al., 2017) of the infected individuals. the vast majority of infections are not contagious from person to person; however, it may be passed person to person during sex. the virus infection is usually diagnosed by a blood test. the disease symptoms are usually mild and short lasting (17 days), and infection may go unrecognized or be misdiagnosed as dengue fever (ecdc). unfortunately, there is no vaccine, antiviral drug, or other modality available to prevent or treat the zika virus infection. zika fever is a preventable but not a curable disease. thus, the only means of controlling the zika virus is to control the mosquitoes that spread the disease and protection during sex. in the past several years, a number of deterministic models for the transmission dynamics of the dengue virus have been studied and analyzed (esteva and vargas, 1998 ferguson et al., 1999; garba et al., 2008 garba et al., , 2010 kautner et al., 1997; wearing and rohani, 2006) . after the zika outbreak, models for zika transmission have been developed (agustoa et al., 2017; maxiana et al., 2017; wiratsudakul et al., 2018) and analyzed. in these models, the authors included the effect of sexual transmission of the disease. in this work, we formulate and study a deterministic model for zika virus transmission including vertical and horizontal transmission of the disease. although esteva (esteva and vargas, 2000) discussed vertical disease transmission it was among vectors in a dengue transmission. our deterministic model for zika virus transmission includes horizontal and vertical transmission in both humans and vectors. as stated previously, it has been suggested that zika virus can spread to newborns from their mothers, and we therefore feel that an accurate model must include the vertical transmission. our work is an extension of our previous model (imran et al., 2017) by including a population group that is using controlling measures. in the previous work, we considered death due to the infection and the total human and vector populations were functions of time. the previous model did not possess global stability for both disease-free and endemic equilibriums. we showed a backward bifurcation phenomenon. the current model has constant population size. since death cases reported from zika fever were negligible, we take disease-induced mortality to be zero. there is no backward bifurcation and the steady states results are global. since the only way to control the disease is to isolate patients who have been infected with the zika virus, we included a new population compartment consisting of hospitalized individuals. we have calculated the basic reproductive number associated with our model that guarantees the elimination of the disease. finally, using optimal control techniques, we also propose and analyze the control strategies for decrease infected individuals while minimizing the costs and resources simultaneously. the rest of this paper is organized as follows, the proposed model is presented in section 2. basic properties and a detailed steady-state analysis of the model are presented in section 3. in section 4, we perform a sensitivity and uncertainty analysis of the model parameters and reproductive number associated with our model. section 5 uses ideas from optimal control theory to propose various controlling strategies to overcome zika are proposed. finally, section 6 presents our conclusions and contains a brief discussion of our results. we consider two types of populations in this model one for the humans and for the mosquitoes. the total human and mosquitoes populations at time t, denoted by n h and n v , are constant. the human population is divided into five mutually exclusive groups, susceptible humans s h (t), exposed humans e h (t), infected humans i h (t), isolated or hospitalized individuals h h (t) and recovered humans r h (t). the total vector population is divided into three mutually exclusive classes comprising of susceptible vectors s t the model assumes that the susceptible human population s h (t) has a recruitment rate μ h n h , where n h is total human population and μ h is the natural birth rate of humans. we assume that the birth rate of human population is same as the natural death rate. susceptible individuals get infected with zika fever virus (due to contact with infected vectors) at a rate λ h and thus enter the exposed class e h . in order to consider vertical transmission in our model, we make the assumption (see, e.g., li et al., 2001 ) that a fraction of newborn individuals from parents in the e h (t) and i h (t) classes will be infected, and thus remain in e h class before becoming infectious. we have assumed that the hospitalized individuals do not contribute to vertical transmission. population in each class is removed at the natural death rate μ h . we assumed a lifelong immunity for humans who recovered from zika virus. the exposed individuals who got an infection, move to infectious class at a rate ξ. the infected population recovers from the zika fever at a rate θ i and some infected individuals are transferred to hospitalized class at a rate τ. the hospitalized population recovered at a rate of θ h . the susceptible vector population s t ( ) v has a recruitment rate μ n v v , and μ v is a natural death rate of vector population. a fraction of offsprings in the e t ( ) v and i t ( ) v classes will be infected, and thus remain in e h class before becoming infectious. because of this vertical transmission, a fraction of susceptible individuals will enter the exposed class. susceptible vectors are infected with zika virus (due to effective contact with infected humans) at a rate of λ v and thus move to the exposed vector class e v . the susceptible, exposed and infected vectors have natural death rate μ . v in addition, exposed vectors develop symptoms and move to the infected vector class i v at a rate of σ v . it is assumed that infected vectors do not recover, and die at the natural death rate of μ v . as mentioned earlier there is no vaccine available for zika fever. the only way to control this disease is to reduce the contact rate either by killing the mosquitoes or using the protective measure like mosquito repellents, nets etc. the effective contacts will be further reduced by isolating the infected humans. isolation of individuals with disease symptoms constitutes what is probably the first infection control measure since the beginning of recorded human history (hethcote, 2000) . over the decades, these control measures have been applied, with varying degrees of success, to combat the spread of some emerging and re-emerging diseases such as leprosy, plague, cholera, typhus, yellow fever, smallpox, diphtheria, tuberculosis, measles, ebola, pandemic influenza and, more recently, severe acute respiratory syndrome (gumel et al., 2003; imran et al., 2013; lipsitch et al., 2003; lloyd-smith et al., 2003) . chavez et al. analyzed a saiqr model in detail, to investigate the effect of isolation on influenza (vivas-barber et al., 2015) . they used the isolation (quarantine) i-q model, where the infected population is isolated. we have included epidemiological factors like permanent or partial immunity after recovery as well as intervention control measures through the inclusion of a hospitalized (or isolated) class, h h . in this case both the total host population and the vector population are constant. the forces of infection λ h and λ v are given as (garba et al., 2008) : here we assumed that an individual in h class still can transmit the disease but with lower rate. the value of modification parameter 0 ≤ η < 1. fig. 1 presents schematic diagram of the model (1). a description of the variables and parameters of the model (1) is given in tables 1 and 2 respectively. the model (1) will be studied on the closed set: is positively invariant and attracting with respect to the model (1). it can be seen that the solutions are always positive. the right sides of (1) are smooth, so that initial value problems have unique solutions that exist on maximal intervals. since paths cannot leave , solutions exist for all positive time. thus the model is mathematically and epidemiologically well posed. in this section, we will perform a detailed steady state and stability analysis of the zika fever model presented in section 2. the model (1) has a disease free equilibrium (dfe) given by in order to investigate the local stability of the dfe ( ) 0 , the next generation operator method (van den driessche and watmough, 2002) will be used. following the notation of van den driessche and watmough (2002), the matrix f (for the new infection terms) and the matrix v (of the transition terms) are given, respectively, by progression rate of humans from exposed to infected class τ hospitalization rate of infected individuals σ v progression rate of vectors from exposed to infected class c hv effective contact rate η modification parameter for relative infectiousness of hospitalized humans the basic reproduction number r 0 for our model is given by (2) when p = q = 0, vertical transmission is not present in the model; the above r 0 reduces to the basic reproduction number r 0 of a seir model for a vector disease (garba et al., 2008) . to get a better understanding of the basic reproduction number r 0 in (2), we rewrite it using taylor expansion about p and q: where ) and . note that r c is the basic reproductive number for horizontal transmission (khan et al., 2014) . the square root means that the two generations required for an infected vector or host to reproduce itself (van den driessche and watmough, 2002) . r p + r q is the sum of the number of infected individuals during the mean latent period and the number of infected individuals during the mean infectious period. similarly, r r + r s is the sum of the number of infected vector offsprings during the mean latent period and the number of infected vector offsprings during the mean infectious period. the expression r = r c r p + r c r q + r c r r + r c r s ) represents the total contribution to the infective class made by the exposed and infective individuals of first generation (li et al., 2001) . the local stability of the disease free equilibrium follows directly from van den driessche and watmough (2002) . we have following result about local and global stability of disease free state: lemma 3.1. the dfe ( ) 0 of the model (2.1), is locally-asymptotically stable if r 0 < 1, and unstable if r 0 > 1. (1) is globally-asymptotically stable in whenever r 0 < 1. be a solution of (1) with x 0 = x(0). a comparison theorem will be used for the proof. the equations for the infected components of (1) can be written as (where the prime denotes the derivative with respect to time), lemma 3.1 established the local asymptotic stability of the dfe when r 0 < 1, or equivalently, ρ(fv −1 ) < 1, which is equivalent to all eigenvalues of f − v having negative real parts when r 0 < 1 (van den driessche and watmough, 2002) . also, f − v has all off-diagonal entries non negative. then this means that the omega limit set of x 0 , ω(x 0 ), is contained in the disease-free space. but, on the other hand, it is straightforward to check that every solution with the initial condition in the disease-free space converges to the epidemiological implication of the above result is that the disease can be eliminated from the population if the basic reproduction number r 0 can be brought down to a value less than unity (that is, the condition r 0 < 1 is sufficient and necessary for disease elimination) irrespective of the size of the initial populations in each class. the stability of the dfe is demonstrated in fig. 2a . if r 0 > 1 the dfe is unstable in this case and the solutions are attracted to an (apparently unique and stable) endemic equilibrium, as depicted in fig. 2b . in this section, the existence and stability of endemic equilibrium (ee) of the model (1) will be discussed. we define endemic equilibrium to be those fixed points of the system (1) in which at least one of the infected compartments of the model are non-zero. theorem 3.3. the endemic state, 1 , of the model (1) exists whenever r 0 > 1. denote an arbitrary endemic equilibrium of the model (1). also, let solving the equations of the model (1) for steady-state by setting right hand asides of the model (1) . where and all k′s are defined above. substituting (4) in (3) and simplifying gives, also, substitute (6) into (5), we have the following equation in λ h . clearly the model (1) has no endemic state if r 0 < 1 and one unique endemic state when r 0 > 1. □ fig. 3 shows the variation in r 0 with the relative fraction of newborn exposed individuals (p) and newborn infected individuals (q). we notice that no less than %5 of newborn exposed individuals (p) and no more than 25% of the fraction of newborn infected individuals to bring r 0 less than 1: fig. 4 shows the effect of hospitalization rate τ of infected individuals on the basic reproductive number r 0 . from this figure, we can see that effective isolation will help to reduce the basic reproductive number. about 25% of infected individuals should be effectively isolated to bring the basic reproductive number to less than 1. this plot shows that the effective isolation is helpful in controlling the epidemic of zika virus. (1) showing the contour plot of r 0 as a function of fraction of newborn exposed individuals (p) and newborn infected individuals (q). theorem 3.4. if r 0 > 1 then the disease is strongly uniformly ρ-persistent: be a solution of model (1). in this case there exists an endemic steady state. let (that is, x is the disease-free subspace). note that x, as well as + x ℝ \ 7 , are positively invariant. also, all solutions originating in x converge to n 0 as t → ∞. n 0 is asymptotically stable in x. hence n 0 is isolated in x. corollary 4.7 in salceanu (2011) )), together with proposition 4.1 and lemma 3.1 in salceanu (2011) , imply that {n 0 } is also uniformly weakly repelling. then, from theorem 8.17 in smith and thieme (2011) we have that the semiflow generated by (2.4) is uniformly weakly ρ-persistent. from the positive invariance of , we have that (2.4) is point dissipative. then, according to theorem 2.28 in smith and thieme (2011) , there exists a compact attractor of points for (1). this, together with uniformly weakly ρpersistent imply (10) (see smith and thieme, 2011, theorem 5.2) . in this case there exists an endemic steady state (smith and thieme, 2011) . □ the local stability of the endemic steady state 1 of the model (1) is given in the lemma below. theorem 3.5. if r 0 > 1, then the endemic state of the model (1) is locally asymptotically stable in for model (1). for the proof of this above local stability theorem see imran et al. (2017) . for the global stability of the endemic steady state 1 , we consider the (1) with no hospitalization and a small incubation period so that we can assume that susceptible individuals after infection move to infected class. in this case, it is easily seen that both for the host population and for the vector population the corresponding total population sizes are asymptotically constant. we assumed that in our model the total population is constant. previous results (thieme, 1992) imply that the dynamics of systems (1) is qualitatively equivalent to the dynamics of system given by: theorem 3.6. if r 0 > 1, then the endemic state of (11) is globally asymptotically stable in . o proof. we will use geometric approach to global-stability method given in li et al. (2001) . let x = (s, i h , i v ) and f(x) denote the vector field of (11). the jacobian matrix and its second additive compound matrix j [2] is (see li et al., 2001) : where now from the system (11), we can write second and third equation, the variation in the values of the parameters of our model (1) is a source of uncertainty and sensitivity. in this section, we carry out a parameter base global uncertainty and sensitivity analyses on r 0 . there are a lot of reasons for the sensitivity of the parameters, for example, inadequate data, lack of information about the vertical transmission. we use the latin-hypercube sampling based method to quantify the uncertainty and the sensitivity of r 0 as a function of the model parameters. we use the latin-hypercube sampling based method to quantify the uncertainty and the sensitivity of r 0 as a function of 13 model parameters, namely μ μ θ θ ξ σ p q r s c τ η , , , , , , , , , , , , . the partial rank correlation coefficient (prcc) measures the impact of the parameters on the output variable using the rank transformation of the data to reduce the effects of nonlinearity. the uncertainty analysis (figs. 5 and 6) yields an estimated value of r 0 = 1.54 with 95% ci (1.3491, 1.7669) for the zika fever. the sensitivity analysis suggests that r 0 is highly sensitive to the parameters c θ θ μ τ , , , , . the accuracy and precision in the values of these parameters is vital for the accurate predictions of the model. the estimated parameters are presented in table 3 . one of the goals of this study is to come up with a time-dependent hospitalization/isolation strategy that would minimize the infected population while keeping the costs to a minimum at the same time. m. imran et al. virus research 255 (2018) 95-104 optimal control is a very useful mathematical technique that can help us to address these questions. here our goal is to put down infection from the population by increasing the recovered class and to minimize the required resources to control zika fever infection using isolation or hospitalization. the optimal control algorithm we use is based on pontrygain's maximum principle which appends the original model to an adjoint system of differential equations with terminal conditions. the optimal objective system, which characterizes the optimal controls, consists of differential equations of the original model (state system) along with the adjoint differential equations (the adjoint system). the number of equations in the adjoint system is same as that of in the state system. the detailed mechanism of forming the necessary conditions for the adjoint and optimal controls are discussed in fleming and rishel (1975) and pontryagin and boltyanskii (1980) . an important decision while formulating an optimal control problem is deciding how and where to introduce the control in the system of differential equations. the form of the optimal control primarily depends on the system being analyzed and the objective function to be optimized. in this paper, we will propose various strategies to eradicate zika fever using optimal control techniques. the first step is to find an optimal hospitalization schedule that minimizes the number of infectious individuals and the overall cost of hospitalization during a fixed time. we define the control set as u= {τ(t) : 0 ≤ τ(t) ≤ ζ, 0 ≤ t ≤ t, 0 < ζ ≤ 1, τ(t) is lebesgue measur-able}. here ζ is a positive number and is defined as the maximum value attained during optimal control procedure. our aim is to minimize the associated cost function which is given as: here a i and a n are positive constants used to balance in the size of i(t) and n t ( ) v . further, we used a nonlinear cost function in order to accommodate the impact of variety of factors associated with hospitalization, documented widely in literature, see for instance kirschner et al. (1997) . w is weight associated with quadratic cost due to hospitalization. moreover a linear function has been chosen for cost incurred by infected individuals and the mosquitoes population. our objective is to find an optimal control for hospitalization rate τ * (t) such that . the lagrangian of the optimization problem is given the associated "hamiltonian" is given by where k i represents the right hand side of the ith equation in our original model. w depends on the relative importance of the control measures in mitigating the spread of the disease as well as the cost incurred (such as material resources and human effort) during the implementation of control measure per unit time. pontrayagin's maximum principal converts model (1) and objective function (16) into minimizing the hamiltonian (17) with respect to τ. now we prove the following theorem to elicit the effect of optimal control of hospitalization. theorem 5.1. there exists a unique optimal control τ * (t) which minimizes j over u. also, there exists an adjoint system of ϕ i 's such that the optimal treatment control is characterized as for some positive number ζ. the adjoint system is given as the above adjoint system also satisfies the transversality condition, proof. we can easily verify that the integrand of j is convex with respect to τ(t). also, the solutions of our model are bounded above. in addition, it is verifiable that the model has the lipschitz property with respect to the state variables. using the properties mentioned above along with corollary 4.1 of fleming and rishel (1975) , the existence of an optimal control is established. now using the pontryagin's maximum principle, we obtain . therefore on the set {t : 0 < τ * (t) < ζ} we obtain now, we discuss the numerical solutions of the optimality system and the corresponding optimal control obtained using ζ = 0.5. the optimal strategy is obtained by solving the optimal system consisting of both the state system as well as the adjoint system. since there are initial conditions present for the state equations, we start solving them with a guess for τ using the fourth-order forward runge-kutta method. the adjoint equations are then solved using the fourth-order backward runge-kutta method because of the presence of final conditions. then, the controls are updated by using a convex combination of the previous control and the value from the characterization given above. this process is repeated until we obtain a desired accuracy of convergence (fig. 7) . fig. 8 represents the optimal isolation(hospitalization) strategy to be employed to minimize the cost and the infected population. considering the practical constraints, an upper bound of 0.5 was chosen for the optimal hospitalization control. the figure shows that initially, the optimal level remains at the upper bound of 0.5 after which it declines steadily to 0. this implies that in the early phase of the endemic breakout, keeping the control at the upper bound would help in decreasing the number of infected individuals. fig. 9 captures the dynamics of the infected population (i h ) by virtue of a comparison between the infected host population under optimal control and constant control. it can be seen that the decrease in the number of infected individuals is greater with optimal control as compared to that with constant control. furthermore, in contrast with a constant control, the infected population remains lower when an optimal control is applied. fig. 10 shows a comparison between the costs associated with optimal and different constant control strategies. it is clear that the cost associated with different control strategies is higher as compared to that of optimal control. it is important to note that high constant isolation rate (τ = 0.4) incurs almost the same cost as the of optimal control. however, practically it is highly unlikely to implement these high constant controls primarily due to the lack of required resources and facilities. fig. 11 captures the effect of the change in effective contact rate over the optimal control strategy. it is clear from the simulation that an increase in the contact rate may or may not lead to higher rates of hospitalization. in this paper, we have presented a zika fever epidemic model comprising of eight compartments consists of vector and human population. the dynamics of zika fever epidemic model have been considered. furthermore, using optimal control theory, we proposed control strategies to eliminate the infection from the population. a vertical and horizontal transmission model for zika fever is constructed in the form of a system of ordinary differential equations. this model features the study of zika fever by considering vertical transmission in both humans as well as vectors. the basic reproductive number r 0 is formulated by using a next-generation matrix. this reproductive number is simplified in order to better understand the effect of vertical transmission parameters. it is shown that the disease-free steady state is globally asymptotically stable when the basic reproductive number (r 0 ) is less than 1. the model has a unique endemic equilibrium when the reproduction number r 0 exceeds unity. this equilibrium is shown to be globally asymptotically stable when the reproduction number r 0 exceeds unity under the reduced model. it is locally asymptotically stable when we consider a full model. we performed a parameter based global uncertainty and sensitivity analysis on r 0 . the uncertainty analysis yields an estimated value of the basic reproductive number r 0 = 1.54 with 95% confidence interval (1.3491, 1.7669). this estimated value of r 0 is close to the calculated value of basic reproductive using real data (villela et al., 2017 ). our previous model had an estimated basic reproductive number r 0 = 1.31 with 95% confidence interval (1.23, 1.39) given in imran et al. (2017) . our sensitivity analysis on the zika model parameters showed that the most influential parameters are the effective contact rates, the recovery rate of the infected individuals and the birth rate of mosquitoes. we proposed an optimal controlling strategy to eliminate zika fever from the population. we observed that optimal control strategy is most effective in terms of eliminating infection as it minimizes our cost and resources at the same time. moreover, the control measures themselves may take time to implement, once the outbreak has been realized. despite these points, our analysis can help public health authorities to determine quasi-optimal strategies they might want to adopt, especially as our work highlights the relative effectiveness of different control strategies. mathematical model of zika virus with vertical transmission zika virus background zika virus (i). isolations and serological specificity estimated incubation period for zika virus disease analysis of a dengue disease transmission model a model for dengue disease with variable human population influence of vertical and mechanical transmission on the dynamics of dengue disease coexistence of different serotypes of dengue virus microcephaly in brazil potentially linked to the zika virus epidemic the effect of antibody-dependent enhancement on the transmission dynamics and persistence of multiple-strain pathogens deterministic and stochastic optimal control backward bifurcations in dengue transmission dynamics effect of cross-immunity on the transmission dynamics of two strains of dengue modelling strategies for controlling sars outbreaks zika virus and sexual transmission: a new route of transmission for mosquito-borne flaviviruses zika virus outside africa the mathematics of infectious diseases a comparison of a deterministic and stochastic model for hepatitis c with an isolation stage transmission dynamics of zika fever: a seir based model dengue virus infection: epidemiology, pathogenesis, clinical presentation, diagnosis, and prevention estimating the basic reproduction number for single-strain dengue fever epidemics optimal control of the chemotherapy of hiv global dynamics of an seir epidemic model with vertical transmission transmission dynamics and control of severe acute respiratory syndrome curtailing transmission of severe acute respiratory syndrome within a community and its hospital zika virus dynamics. when does sexual transmission matter the mathematical theory of optimal processes pan american health organization, zika cumulative cases robust uniform persistence in discrete and continuous dynamical systems using lyapunov exponents dynamical systems and population persistence zika virus in an american recreational traveler convergence results and a poincar bendixson trichotomy for asymptotically autonomous differential equations zika in rio de janeiro: assessment of basic reproduction number and comparison with dengue outbreaks dynamics of an saiqr influenza model ecological and immunological determinants of dengue epidemics dynamics of zika virus outbreaks: an overview of mathematical modeling approaches the authors declare that there is no conflict of interests regarding the publication of this article. we study the following feasible region of the new system (11) key: cord-267362-l4288mxw authors: shi, xibao; wang, li; zhi, yubao; xing, guangxu; zhao, dong; deng, ruiguang; zhang, gaiping title: porcine reproductive and respiratory syndrome virus (prrsv) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in marc-145 cells date: 2010-08-06 journal: virus res doi: 10.1016/j.virusres.2010.07.028 sha: doc_id: 267362 cord_uid: l4288mxw porcine reproductive and respiratory syndrome virus (prrsv) causes an economically important disease in swine-producing area, and interferon beta (ifn-β) is the first responder against the animal virus infection. however, whether prrsv could induce the production of ifn-β is controversial. in this paper, we first time found that prrsv could phosphorylate ifn-regulatory factor 3 (irf-3) and weakly activate the ifn-β promoter in marc-145 cells in early infection, but the activations of irf-3 and ifn-β promoter were rapidly inhibited in the following infection. furthermore, which components or products of the invading prrsv cause prrsv to inhibit ifn-β promoter activity attracted our attentions. the obtained results showed that prrsv nsp1 could inhibit poly(i:c)-induced ifn-β promoter activity in marc-145 cells by down-regulating the protein level of irf-3 and inhibiting the phosphorylation of irf-3. in conclusion, our results suggested that prrsv could be sensed by professional ifn-β-producing system and had mechanisms to inhibit this action in marc-145 cells. prrsv, a positive-stranded rna virus, is a member of family arteriviridae (meulenberg, 2000) . since it was firstly identified in the united states in 1987 and in europe in 1990, prrsv has caused one of the most economically important diseases of swine which is characterized by severe reproductive failure in sows and respiratory distress in piglets and growing pigs (rossow, 1998) . infection with prrsv also predisposes pigs to secondary infection by bacterial and viral pathogens, which may be due to the immunosuppression induced by the virus (feng et al., 2001; mateu and diaz, 2008) . ifn-␤ is the first responder against animal virus infection (muller et al., 1994; weber et al., 2004) . when virus infects, the virus could be recognized by the pathogen-associated molecular patterns (pamps) such as membrane bound toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i). these pamps recruit different adaptor proteins, for example, tlrs recruits the adaptor molecule myeloid differentiation primary-response gene 88(myd88) and toll/il-1 receptor domain-containing adaptor inducing ifn(trif) while rig-i recruits virus-induced signaling adapter (visa), to make tank-binding kinase 1 (tbk1) or ib kinase-(ikk-) phosphorylate irf-3 and finally to induce ifn-␤ transcription (bowie and unterholzner, 2008) . then, ifn-␤ induces the ifn-regulated genes responsible for the antiviral response (sadler and williams, 2008) . however, during the co-evolution with the host cells, many viruses have developed defensive mechanisms to inhibit ifn-␤ production, making it difficult for host cells to defeat viral infection (bowie and unterholzner, 2008; weber et al., 2004) . luo et al. (2008) and miller et al. (2004) concluded that prrsv does not induce ifn-␤ in marc-145 cells infected with prrsv, but luo et al. did not detect the level of ifn-␤ mrna by rt-pcr, and in miller's paper, the level of ifn-␤ appears a little higher in marc-145 cells infected by prrsv than that in control group, which may lead to a suspicion that whether prrsv could induce ifn-␤ production or not may be valued for verifying. furthermore, genini et al. (2008) and loving et al. (2007) reported that prrsv could induce the production of ifn-␤ in primary swine cells, which supply a clue that maybe prrsv could also induce the production of ifn-␤ in marc-145 cells. previous studies have documented that sars-cov nsp3 could inhibit the ifn-␤ production by its papain-like protease domain (devaraj et al., 2007) and sars-cov n was capable of inhibiting ifn-␤ production (kopecky-bromberg et al., 2007) . it is a coincidence that prrsv nsp1 also contained papain-like protease domain (den boon et al., 1995) and the crystal structure of prrsv n protein was similar to that of sars-cov n protein (yu et al., 2006) . so, the purpose of the present experiments is to analyze the patterns of ifn-␤ promoter activity in marc-145 cells during infection with prrsv and to analyze whether prrsv nsp1 and n protein could inhibit ifn-␤ production. marc-145 cell, a fetal green monkey fibroblast cell line derived from ma-104 (kim et al., 1993) , was maintained in dulbecco's modified eagle medium (gibco) supplemented with 10% fetal bovine serum (hyclone). prrsv strain bj-4, a kind gift from dr. hanchun yang (china agricultural university), was propagated in marc-145 cells, which after 96 h post-infection (p.i.), the cells were frozen and thawed and clarified by low-speed centrifugation, and then the supernatants were stocked at −80 • c. by the same methods, the supernatants of cells which were not infected with prrsv were also prepared as the sham virus infection in the experiment. primary antibodies used for this study were anti-irf-3, anti-serine 396-phosphorylated species of irf-3 (pirf-3) (cell signaling technology), anti-actin and anti-his tag (beijing zhongshan goldenbridge biotechnology company, china). the prrsv nsp1 and n, which contained 6× his-tag in cterminus in their reverse primers, were cloned from prrsv rna, and the pcr products were cloned into pmd19-t vector (takara) and then ligated into pcdna3.1 (invitrogen). tbk1, visa, and trif genes were cloned from marc-145 cells by rt-pcr and were ligated into pcdna3.1. rig-n gene, the constitutive active caspase recruitment domain in rig-i, was cloned from pef-bos-flag rig-n which was kindly provided by dr. takashi fujita (institute for virus research kyoto university, tokyo, japan) (yoneyama et al., 2004) and was ligated into pcdna3.1. pcdna3.1-irf-3(5d) was constructed from the irf-3(5d)-flag expression plasmid which was obtained from dr. rongtuan lin (lady davis institute for medical research, mcgill university, canada) (lin et al., 1998) . the firefly-luciferase plasmid for monitoring ifn-␤ promoter activation (p-284 luc) was cloned from genetic dna of marc-145 cells and was ligated into pgl-417 (prpmega). p55c1b luc (devaraj et al., 2007; yoneyama et al., 1996 yoneyama et al., , 2004 , a firefly-luciferase reporter gene plasmid containing repetitive pirf-3 binding sites, was kindly provided by dr. takashi fujita. phrl-tk (promega), contained a renilla-luciferase reporter gene, was used as an internal control in dual luciferase reporter assay system. pcdna3.1-his was constructed by using his primers. all primers for constructing plasmids above were listed in table 1 . all newly prepared plasmids were verified by sequencing. transient transfection was performed by using lipofectamine2000 (invitrogen). cells grown in 24-well plates were transfected in triplicate with the indicated reporter plasmid (400 ng), phrl-tk (200 ng), and expression vector (1000 ng) or supplied with an equivalent control vector. at the appointed time, cells were harvested and the luciferase activity was measured using the dual luciferase reporter assay system (promega) in microbeta ® trilux liquid scintillation and luminescence conters (microbeta-1450) (wallac). marc-145 cells grown in 24-well plates were transfected in triplicate with the pcdna3.1-his (control vector) or pcdna3.1-nsp1 (1000 ng), and 24 h later, transfected cells were mock-treated table 1 the primers used in plasmid construction and rt-pcr. sequence ( or treated with poly(i:c) for 6 h and lysed in lysing buffer (1% nonidet p-40, 0.1% sodium deoxycholate, 0.1% sds, 50 mm tris-hcl ph 7.4, 150 mm nacl, 2 mm edta, 2 mm na3vo4, 2 mm naf and a protease inhibitor cocktail). the detailed procedure for immunoblots has been described (shi et al., 2008) . briefly, the total protein concentration was quantified with the bradford protein assay (biocolor bioscience & technology company, shanghai). equal proteins were separated on sds-page gels and transferred to polyvinylidene difluoride membranes (millipore company), and then probed with appropriate antibodies. proteins were detected by using an ecl detection system (cell signaling technology). marc-145 cells were transfected with pcdna3.1-his (control vector) and pcdna3.1-nsp1. thirty hours later, the total cellular rna was extracted by using trizol reagent (invitrogen), and treated with dnase i (takara) to remove genomic dna contamination. then, the rna was reverse-transcribed to cdna by reverse transcriptase m-mlv (takara). the irf-3 rt primers used in semiquantitative analysis of the irf-3 mrna were listed in table 1 . statistical analyses was performed by student's t-test, and the comparisons were considered as statistical significance when p < 0.05. it has been extensively documented that cytopathic effect (cpe), induced by prrsv infection in marc-145 cells, only appeared after 72 h p.i. (cafruny et al., 2006; kim et al., 2002) , which was also confirmed in our experiment (data not shown). cell viability was assessed by trypan blue staining, and approximately 93% of prrsvinfected cells were viable at 48 h p.i. (data not shown). to investigate whether prrsv could induce the ifn-␤ response in cell culture, marc-145 cells were infected with prrsv at an moi of 0.05. at a certain time, cells were co-transfected with p-284 luc and phrl-tk, and 18 h later, the cells were harvested and subjected to a dual luciferase reporter assay system. as shown in fig. 1a , prrsv could activate ifn-␤ promoter in marc-145 cells at 24 h p.i., but the activity of ifn-␤ promoter was much lower than that triggered by poly(i:c). and the activity of ifn-␤ promoter was rapidly inhibited in the following infection. the expression of ifn-␤ mrna was also analyzed by semi-quantitative rt-pcr at the different time points after prrsv infection and gotten the results similar to that of fig. 1a (data not shown) . to confirm this result, the level of pirf-3 was also detected because pirf-3 was a necessary transcription factor to the activation of ifn-␤ promoter (peters et al., 2002; yoneyama et al., 1998) . in consistent with the results of fig. 1a , prrsv could phosphorylate irf-3 in early stage of infection (24 h p.i.), but after that, no detectable levels of pirf-3 showed at certain time interval (fig. 1b) . previous studies have documented that sars-cov nsp3 could inhibit the ifn-␤ production by its papain-like protease domain (devaraj et al., 2007) and sars-cov n was capable of inhibiting ifn-␤ production (kopecky-bromberg et al., 2007) . since prrsv nsp1 also contains papain-like protease domain (den boon et al., 1995) and the crystal structure of prrsv n protein was similar to that of sars-cov n protein (yu et al., 2006) , prrsv nsp1 and n proteins were chosen to probe which component of prrsv could inhibit ifn-␤ production. the expressions of pcdna3.1 nsp1 and pcdna3.1 n in marc-145 cells were confirmed by rt-pcr ( fig. 2a) and western blot (fig. 2b ). since nsp1 should be cleaved into nsp1␣ and nsp1␤ subunits (den boon et al., 1995) and the 6× his tag was ligated to the c-terminal of nsp1 in this study, here only nsp1␤ could be detected (fig. 2b) . marc-145 cells were co-transfected with pcdna3.1-nsp1 or pcdna3.1-n, p-284 luc and phrl-tk plasmids. as control group, pcdna3.1-his was in place of pcdna3.1 nsp1. twenty four hours later, marc-145 cells were either mock-treated or poly(i:c)treated for 6 h, and then the cells were harvested for luciferase reporter assay. the experimental data showed that only nsp1 suppressed the activation of ifn-␤ promoter (fig. 2c ), but n protein did not. because the pirf-3 was a necessary component to the activation of ifn-␤ promoter, after the poly(i:c) treatment or the mock treatment, pirf-3-dependent synthetic promoter, p55c1b-luc (devaraj et al., 2007; yoneyama et al., 1998 yoneyama et al., , 2004 , was detected with luciferase reporter assays,. as shown in fig. 2d , only nsp1 could inhibit the activation of p55c1b-luc, while n protein could not. the results in fig. 2d confirmed the results in fig. 2c. 3.3. prrsv nsp1 inhibited the activity of ifn-ˇpromoter induced by rig-n, visa, trif or tbk1, but did not inhibit the activity of ifn-ˇpromoter induced by irf-3(5d) rig-i, visa, trif and tbk1 were the upstream signaling proteins of irf-3 in the signal pathway of ifn-␤ production. the overn (n) . the control cells were transfected with pcdna 3.1-his (con). since the nsp1 should be cleaved into nsp1␣ and nsp1␤ subunits, the arrow-pointed band was only the nsp1␤ subunits. (c) marc-145 cells were co-transfected with p-284 luc, phrl-tk and different expression plasmid, and 24 h later, cells were either mock-treated (con) or transfected with poly(i:c) for 6 h. cells were analyzed by dual luciferase reporter assay. (d) marc-145 cells were co-transfected with p55c1b luc, phrl-tk and different expression plasmid. twenty four hours later, cells were either mock-treated (con) or transfected with poly(i:c) for 6 h. cells were analyzed by dual luciferase reporter assay. lcps was the luminescence counts per second detected by luminescence counting on the microbeta-1450. bars showed relative luciferase activity (relative lcps) normalized to phrl-tk activity (relative lcps = lcps of firefly-luciferase/lcps of renilla-luciferase). data represented means of three replicates, and experiments were repeated three times. vector (pcdna3.1-his) showed in figure was the control plasmid. *: p < 0.05 compared to the cells transfected with pcdna3.1-his (con). expression of rig-i, visa, trif or tbk1 could induce the activation of irf-3 and promote the activation of ifn-␤ promoter (devaraj et al., 2007; yoneyama et al., 2004; zhong et al., 2008) . rig-n was the constitutive active caspase recruitment domain in rig-i (yoneyama et al., 2004) . prrsv nsp1 strongly suppressed the activation of ifn-␤ promoter induced by over-expression of rig-n or ectopic expression of visa, trif, tbk1 (fig. 3a) . in contrast, irf-3(5d), a constitutively active and phospho-mimetic irf-3 mutant (lin et al., 1998) , activated the ifn-␤ promoter, and that was no difference between cells expressing nsp1 and cells transfected with a control vector (fig. 3a) . similar results were obtained when p55c1b luc was in place of p-284 luc (fig. 3b) . immunoblot analyzed the activation and latent status of irf-3. and the results showed that nsp1 not only down-regulated the protein level of irf-3, but also inhibited the phosphorylation of irf-3 induced by poly(i:c) (fig. 4a) . the expression of irf-3 mrna was analyzed by semiquantitative rt-pcr. the results in fig. 4b demonstrated that nsp1 have no effect on the transcription of irf-3. our present work was for the first time to analyze the dynamic activities of ifn-␤ promoter and irf-3, a necessary transcription factor to the activity of ifn-␤ promoter (yoneyama et al., 1998) during prrsv infection in marc-145 cell line which was capable of producing ifn-␤ (mckimm-breschkin and holmes, 1982) and supported the replication of prrsv (kim et al., 1993) . the results indicated that prrsv could trigger the activation of irf-3 as well as induce the activation of ifn-␤ promoter at 24 h p.i., but the activities were much lower than that triggered by the poly(i:c). and their activities were rapidly inhibited in following infection (fig. 1) . so it is reasonable to speculate that prrsv could be sensed by professional ifn-␤-producing system and had mechanisms to inhibit this action in marc-145 cells. prrsv nsp1 contained papain-like protease domain (den boon et al., 1995) and the crystal structure of prrsv n protein was similar to that of sars-cov n (yu et al., 2006) . previous studies have documented that sars-cov nsp3 could inhibit the ifn-␤ production by its papain-like protease domain (devaraj et al., 2007) and sars-cov n was capable of inhibiting ifn-␤ production (kopecky-bromberg et al., 2007) . our present work indicated that prrsv nsp1 inhibited the ifn-␤ production induced by poly(i:c), while n protein did not (fig. 2) . it's unexpected that prrsv n protein could not inhibit the ifn-␤ production, because a protein's function generally depended on its three-dimension structure or conformation. perhaps, sars-cov n protein could inhibit ifn-␤ production by its peptide or other unclear mechanism. poly(i:c), a double-stranded rna, could be recognized by tlr3 (yamamoto et al., 2003) and rig-i (kato et al., 2006) . then through tbk1, tlr3 recruited trif and rig-i recruited visa to phosphorylate irf-3 and finally activate ifn-␤ promoter (bowie and unterholzner, 2008) . over-expression of rig-i, visa, tbk1 or trif could induce the activation of irf-3 and promote the activation of ifn-␤ promoter (devaraj et al., 2007; yoneyama et al., 2004; zhong et al., 2008) . our data showed that nsp1 antagonized the ifn-␤ production induced by rig-i, visa, tbk1 or trif, which indicated that nsp1 inhibited the ifn-␤ production induced by both tlr3 and rig-i pathways (fig. 3) . nevertheless, nsp1 had no effect on the activity of ifn-␤ promoter induced by irf-3(5d), which suggested that nsp1 inhibited ifn-␤ production by suppressing the activation of irf-3. this speculation has been confirmed. prrsv nsp1 downregulated the protein level of irf-3 and inhibited poly(i:c)-induced phosphorylation of irf-3, but had no effect on the level of irf-3 mrna (fig. 4) . perhaps, nsp1 inhibited the translation of irf-3 or induced the degradation of irf-3 protein or inhibited the activation of kinase to phosphorylate irf-3. sars-cov nsp3 inhibited phosphorylation of irf-3 through its papain-like protease domain to be interacted with irf-3 but did not down-regulated the protein level of irf-3 (devaraj et al., 2007) , while the npro protein of classical swine fever virus, similar to the group of papain-like proteases (stark et al., 1993) , could antagonize irf-3 activity by inducing its proteasomal degradation (bauhofer et al., 2007) . prrsv nsp1 also contains papain-like protease domain (den boon et al., 1995) and prrsv nsp1 down-regulated the protein level of irf-3. so it is possible that prrsv nsp1 inhibits the irf-3 activation by inducing irf-3 degradation or inhibits the activation of kinase to phosphorylate irf-3. and this suppositional mechanism deserves a further study. based on the experimental data discussed above, we propose a model to illustrate how prrsv nsp1 negatively regulates ifn-␤ signaling pathways (fig. 5) . first, prrsv could be sensed by the professional beta interferon-producing system, although it is unclear that prrsv could be recognized by tlrs or rig-i-like receptors (rlrs) or by the both. second, prrsv nsp1 could negatively regulate ifn-␤ production by inhibiting the phosphorylation of irf-3. it is known that type i ifn system (ifn-␣/␤) provides an important first-host response to viral intruders (weber et al., 2004) , and type i ifn is essential to the initial control of virus infection and the establishment of an adaptive immune response (muller et al., 1994) . many viruses developed defensive mechanisms to inhibit ifn-␤ production (bowie and unterholzner, 2008; devaraj et al., 2007; weber et al., 2004) . the influenza virus ns1 protein, a multifunctional facilitator of virus replication, could antagonize the ifn-␤ production (hale et al., 2008) . deletion of the ifn-␤ antagonist ns1 could produce a novel type of influenza vaccine (wacheck et al., 2010) . novel antagonists against influenza virus ns1, only significantly and specifically inhibiting influenza a replication in cell capable of interferon production, could be a potential drug for influenza virus-treatment (basu et al., 2009) . previous studies also showed intriguing observations that recombinant ifn-␤ not only protected swine alveolar macrophages and marc-145 cells from infection with prrsv (overend et al., 2007) , but also could reduce the yield of prrsv in vivo (buddaert et al., 1998) . perhaps, inhibition of ifn-␤ production leads to the persistent infection of prrsv. our data demonstrated that prrsv could inhibit the ifn-␤ production by prrsv nsp1. in the future, it will be interesting to research on the function or characterization of prrsv nsp1 in virus-induced immunosuppression and virus replication, and prrsv nsp1 may be used as a potent target for exploiting new drags for prrsv treatment or prrsv vaccine. novel influenza virus ns1 antagonists block replication and restore innate immune function classical swine fever virus npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation viral evasion and subversion of patternrecognition receptor signalling vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) porcine 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syndrome virus-induced cell death exhibits features consistent with a nontypical form of apoptosis severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists virus-dependent phosphorylation of the irf-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferonbeta production by interfering with the rig-i signaling pathway the challenge of prrs immunology conditions required for induction of interferon by rotaviruses and for their sensitivity to its action prrsv, the virus interferon type i response in porcine reproductive and respiratory syndrome virus-infected marc-145 cells functional role of type i and type ii interferons in antiviral defense recombinant swine beta interferon protects swine alveolar macrophages and marc-145 cells from infection with porcine reproductive and respiratory syndrome virus irf-3-dependent, nfkappa band jnk-independent activation of the 561 and ifn-beta genes in response to double-stranded rna porcine reproductive and respiratory syndrome interferon-inducible antiviral effectors dynamic balance of pstat1 and pstat3 in c57bl/6 mice infected with lethal or nonlethal plasmodium yoelii processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus a novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist ns1 inverse interference: how viruses fight the interferon system role of adaptor trif in the myd88-independent toll-like receptor signaling pathway the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf-3 and cbp/p300 autocrine amplification of type i interferon gene expression mediated by interferon stimulated gene factor 3 (isgf3) crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation we thank takashi fujita, rongtuan lin for providing reagents. this work was supported by the key program national natural science foundation of china (30730068). key: cord-330508-uilejxmi authors: van den hoogen, bernadette; santoni, angela; sciumé, giuseppe; bowie, andrew; o’farrelly, cliona; o’neill, luke; anthonsen, marit; pardalli, katerina; young, simon; bergthaler, andreas; manel, nicolas; zahn, ronald; kikkert, marjolein; snijder, eric; van kuppeveld, frank; fouchier, ron; hiscott, john title: immunometabolism pathways as the basis for innovative anti-viral strategies (initiate): a marie sklodowska-curie innovative training network date: 2020-07-28 journal: virus res doi: 10.1016/j.virusres.2020.198094 sha: doc_id: 330508 cord_uid: uilejxmi the past century has witnessed major advances in the control of many infectious diseases, yet outbreaks and epidemics caused by (re-) emerging rna viruses continue to pose a global threat to human health. as illustrated by the global covid19 pandemic, high healthcare costs, economic disruption and loss of productivity reinforce the unmet medical need to develop new antiviral strategies to combat not only the current pandemic but also future viral outbreaks. pivotal for effective anti-viral defense is the innate immune system, a first line host response that senses and responds to virus infection. while molecular details of the innate immune response are well characterized, this research field is now being revolutionized with the recognition that cell metabolism has a major impact on the antiviral and inflammatory responses to virus infections. a detailed understanding of the role of metabolic regulation with respect to antiviral and inflammatory responses, together with knowledge of the strategies used by viruses to exploit immunometabolic pathways, will ultimately change our understanding and treatment of pathogenic viral diseases. initiate is a marie sklodowska-curie actions innovative training network (msca-itn), with the goal to train 15 early stage phd researchers (esrs) to become experts in antiviral immunometabolism (https://initiate-itn.eu/). to this end, initiate brings together a highly complementary international team of academic and corporate leaders from 7 european countries, with outstanding track records in the historically distinct research fields of virology, immunology and metabolism. the esrs of initiate are trained in these interdisciplinary research fields through individual investigator-driven research projects, specialized scientific training events, workshops on academia-industry interactions, outreach & communication. initiate will deliver a new generation of creative and entrepreneurial researchers who will be able to face the inevitable future challenges in combating viral diseases. pivotal for effective anti-viral defense is the innate immune system, a first line host response that senses and responds to virus infection. while molecular details of the innate immune response are well characterized, this research field is now being revolutionized with the recognition that cell metabolism has a major impact on the antiviral and inflammatory responses to virus infections. a detailed understanding of the role of metabolic regulation with respect to antiviral and inflammatory responses, together with knowledge of the strategies used by viruses to exploit immunometabolic pathways, will ultimately change our understanding and treatment of pathogenic viral diseases. initiate is a marie sklodowska-curie actions innovative training network (msca-itn), with the goal to train 15 early stage phd researchers (esrs) to become experts in antiviral immunometabolism (https://initiate-itn.eu/). to this end, initiate brings together a highly complementary international team of academic and corporate leaders from 7 european countries, with outstanding track records in the historically distinct research fields of virology, immunology and metabolism. the esrs of initiate are trained in these interdisciplinary research fields through individual investigator-driven research projects, specialized scientific training events, workshops on academia-industry interactions, outreach & communication. initiate will deliver a new generation of creative and entrepreneurial researchers who will be able to face the inevitable future challenges in combating viral diseases. key words: virology, innate immunity, immunometabolism, coronavirus, influenza virus, pneumovirus, innovative training network the past century has witnessed major advances in the control of many infectious diseases, as well as the mechanistic basis of the immune responses that limit virus replication and pathogenesis. nevertheless, viral outbreaks and epidemics caused by (re-) emerging rna viruses continue to pose an imminent global threat to human and animal health. during the early years of the 21 st century, prominent examples of such viral outbreaks included the h1n1 influenza pandemic of 2009, sars-and mers-coronavirus outbreaks in 2003 and 2012 respectively, ebola virus in 2014, 2018 and 2019, zika virus in 2016 and annual outbreaks of dengue viruses in tropical regions -each of which resulted in a dramatic strain on health care infrastructure and major societal disruptions. but since the start of 2020, the global community has been engulfed by the current pandemic of coronavirus induced disease (covid-19) caused by the novel sars-cov-2. at the time of writing, the pandemic rages on, and in the absence of effective therapeutics or a vaccine, as well as a strong international political strategy, the only defence available to humankind is the use of masks, social distancing and personal hygiene. despite an unparalleled effort by the global biomedical community, knowledge of the mechanisms of pathogenesis remains limited [1, 2] . such outbreaks are expected to continue because the environmental, demographic and behavioural drivers of infectious disease emergence are likely to remain intact for the foreseeable future. as illustrated by the current covid-19 pandemic, high healthcare costs, economic disruption and loss of productivity are additional pandemic consequences that reinforce the urgent medical need to develop new antiviral strategies and therapeutics to combat, not only the current pandemic, but also future viral outbreaks [3] . the innate immune system is pivotal in host defense against virus infection and this field is now being revolutionized by the recognition that cell metabolism is also critical to host defense against several diseases [4, 5] . immunometabolism is defined as "the changes in intracellular metabolic pathways in immune cells that alter their function" or "the interface of immune and metabolic responses in disease" [6] . it is now clear that immunometabolism pathways also have a major impact on the host antiviral and inflammatory response to virus infections. to drive the emerging field of antiviral immunometabolism, a new generation of scientists is needed with expertise in the interrelationships between viral pathogenesis, host metabolism and immune defences. initiate msca-itn addresses this unmet need by bringing together multidisciplinary european academics and private sector partners with a passion to understand interrelationships between antiviral innate immunity and host metabolism. initiate includes researchers from 7 academic institutions and 3 industrial partners from 7 european countries ( figure 1 ); 15 phd students -early stage researchers (esrs) have been recruited to initiate and each esr is pursuing an individual research project at one of the partner institutions ( table 1 ). the research components of initiate are complemented with specialized scientific training events, workshops on academia-industry interactions, outreach, communication and public engagement activities. these components of the network are organized by outstanding partners -agilent, biocrates, elsevier and sovalaccwho will provide essential training to the esrs. the initiate training program will deliver a new generation of creative biomedical entrepreneurial researchers at the forefront of antiviral immunometabolism. to meet the esrs and the research projects, visit: https://initiateitn.eu/early-stage-researchers/. antiviral immunity is initiated with the sensing and recognition by a set of patternrecognition receptors (prrs) of pathogen-associated molecular patterns (pamps). beststudied are the toll-like (tlrs), rig-i-like (rlrs), and nod-like (nlrs) receptors, cgas-sting and the pyhin proteins. activation of prrs triggers downstream signaling through a myriad of adapters (myd88, sting and mavs), protein kinases (jaks, tbk1, ikks) and transcriptional regulators (stat, irf and nf-b) to trigger the induction of type-i and iii interferons (ifn), and hundreds of ifn stimulated genes (isgs) that culminate in the generation of the so-called antiviral state; inflammatory signaling converges on the assembly and activation of the inflammasome (nlrp3, asc, pyhin). however, what has emerged more recently, is that cells activated by pamps also undergo profound metabolic changes. these changes are required for biosynthesis and energy production, but also drive key changes in immune signaling processes [7, 8] . the innate immune response also relies on a wide range of innate lymphocytes (ilcs), which are now considered as critical players of the immune response. natural killer (nk) cells are the founding member of this family and have been studied for decades and their role in providing early protection against viral infections. the ilc family comprises other four prototypical subsets including ilc1, ilc2, ilc3, and lymphoid tissue-inducer (lti) cells. independence from antigen recognition, tissue residency, and poised nature of key genomic loci (e.g. those encoding cytokines and other effector molecules) are the main features that make ilcs unique in regulating the early events of viral infection [9] . increased glycolysis is the hallmark metabolic switch in most immune cells undergoing rapid activation in response to detection of viruses and stimulation of prrs, cytokine receptors or antigen receptors [10] , [11] . in addition, viruses are likewise dependent on host metabolism for energy production and macromolecular synthesis, in order to complete efficient replication. while many mammalian viruses have evolved ingenious strategies to reprogram immune pathways to secure their maintenance in the infected host [12, 13] , few studies have investigated the crosstalk between virus infection, innate antiviral activity and metabolic pathways [14] . in addition, by-products from the krebs cycle such as succinate and itaconate act to stimulate or repress the antiviral and inflammatory response to infection [15] . the research program is divided into 3 work packages (figure 2 the objective of wp1 is to obtain in-depth knowledge of the roles of different organelles and signaling complexes (such as mitochondria, signalosomes, stress granules, replication organelles) in modulation of immune-and metabolic pathways in response to rna virus infections, including influenza virus, pneumoviruses and coronaviruses. pathways. the objective of wp2 is to obtain in-depth knowledge of the interaction of viruses, viral products and viral mechanisms that modulate the immune-and metabolism pathways and the interaction between these pathways. viral interaction with these pathways will elucidate new functions of viral proteins and modulatory interactions. in wp3, innate lymphoid cells, natural killer cells, hepatocytes, dendritic cells and in vivo animal models relevant for rna virus infection will be used to investigate the regulatory interface between immune and metabolic pathways on antiviral immunity. to unravel the innate and metabolic signature of mucosal vaccination by adenoviral vectors immunology of covid-19: current state of the science imbalanced host response to sars-cov-2 drives development of covid-19 the global impact of the coronavirus pandemic coupling krebs cycle metabolites to signalling in immunity and cancer cytokine-like roles for metabolites in immunity a guide to immunometabolism for immunologists crosstalk between cytoplasmic rig-i and sting sensing pathways self-rna sentinels signal viral invasion innate lymphoid cells: 10 years on control strategies in systemic metabolism immunometabolism of infections innate immune evasion by human respiratory rna viruses ten strategies of interferon evasion by viruses flavivirus modulation of cellular metabolism itaconate is an anti-inflammatory metabolite that activates nrf2 via alkylation of keap1 key: cord-268010-1m5h3krw authors: jung, kwonil; hu, hui; saif, linda j. title: porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis date: 2016-12-02 journal: virus res doi: 10.1016/j.virusres.2016.04.009 sha: doc_id: 268010 cord_uid: 1m5h3krw porcine deltacoronavirus (pdcov) (family coronaviridae, genus deltacoronavirus) is a novel swine enteropathogenic coronavirus that causes acute diarrhea/vomiting, dehydration and mortality in seronegative neonatal piglets. pdcov diarrhea was first reported in the us in early 2014, concurrently with co-circulation of porcine epidemic diarrhea virus (pedv) (family coronaviridae, genus alphacoronavirus). the origin of pdcov in pigs and also its sudden emergence or route of introduction into the us still remains unclear. in the us, since 2013–2014, the newly emerged pdcov and pedv have spread nationwide, causing a high number of pig deaths and significant economic impacts. the current us pdcov strains are enteropathogenic and infect villous epithelial cells of the entire small and large intestines although the jejunum and ileum are the primary sites of infection. similar to pedv infections, pdcov infections also cause acute, severe atrophic enteritis accompanied by transient viremia (viral rna) that leads to severe diarrhea and/or vomiting, followed by dehydration as the potential cause of death in nursing piglets. at present, differential diagnosis of pdcov, pedv, and transmissible gastroenteritis virus (tgev) is essential to control viral diarrheas in us swine. cell culture-adapted us pdcov (tc-pdcov) strains have been isolated and propagated by us and in several other laboratories. tc-pdcov strains will be useful to develop serologic assays and to evaluate if serial cell-culture passage attenuates tc-pdcov as a potential vaccine candidate strain. a comprehensive understanding of the pathogenesis and epidemiology of epidemic pdcov strains is currently needed to prevent and control the disease in affected regions and to develop an effective vaccine. this review focuses on the etiology, cell culture isolation and propagation, molecular epidemiology, disease mechanisms and pathogenesis of pdcov infection. porcine deltacoronavirus (pdcov), a member of the genus deltacoronavirus in the family coronaviridae of the order nidovirales, causes acute diarrhea, vomiting, dehydration and mortality in neonatal piglets (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . pdcov was first reported in pigs in hong kong in 2012 (woo et al., 2012) . among a total of 7140 rectal, tracheal, or cloacal swab samples from wild asian leopard cats, bats, wild birds, domestic cats, cattle, chickens, dogs, pigs, rodents, monkeys, and humans in china and hong kong in 2007-2011, dcovs were detected only in pigs and wild birds, with a 10.1% positive rate for pdcov in the 169 swine rectal swab samples tested (woo et al., 2012) . however, it appears that dcovs were present in rectal swabs of small mammals, such as asian leopard cats and chinese ferret badgers, at live-animal markets in china since 2005 -2006 (dong et al., 2007 . pdcov diarrhea was first reported in the us in early 2014 (li et al., 2014; marthaler et al., 2014b; wang et al., 2014a, b) , concurrently with porcine epidemic diarrhea virus (pedv) (family coronaviridae, genus alphacoronavirus), which causes similar, but more severe clinical disease in neonatal piglets (cima, 2013; stevenson et al., 2013) . recent studies reported that pdcov chn-ah-2004 and ch/sichuan/s27/2012 strains, genetically related to the current us pdcov strains, were previously present in diarrheic piglets in china in 2004 and 2012, respectively, prior to the first outbreak of pdcov infection in the us in early 2014 (dong et al., 2015; wang et al., 2015b) . the family coronaviridae can be genetically divided into the four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (woo et al., 2012) . bats are the projected host for the gene source of alphacoronaviruses and betacoronaviruses, while birds are the suspect host for gammacoronaviruses and deltacoronaviruses (woo et al., 2012) . the other swine enteropathogenic coronavirus, transmissible gastroenteritis virus (tgev), also belongs to alphacoronavirus. similar epidemiological, clinical, and pathological features among pdcov, pedv and tgev have complicated their diagnosis, requiring differential laboratory tests . in the us, since 2013-2014, the newly emerged pedv and pdcov have spread nationwide and affected pigs alone or jointly, causing a high number of pig deaths and significant economic impacts and hindering the control of both viral infections in farms dually affected by these two viruses (anon., 2014; jung and saif, 2015) . at present, differential diagnosis of pedv, pdcov, and tgev is critical to control the coronaviral epidemic diarrheas in us pig farms. cultivable, cell culture-adapted pdcov (tc-pdcov) strains include the two isolates, usa/il/2014 and michigan/8977/2014, isolated at the usda national veterinary services laboratories (chen et al., 2015b; ma et al., 2015) and tc-pdcov strain oh-fd22 isolated in our lab (hu et al., 2015) . the tc-pdcov oh-fd22 has been serially passaged >90 times on llc-pk cells, with high sustained infectious titers (hu et al., 2015) . the tc-pdcov strains are useful for pdcov pathogenesis studies, virological and serological assays, and vaccine development. this review focuses on the current understanding of the etiology, molecular epidemiology, disease mechanisms and pathogenesis of pdcov, and cell culture isolation and propagation of the virus. pdcov is enveloped and pleomorphic with diameter of 60-180 nm, excluding the projections (ma et al., 2015) . pdcov has a single-stranded positive-sense rna genome of approximately 25.4 kb in size (excluding the poly a-tail) that encodes four structural proteins, namely, spike (s), envelope (e), membrane (m), and nucleocapsid (n), and four nonstructural proteins (lee and lee, 2014; li et al., 2014; ma et al., 2015; marthaler et al., 2014a; wang et al., 2015b) . the pdcov genome organization and arrangement consist of: 5 untranslated region, open reading frame 1a/1b (orf1a/1b), s, e, m, nonstructural protein 6 (ns6), n, nonstructural protein 7 (ns7), and 3 utr. although general characteristics of the structural and nonstructural proteins of coronaviruses and their roles in viral replication have been identified saif et al., 2012) , the detailed functions and roles of structural and nonstructural proteins of pdcov in host cells are unknown. a recent study reported multifunctional roles of pdcov n protein similar to those of other coronaviruses. the n protein was identified in both the nucleus and cytoplasm of stably n-expressing porcine kidney cells, suggesting rna synthesis or ribosome biogenesis through its interactions with ribosomal subunits or nucleolar proteins . there was no cross-reactivity of pdcov usa/il/2014 strain with antibodies to either pedv or tgev, as determined by indirect immunofluorescence assays of virus-infected cells using a rabbit antiserum against synthetic m protein of pdcov, a pedv monoclonal antibody (sd6-29) targeting the n protein, and a tgev polyclonal antiserum (chen et al., 2015b) . this finding was also supported by other reports (jung et al., 2015c; ma et al., 2015) , showing no cross-reactivity of pig hyperimmune or convalescent antiserum to pdcov oh-fd22 or cvm1 strains with pedv, as determined by immunohistochemical staining of frozen or formalin-fixed intestinal tissues of pedv-infected pigs. however, a recent study reported evidence of antigenic cross-reactivity between pdcov michigan/8977/2014 strain and pedv, possibly sharing at least one conserved or similar epitope on their n proteins, as determined by enzyme-linked immunosorbent assay (elisa) and western blot using monoclonal pedv and pdcov n-specific antibodies, whereas no cross-reactivity was detected when virus neutralization, indirect immunofluorescence, and immunostaining assays were conducted on either virus-infected cells or intestinal tissues using pig hyperimmune antisera to pedv or pdcov (ma et al., 2016) . llc porcine kidney (llc-pk) and swine testicular (st) cells support the isolation and serial propagation of pdcov in cell cultures supplemented with exogenous protease trypsin or pancreatin (hu et al., 2015) . trypsin contributed to a significant increase in pdcov growth after several passages in llc-pk cells. however, even without supplemental trypsin, pdcov replicated in llc-pk cells, but without cytopathic effects (cpe) and at relatively lower virus titers compared to the trypsin supplemented cell cultures (hu et al., 2015) . thus, trypsin is not essential, but is beneficial for the propagation of pdcov in llc-pk cells. the cell-culture conditions required for the propagation of pdcov in st cells differ from those in llc-pk cells. trypsin did not support pdcov propagation in st cells. in contrast, pdcov could be serially propagated in st cells supplemented with pancreatin or small intestinal contents from gnotobiotic (gn) piglets (hu et al., 2015) . the addition of trypsin and pancreatin in pdcov-inoculated llc-pk and st cells, respectively, resulted in similar cpe that consisted of enlarged, rounded, and densely granular cells that occurred singly or in clusters and then, cell shrinkage and detachment as a result of apoptotic cell death hu et al., 2015) . the roles of trypsin or pancreatin in cell entry and release of pdcov virions in llc-pk or st cells remain unknown. coronaviruses in the genus deltacoronavirus have been identified recently in a variety of wild small mammals and avian species (dong et al., 2007; woo et al., 2009a; woo et al., 2009b; woo et al., 2012) . in 2006, novel coronaviruses were detected in feces of wild asian leopard cats (prionailurus bengalensis) and chinese ferret badgers (melogale moschata) during a virological surveillance for severe acute respiratory syndrome (sars) coronavirus in southern china. the isolates were genetically similar to each other and commonly had low amino acid similarity and unique phylogeny distinct from other coronavirus groups (dong et al., 2007) . the two isolates were subsequently shown genetically to belong to deltacoronavirus (woo et al., 2012) . another molecular epidemiology study was conducted in dead wild birds for a 7-month period (december 2006 to june 2007 in hong kong (woo et al., 2009b) . three novel avian coronaviruses (bucov hku11, thcov hku12, and mucov hku13) were identified and proposed as a novel coronavirus subgroup 3c under the group 3 coronaviruses (woo et al., 2009b) . recently, the three isolates were further identified genetically to belong to deltacoronavirus (king et al., 2012) . a coronavirus surveillance study was conducted in a variety of domestic and wild animal species from 2007 to 2011, and the presence of dcovs in the feces of domestic pigs was first reported in 2012 (woo et al., 2012) . seven dcovs in pigs and wild birds were identified, which were designated porcine coronavirus hku15 and the avian coronavirueses, white-eye coronavirus hku16, sparrow coronavirus hku17, magpie robin coronavirus hku18, night heron coronavirus hku19, wigeon coronavirus hku20, and common moorhen coronavirus hku21 (woo et al., 2012) . this study first verified that pdcov was present in the feces of pigs in mainland china and hong kong in 2007-2011, with a positive rate of 10.1%. two chinese pdcov complete genome sequences were generated from two different isolates, hku15-44 and hku15-155 (genbank accession no. jq065042 and jq065043, respectively) (woo et al., 2012) . the helicase, s and n genes of pdcov hku15-44 and hku15-155 were closely related to those of the coronaviruses isolated from the asian leopard cats and chinese ferret badgers [nucleotide (nt) similarity ≥99.8%] (woo et al., 2012) (fig. 1a) , implying the potential interspecies transmission of a dcov between these wild small mammals and pigs. further molecular surveillance studies are needed to define the potential role of the small mammals as an intermediate host of pdcov and the mechanisms of interspecies transmission of dcovs between the small mammals and domestic pigs or wild birds, and also between domestic pigs and wild birds. on february 11, 2014, the ohio department of agriculture first announced the presence of pdcov in us swine. in early february 2014, the animal disease diagnostic laboratory at the ohio department of agriculture received 42 fecal or intestinal samples from diarrheic sows and piglets on 5 ohio farms, and 39 (92.9%) were positive for pdcov by rt-pcr (wang et al., 2014a) . the pdcov hku15-oh1987 strain identified (genbank accession no. kj462462) had a 99% nt identity to pdcov hku15-44 and hku15-155 (wang et al., 2014a) (fig. 1b) , implying a possible importation of a chinese pdcov into us swine. during a similar period, the university of minnesota veterinary diagnostic laboratory also received fecal swabs from diarrheic pigs. the causative agent of the diarrhea was identified as the pdcov sdcv/usa/illinois121/2014 strain (genbank accession no: kj481931.1) that also had approximately 99% nt identity to the two hong kong pdcov strains (marthaler et al., 2014a) . the iowa state university veterinary diagnostic laboratory also identified another us pdcov strain usa/ia/2014/8734 (genbank accession no: kj567050) that had 98.9% nt identity to the hku15-44 strain and 99.2% nt identity to the hku15-155 strain (li et al., 2014) . pdcovs were further detected in other us states (minnesota, south dakota, nebraska, illinois, indiana, michigan, kentucky, pennsylvania, and ohio) (wang et al., 2014b) . of the 435 samples tested, 109 (25%) were positive for pdcov by rt-pcr, and they shared high nt similarity (≥99.8%) with each other and also 98.9-99.2% nt similarity with pdcov hku15-44 and hku15-155 (wang et al., 2014b) . as of march 2016, pdcov has been detected in 18 us states according to a report of the usda animal and plant health inspection service (www.aphis.usda.gov/ animal-health/secd). several retrospective studies were conducted to determine the earliest date when pdcov existed in us swine. a retrospective study using real-time rt-pcr was performed on swine samples that were submitted to the iowa state university veterinary diagnostic laboratory for investigation of enteric diseases between october 2012 and december 2013 (sinha et al., 2015) . a total of 5 samples were positive for pdcov rna among the 1734 clinical samples from 18 states tested. pdcov rna was detected in fecal samples collected on august 19th 2013 from minnesota, august 20th and august 27th from iowa, and august 29th 2013 from illinois (sinha et al., 2015) . based on these data, pdcov has been present in us swine since at least august 2013. another retrospective study using an indirect elisa for the detection of pdcov igg antibodies demonstrated their prevalence in us pigs (thachil et al., 2015) . a total of 968 serum samples collected between 2006 and 2014 were tested. the pdcov igg antibodies were first detected in the archival serum samples collected in 2010 (thachil et al., 2015) . however, the origin of us pdcov in us swine remains unclear. to date, pdcov also has been reported in canada, korea, china, and thailand. epidemic diarrhea related to pdcov was first detected in 6 ontario farms in canada in march 2014 (marthaler et al., 2014b) . in april 2014, pdcov (kun14-04 strain, genbank accession no. km820765) was first identified in feces of diarrheic piglets in south korea. the korean strain had nt identities of 98.8-99.0% to hku15-44 and hku15-155 and 99.6-99.8% to us pdcov strains (lee and lee, 2014) . a recent survey conducted in south korea reported that of 691 diarrheic fecal samples collected on 59 pig farms in january 2014 to march 2015, only 2 samples from 1 pig farm (march 2015) were pdcov-positive by rt-pcr (lee et al., 2016) . two korean pdcov strains sl2 and sl5 isolated in the study were also closely related to us pdcov strains, but they appeared to genetically differ from the previous korean strain knu14-04 isolated in 2014 (lee et al., 2016) . the first identification of pdcov hku15-44 and hku15-155 strains in swine in hong kong, china for the period 2007-2011 were documented in 2012 (woo et al., 2012) . since then, pdcov has been identified in diarrheic pigs in mainland china (dong et al., 2015; song et al., 2015; wang et al., 2015b) . a study also found pdcov rna in diarrheic pig samples collected in 2004 in china (dong et al., 2015) . the prevalence of pdcov in mainland china was high (>30%), and co-infections with pdcov and pedv (51%) were also common (dong et al., 2015; song et al., 2015) . all pdcov strains reported from china shared high nt identities (≥98.9%) with each other and with the other pdcov strains found globally (dong et al., 2015; song et al., 2015) (fig. 1b) , implying a possible global circulation of a single genotype. pdcov strains with multiple mutation or deletion sites in their s, nonstructural, or 3 utr genes have been also found in china, whereas the mutations were not found in the genomes of us pdcov strains (wang et al., 2015a; wang et al., 2015b) . in june 2015, a pdcov-related diarrhea outbreak was identified on a pig farm in thailand (janetanakit et al., 2016) . pdcov strains detected in thailand shared the highest nt identities (≥98.4%) with the chinese pdcov strain chn-ah-2004 (janetanakit et al., 2016) . like pedv and tgev saif et al., 2012) , the fecal-oral route may be the main means of pdcov transmission. since aerosolized pedv remains infectious (alonso et al., 2014) , whether aerosolized pdcov is also infectious should be investigated. like pedv transmission, diarrheal feces and/or vomitus and other contaminated fomites, such as transport trailers (lowe et al., 2014) and feed (dee et al., 2014) , may be major transmission sources of the virus. another possible reservoir for pdcov includes carriers, such as older pigs with subclinical infection, similar to pedv infection in weaned pigs (jung et al., 2015a) . like pedv and tgev saif et al., 2012) , pdcov is cytolytic, and infected enterocytes rapidly undergo acute necrosis, leading to marked villous atrophy in the small but not in the large intestine ( fig. 2a) (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . like pedv and tgev (kim et al., 2000) , pdcov may not induce apoptotic death of enterocytes in the small intestine of infected pigs . pdcov antigens ( fig. 2b and c) and nucleic acids (fig. 2d ) are observed mainly in villous enterocytes of the small (duodenum to ileum) and large intestines (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . as tested at post-inoculation days (pid) 3-7, pdcov antigens were detected mainly in the villous epithelium of the atrophied mid-jejunum to ileum of experimentally infected pigs and to a lesser extent, in duodenum, proximal jejunum and cecum/colon (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . occasionally, a few pdcov antigenpositive cells are detected in the intestinal crypts . porcine small intestinal villous enterocytes express large amounts of aminopeptidase n (apn), a 150-kda glycosylated transmembrane protein, identified as the cellular receptor for pedv and tgev (delmas et al., 1992; li et al., 2007) . whether apn also functions as the cellular receptor for pdcov or if other cellular receptors are involved in cell entry of this virus is unknown. in addition, a few pdcov antigen-positive cells were also detected in the intestinal lamina propria, peyer's patches, and mesenteric lymph nodes (mln) during the early stages of infection jung et al., 2016) . as tested at pid 23-24 after experimentally infected pigs had recovered from clinical disease and ceased fecal virus shedding, larger numbers of pdcov antigen-positive cells were detected in these tissues . the pdcov antigen-positive cells might include antigen-presenting cells, such as macrophages. pdcov antigens were not detected in other organs, such as stomach, lung, heart, tonsil, spleen, liver and kidney (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . however, by qrt-pcr, pdcov rna could be detected in low to moderate quantities in multiple organs, possibly due to the effect of viremia (chen et al., 2015b; ma et al., 2015) . lung tissues of orally infected pigs were negative for pdcov antigen (chen et al., 2015b; hu et al., 2016; jung et al., 2015c) . in our preliminary study, four 10-to 14-day-old gn pigs orally and nasally inoculated with tc-pdcov oh-fd22 at llc-pk cell-culture passages 5 (p5) or 80 (p80) were negative for pdcov rna in nasal swabs and bronchoalveolar lavage fluids at pid 3-10, although the inoculated pigs were positive for both clinical disease and fecal virus shedding for the period, suggesting lack of pdcov replication in the respiratory tract. however, further studies are still needed to investigate whether extra-intestinal replication of pdcov occurs. based on clinical observations in experimentally infected gn or conventional nursing pigs, like pedv and tgev saif et al., 2012) , pdcov also induces acute, watery diarrhea, frequently accompanied by acute, mild to moderate vomiting, ultimately leading to dehydration, loss of body weight, lethargy, and death (jung et al., 2015c; ma et al., 2015) . similar to pedv infection (jung et al., 2014) , in a controlled experimental setting, clinical pigs exhibited dehydration, loss of bodyweight, and lethargy, but frequently maintained their appetite until sudden death (ma et al., 2015) . among the different clinical signs identified in gn or conventional pigs, only diarrhea could be observed in experimentally infected conventional nursing pigs (chen et al., 2015b) , indicating that a variety of disease outcomes are expected based on different experimental conditions. however, conventional pigs appeared to be more susceptible to pdcov infection, compared to gn pigs, under similar experimental conditions, such as when the same virus and inoculation dose and age of pigs were used (ma et al., 2015) . based on field observations in us swine (anon., 2014) , pdcov caused a number of deaths (up to a 40% mortality rate) among suckling pigs, a lower rate than that found in pedv infections. the clinical impact and disease severity of pdcov in the field may be less than that of pedv (anon., 2014) . on the other hand, a pdcov-related diarrhea outbreak in a breeding farm in china resulted in a higher mortality rate (more than 80%) among suckling piglets (song et al., 2015) . like pedv and tgev saif et al., 2012) , diarrhea induced by pdcov may be a consequence of malabsorption due to massive loss of absorptive enterocytes. functional disorders of infected enterocytes may also contribute to the malabsorptive diarrhea. like pedv , mild vacuolation observed in the infected colonic epithelial cells may interfere with the vital reabsorption of water and electrolytes (jung et al., 2015c) . as a consequence of extensive loss of enterocytes, brush border membrane-bound digestive enzymes such as disaccharidases (lactase, sucrase, and maltase), leucine apn, and alkaline phosphatase were significantly decreased in the small intestine of diarrheic piglets after pedv infection jung et al., 2006) , resulting in maldigestive diarrhea. because of the similar pathological features between pdcov and pedv, pdcov infection may also result in maldigestive diarrhea. dehydration is exacerbated by vomiting, but the mechanisms by which vomiting is induced in pdcov infection are poorly understood. whether pdcov infection induces metabolic acidosis, similar to hyperkalemia and acidosis in acute tgev and pedv infections saif et al., 2012) , needs to be studied. based on experimental findings (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) , the onset of diarrhea after pdcov infection appears to be acute, which coincided mostly with or was detected 1-2 days later than the detection of viral rna in feces. fecal viral rna titers peaked 1-3 days later. the findings of these studies are summarized in table 1 . five 14-day-old gn pigs inoculated with 8.8 or 11.0 log 10 ge of two field pdcov strains oh-fd22 and oh-fd100 all showed severe diarrhea and/or vomiting at post-inoculation hours (pih) 21-24, which coincided with the detection of viral rna in feces (jung et al., 2015c) . six 14-day-old gn pigs inoculated with 10.1-10.8 log 10 ge [≈11 log 10 50% tissue culture infectious dose (tcid 50 ) or 9 log 10 plaque forming units (pfu) per pig (hu et al., 2015) ] of the tc-pdcov oh-fd22 p5, p20, and p40 all showed severe diarrhea and/or vomiting at pid 2, which also coincided with the detection of viral rna in feces . in this study, fecal viral rna titers peaked, ranging from 7.8 to 8.9 log 10 ge/ml, at pid 2-5 and then decreased gradually thereafter . another study using conventional 5-day-old pigs and a cell culture-adapted pdcov usa/il/2014 strain (p11) reported the onset of diarrhea at pid 5 in 5 of 5 pigs orally inoculated with 3 × 10 4 tcid 50 /pig of the virus, which was 1 day later or coincided with the detection of viral rna in feces at pid 4 (3/5 pigs tested) or 5 (2/5 pigs tested) (chen et al., 2015b) . fecal viral rna peaked at approximately pid 5-7 (chen et al., 2015b) . in another study using another strain genetically similar to the usa/il/2014 strain, four 10-dayold conventional and three 10-day-old gn pigs orally inoculated with 10 6 pfu (≈10 9 genomic rna copies) of a cell culture-adapted pdcov michigan/8977/2014 strain (p15) all exhibited moderate to severe diarrhea at pid 1 (conventional pig) and pid 3 (gn pig), respectively (ma et al., 2015) . fecal shedding occurred at pid 1 (1/4 pigs tested) or pid 2 (3/4 pigs tested) in conventional pigs, whereas fecal shedding was detected at pid 1 (1/3 pigs tested) or pid 2 (2/3 pigs tested) in gn pigs prior to the onset of diarrhea (ma et al., 2015) . in the infected conventional pigs, fecal viral rna titers peaked, ranging from 6 to 7 log 10 rna copies/gram, on pid 7 and then decreased gradually thereafter (ma et al., 2015) . based on experimental findings jung et al., 2015c; ma et al., 2015) , persisting diarrhea in infected nursing pigs is observed for approximately 5-10 days. prolonged fecal virus rna shedding was also evident in pdcov-infected nursing pigs. after recovery from disease, pigs continued to shed pdcov rna in the feces. the findings of these studies are summarized in table 1 . the gn pig inoculated with 8.8 log 10 ge of the field pdcov strain oh-fd22 developed diarrhea at pih 21-24, which was consistently observed until pid 7 (jung et al., 2015c) . four 10-day-old conventional pigs orally inoculated with 10 6 pfu of the michigan/8977/2014 strain all exhibited diarrhea at pid 1, which then persisted for another 6-9 days (ma et al., 2015) . the gn pigs inoculated with 10.1-10.8 log 10 ge of the tc-pdcov oh-fd22 p5, p20, and p40 all showed diarrhea at pid 2, which then persisted for another 4-6 days . hu et al. (2016) reported that similar to prolonged fecal virus rna shedding (until pid 20) observed in the wild-type pdcov oh-fd22-inoculated pigs, fecal virus shedding in the tc-pdcov oh-fd22 p5 or p20-inoculated pigs was detectable until pid 18 or 20, respectively . the tc-pdcov oh-fd22 p40-inoculated pig also had pdcov rna-positive feces until pid 15 . one of the four 10-day-old conventional pigs (25%) inoculated with the michigan/8977/2014 strain had detectable pdcov rna in the feces until pid 21 (ma et al., 2015) . there is little information on intestinal replication of pdcov during disease progression in pigs of varying ages. in previous experimental infection studies, most of the infected gn or conventional suckling pigs were euthanized at pid 3-7 (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . thus, the earlier, pathological characteristics related to pdcov infection at pid 1-3 are unknown. in experimental pedv infections in seronegative suckling pigs, during the incubation period, i.e. at approximately pih 12-24, pedv antigen-positive cells were seen throughout the small intestine and as many as 30-100% of the absorptive epithelial cells were positive (debouck et al., 1981; madson et al., 2015) , consistent with fecal virus shedding in subclinical pigs. after that, during the clinical period, moderate to large numbers of pedv antigen-positive cells were observed throughout the small intestine (debouck et al., 1981; jung et al., 2015a; madson et al., 2015) . whether pdcov also replicates similarly in the small intestine of nursing pigs, needs to be studied in well-controlled experimental settings with large numbers of pigs and multiple time-points. more information on the pathogenesis of pdcov in pigs at earlier timepoints (pids 1-2) is needed to fully understand disease progression. relative to experimental pedv infections (jung et al., 2015a; jung et al., 2014; madson et al., 2015) , pdcov-infected pigs also appeared to shed less pdcov rna and had lower peak shedding titers in the feces (chen et al., 2015b; jung et al., 2015c) , implying a potentially lower replication rate of pdcov in the gastrointestinal tract of pigs. studies are also needed to investigate if this might contribute to the lower mortality of pdcov-infected nursing pigs, as compared to pedv infections. based on field observations (anon., 2014; song et al., 2015) , pdcov caused a number of deaths (up to a 40% mortality rate in us and >80% in china) among suckling pigs. similarly, ped is the most devastating in nursing piglets causing 100% morbidity and 50-100% mortality saif et al., 2012) . several mechanisms by which pedv infection induces greater disease severity in nursing versus weaned pigs, have been defined (annamalai et al., 2015; jung et al., 2015a) . compared to 9-day-old nursing pigs that began to show severe clinical disease, villous atrophy and fecal virus shedding at pid 1, a longer incubation period of pedv was evident before 26-day-old weaned pigs shed fecal virus rna (by 1 more day) or developed lesions and clinical disease (by 2 more days) (annamalai et al., 2015; jung et al., 2015a) . there were significantly decreased functional natural killer cells in the ileum and blood of nursing piglets, compared to weaned pigs, which may have contributed to the greater susceptibility of nursing pigs to pedv infection (annamalai et al., 2015) . studies are needed to confirm if nursing pigs also have higher susceptibility to pdcov infection. anatomical and physiological factors that may contribute to the longer recovery of nursing pigs from ped include: 1) the slower turnover of enterocytes (5-7 days) in neonatal piglets compared to 2-3 days in 3-week-old weaned pigs (moon et al., 1973) ; and 2) the anatomically underdeveloped large intestine of nursing piglets that may increase their vulnerability to dehydration, compared to weaned pigs (jung et al., 2015a) . there was also a lack of crypt stem cells and lower numbers of proliferating crypt cells in the small intestine of nursing pigs, compared to weaned pigs (jung et al., 2015a) . this could lead to the slower turnover of enterocytes in nursing vs weaned pigs, contributing to a slower recovery from ped in nursing piglets and the greater susceptibility of nursing pigs to ped (diarrhea and dehydration). because of the similar pathological features between pdcov and pedv, studies are needed to confirm if pdcov infection may also induce greater disease severity and deaths of nursing piglets vs. weaned pigs by mechanisms similar to those contributing factors to pedv infection. viremia where viral rna in serum ranged from 10 0.5 -10 3 tcid 50 /ml was detected in conventional 5-day-old pigs (15/15; 100%) inoculated with the usa/il/2014 strain at pid 4 (incubation period) and pid 5-7 (clinical period) (chen et al., 2015b) . simi-larly, viral rna in serum ranged from 3.4-4.1 log 10 copies of pdcov rna/ml in 19-day-old gn pigs inoculated with the ohio wild-type field cvm1 strain at pid 1-3 (clinical period) (ma et al., 2015) . in another study, viral rna in serum ranged from 4.8-5.2 log 10 ge/ml in gn piglets (6/6; 100%) inoculated with the us tc-pdcov oh-fd22 during the clinical period at pid 3 . the early, severe diarrhea/vomiting might be accompanied by acute, transient viremia, but no one has yet confirmed the presence of infectious virus in the serum. similar to pedv infection of nursing pigs that was accompanied by early viremia (viral rna, up to 100%), viremia in pdcov-infected pigs might also be related to the severity of atrophic enteritis and structural alteration of tight and adherens junctions in the jejunal and ileal villous epithelium in the small intestine (jung et al., 2015b; jung et al., 2014) . there is a dearth of information on the innate and adaptive immune responses to pdcov. after pdcov infection, infiltration of macrophages, lymphocytes, eosinophils, and neutrophils were observed in the lamina propria of the small intestine (chen et al., 2015b; ma et al., 2015; wang et al., 2015a) . our study reported the development of pdcov-specific antibodies in serum of pdcovinfected pigs . the 14-day-old gn pigs orally inoculated with the original oh-fd22 and tc-pdcov oh-fd22 (p5, p20, and p40) had detectable serum igg, iga and virus neutralization (vn) antibodies at pid 14. thereafter, pdcov-specific igg, iga and vn antibody titers increased and remained high at the end of experiment, pid 23/24, when the pigs were fully recovered from clinical disease and fecal virus rna shedding. similarly, in pedv cv777-infected pigs, serum pedv-specific igg antibody was first detected at approximately pid 7-12 (de arriba et al., 2002) . 6.9. virulence of high cell culture-passaged pdcov (p80) the tc-pdcov oh-fd22 has been serially passaged >p90 on llc-pk cells. our study confirmed the enteropathogenicity of tc-pdcov oh-fd22 p5, p20, and p40 in gn pigs . the virulence appeared to be similar to that of the parent wild-type virus, as evident by severe diarrhea/vomiting, atrophic enteritis, and high levels of serum igg, iga and vn antibodies in inoculated gn pigs. our pilot study also revealed that two 10-day-old gn pigs inoculated orally with 10.2 log 10 ge of the tc-pdcov oh-fd22 p80 (passage 80) developed delayed (by 1-3 days), but severe clinical signs with high viral rna shedding titers in the feces, ranging from 7.7 to 8.8 log 10 ge/ml at pids 5 and 6, which is similar to those in gn pigs inoculated with the original field strain oh-fd22 or tc-pdcov oh-fd22 (cell passages 5-40). therefore, the tc-pdcov oh-fd22 needs to be further passaged to evaluate if higher cell-culture passaged virus becomes attenuated, because attenuation of the virulence of pedv strains has been induced through high cell-culture passages (93rd-144th) (chen et al., 2015a; kweon et al., 1999; sato et al., 2011; song et al., 2003) . the severity of clinical disease caused by pdcov in nursing piglets may be affected by co-infections with other viruses including rotavirus (rv) and pedv. however, the impact of dual pdcov and pedv or rotavirus infection on the disease outcome in pigs needs to be delineated. a survey conducted in the us reported that of 293 samples collected from diarrheic pigs in the us and canada in early 2014, 89 (30%) were pdcov-positive by rt-pcr (marthaler et al., 2014b) . of the 89 pdcov-positive samples, 69 (78%) were positive for pedv, group a rv (rva), rvb, or rvc. co-infections with pdcov and rvc were the most common [52 (58%) samples], and 29 (29/89; 33%) were also positive for pedv. our lab also identified that seven (41.2%) of 42 pdcov-positive samples, collected on different farms with diarrhea outbreaks in ohio and indiana, were also positive for pedv (3 samples) or rvc (4 samples) by rt-pcr (hu et al., 2015) . in mainland china, a surveillance study based on nested rt-pcr results reported that of 356 porcine diarrheic samples collected in jiangxi during 2012-2015, 120 (33.7%) were pdcov-positive; 231 (64.9%) were pedv-positive; and 281(78.9%) were positive for either pedv or pdcov (song et al., 2015) . coinfections with pdcov and pedv (19.7%) were common in diarrheic pigs in jiangxi, china. another study tested by rt-pcr reported that of 215 diarrheic samples collected in anhui, guangxi, hubei, and jiangsu during 2004-2014, 14 (6.51%) were positive for pdcov, 110 (51.2%) were positive for pedv, and 5 (2.3%) were positive for tgev. of the 14 pdcov-positive samples, 7 (50%) were also positive for pedv; and 2 were co-infected with pedv, tgev, and pdcov. in infected nursing pigs, similar to pedv and tgev infections saif et al., 2012) , gross lesions are limited to the gastrointestinal tract and are characterized by thin and transparent intestinal walls (proximal jejunum to colon) with accumulation of large amounts of yellow fluid in the intestinal lumen (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . the stomach is filled with curdled milk. the transparency and fragility of affected intestines appeared milder, as compared to pedv infections. in infected nursing pigs, similar to pedv and tgev infections saif et al., 2012) , histological lesions are characterized by acute, multifocal to diffuse, mild to severe atrophic enteritis in the proximal jejunum to ileum (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015; wang et al., 2015a) , occasionally accompanied by mild vacuolation of superficial epithelial cells in the cecum and colon that coincided with a few pdcov antigen-positive cecal and colonic epithelial cells (jung et al., 2015c) . no villous atrophy or histologic lesions were evident in the remainder of the small intestine, duodenum (chen et al., 2015b; hu et al., 2016; jung et al., 2015c) , which coincided with few pdcov antigen-positive duodenal epithelial cells. during acute infection, vacuolated enterocytes or massive cell exfoliation were seen on the tips or the entire villi in the jejunum and ileum. atrophied villi are frequently fused and covered with a degenerated or regenerated flattened epithelium. infiltration of inflammatory cells, such as macrophages, lymphocytes, neutrophils, and eosinophils, was evident in the lamina propria (chen et al., 2015b; ma et al., 2015; wang et al., 2015a) . no lesions were seen in other organs, such as stomach, lung, spleen, liver, kidney, and mln of infected pigs, all of which were also negative for pdcov antigen (chen et al., 2015b; hu et al., 2016; jung et al., 2015c; ma et al., 2015) . however, one study found an acute, focal, mild degeneration or necrosis of gastric epithelial cells in the gastric pits of stomach (ma et al., 2015) . pdcov is a novel enteropathogenic coronavirus in pigs. the disease related to pdcov was first reported in the us in early 2014, when pedv infections were also epidemic. however, since 2005-2006, dcovs already had been identified in rectal swabs of wild small mammals (asian leopard cats and chinese ferret badgers), domestic pigs, and wild birds. moreover, pdcov (chn-ah-2004 and ch/sichuan/s27/2012 strains), genetically related to the current us pdcov strains, has been present in chinese diarrheic pigs since 2004, prior to the first outbreak of pdcov infection in us swine. although birds are thought to be the suspect host for deltacoronaviruses, the detailed genetic relatedness of pdcov with other dcov isolates from wild birds and other mammal species should be studied further to clarify the origin of pdcov in pigs and also the source of the sudden emergence of us pdcov in us swine. us pdcov strains are enteropathogenic and infect villous epithelial cells of the entire small and large intestines, although the jejunum and ileum are the primary sites of infection. similar to pedv (alphacoronavirus) infections, pdcov (deltacoronavirus) infections also cause acute, severe atrophic enteritis accompanied by viremia (viral rna) that leads to severe diarrhea and/or vomiting, followed by dehydration as the potential cause of death in nursing piglets. differential diagnosis of pedv, pdcov and tgev is essential to control viral diarrheas in us pig farms. currently, cultivable, us tc-pdcov strains have been isolated and propagated in several laboratories. tc-pdcov strains will be useful to evaluate if high level cell-culture passaged viruses become attenuated and can be potential vaccine candidate strains. relative to pedv infections, causing similar, but more severe clinical disease and lesions, the lack of comprehensive understanding of the pathogenic characteristics of pdcov and effective preventive (pdcov vaccine) and therapeutic interventions has impeded biosecurity and development of methods for prevention and control of pdcov in us swine. it is also critical to understand the pathogenic aspects of pdcov infection distinct from those of pedv, possibly clarifying why pdcov infection results in distinctively lower mortality rates 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epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences development and application of an elisa for the detection of porcine deltacoronavirus igg antibodies detection and genetic characterization of deltacoronavirus in pigs porcine coronavirus hku15 detected in 9 us states porcine deltacoronavirus: histological lesions and genetic characterization complete genome sequence of porcine deltacoronavirus strain ch/sichuan/s27/2012 from mainland china coronavirus diversity: phylogeny and interspecies jumping comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this paper was in part supported by a grant from the oardc seeds grants program, grant ohoa1536 (jung k, pi). none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. the authors have no conflict of interest to declare. key: cord-320331-wtxja5i9 authors: cabbab, iris louise n.; manalo, rafael vincent m. title: anti-inflammatory drugs and the renin-angiotensin-aldosterone system: current knowledge and potential effects on early sars-cov-2 infection date: 2020-10-08 journal: virus res doi: 10.1016/j.virusres.2020.198190 sha: doc_id: 320331 cord_uid: wtxja5i9 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the etiologic agent of coronavirus disease 19 (covid-19), and is genetically related to the 2003 sars and middle east respiratory syndrome (mers-cov) coronaviruses. recent studies have reported that similar to sars-cov, this strain expresses a spike protein (s) with a receptor binding domain (rbd) that binds to angiotensin-converting enzyme 2 (ace2) – an enzyme expressed mostly in the endothelium, kidneys, heart, gastrointestinal tract and lungs – to facilitate viral entry and intracellular replication. incidentally, the renin-angiotensin-aldosterone system (raas) is integral to physiologic control of both ace and ace2 expression, and is an essential system utilized by sars-cov-2, albeit with varying schools of thought on how it can affect viral entry. in this paper, we will review current knowledge on the raas and how it can be affected by non-steroidal anti-inflammatory drugs (nsaids) and corticosteroid use at the organ and cellular levels. we will then discuss the relevance of these interactions on organ-specific ace2 expression, and provide scientific insights on how this mechanism can potentially affect sars-cov-2 infection in the early phases of disease. from the standpoint of other known viruses, we will then aim to discuss the potential uses or restrictions of these drugs in viral infection, and provide an update on relevant studies about covid-19. coagulation (dic). likewise, it is prudent to assume that cfr is better interpreted after agestandardization is done, or when it is adjusted for the presence of comorbidity, which has been done in some reports including that of the centers for disease control and prevention (cdc) [68] . a possible contributor to the risk of severity or to variations in the cfr, however, may also come from the patient's medications. there have been studies proposing that covid-19 morbidity and mortality are a result of excessive inflammation, such as airway inflammation and cytokine storms [114] , and infected patients in the early phases of disease may experience malaise or fever and may seek symptomatic relief through analgesics like paracetamol and non-steroidal anti-inflammatory drugs (nsaids) such as ibuprofen. similarly, patients with chronic inflammatory diseases may take nsaids or corticosteroids as management for their disease, which may increase their susceptibility to microbial infection. since covid-19 is a viral illness, there have been concerns that anti-inflammatory drug use can aggravate sars coronavirus infections [20, 45] , but evidence on this speculation has not been unified and is even contradicting. interestingly, the membrane receptor of sars-cov-2, which is ace2, is an essential component of the regulatory mechanism of the renin-angiotensin-aldosterone system (raas), and this hormone pathway may interact with the pathophysiology of covid-19. to this end, this paper will review the mechanisms of the raas and its coincidence with the anti-inflammatory pathways of nsaids and corticosteroids both at the cellular and organ levels. we will discuss current evidence on the effects of these anti-inflammatory drugs on sars-cov-2 infection, as well as the potential interactions of pathways upstream or downstream of ace2 on the raas, in an effort to bridge these pathways in the setting of covid-19. based on the evidence collated, we will then compare the anti-inflammatory drug classes included in this review to determine the potential uses or restrictions of these drugs in early sars-cov-2 infection. for this review, we did a literature search at ncbi pubmed and medline databases to gather current studies on the covid-19-raas-anti-inflammatory drug interactions using the terms 'sars-cov-2' or 'covid-19', matched with 'renin-angiotensin system', 'renin-angiotensin-aldosterone-system', 'raas' or 'ras', which yielded 374 unique results. we further narrowed the search by including only the studies which investigated the effects of anti-inflammatory drugs or corticosteroids on covid-19, using additional terms for the string such as 'corticosteroids', 'nsaid', 'steroids', 'anti-inflammatory' or 'inflammation', and by narrowing the publication type to 'journal article', 'clinical trial', 'review', 'systematic review', 'meta-analysis', or 'randomized controlled trial'. we then analyzed each article to include only those that proposed either the mechanisms of action of sars-cov-2 on the raas or of the drugs that interact with sars-cov-2 or the raas, to a final count of 63. from the following studies, all journal articles, clinical trials, systematic reviews and meta-analyses, excluding literature reviews, were included only if they provided experimental or clinical evidence, which narrowed the inclusion to five articles (two retrospective cohort studies, one case series, one in silico analysis, and one in vitro study). a second, less-stringent search was done which included studies on anti-inflammatory or corticosteroid drug use for covid-19 with or without associations to the raas, using the similar terms as mentioned above. this search returned seven studies which were either systematic reviews or meta-analyses or both, and were included in this review (supplementary file 1). for the said searches, we considered articles that are not only in the english language, but also those written in chinese, dutch, french and russian. the two literature searches were accomplished on august 6. on the other hand, we included studies on the epidemiology of covid-19 and the molecular biology of sars-cov-2 based on chronology to reflect the turn of events during the pandemic. for most of the review, basic science researches that explain or complete mechanistic pathways on the physiology and biology of the raas or of the mechanisms of anti-inflammatory drugs and corticosteroids were referenced from pubmed and google scholar at our discretion. in addition, pertinent references j o u r n a l p r e -p r o o f suggested by the reviewers were included as needed, especially those that were not covered at the time of the literature search. it is important to note that since the approach of this paper is to provide current knowledge on the anatomic, physiologic and molecular bases of anti-inflammatory drug and corticosteroid action on the raas, this paper will not demonstrate a systematic review or meta-analysis of current clinical evidence, but will only provide insight on the probable influences of the discussed pathways on early sars-cov-2 infection. further, since there is a present lack of definitive studies investigating the association between nsaid or corticosteroid drug use and covid-19 outcome severity, such clinical analyses are only warranted as more evidence is obtained. in late of december 2019, dr. wang and researchers from the institute of pathogen biology in china obtained the complete sequence of sars-cov-2 from bronchoalveolar lavage specimens of patients in wuhan presenting with acute respiratory distress syndrome (ards) [7] . a subsequent study then extracted sars-cov-2 dna from nine patients with covid-19, which found a 79% and 50% identity with sars-cov and mers-cov, respectively [8] . similarly, another study reported an 86% similarity between the sars-cov and sars-cov-2 genomic sequences, with high degrees of homology to the sars-like coronaviruses isolated from bats [9] . since sars-cov was known to bind to human angiotensinconverting enzyme 2 (ace2) for viral entry, these data suggested that covid-19 may also utilize its high affinity binding for human ace2 to enter host cells, by virtue of its conserved receptor binding domain (rbd), which was later confirmed to be true [9, 10, 11] . in 2017, khan et. al. developed a pilot clinical trial using human recombinant soluble ace2, also known as gsk2586881, as a potential treatment for acute lung injury. the two-part clinical trial, although showed a successful interaction of hrsace2 and its targets by evaluating the modulation of ras, did not elaborate any clinical changes or improvements j o u r n a l p r e -p r o o f upon administration of gsk2586881 [73] . it did, however show an increase in ang(1-7) synthesis from ang ii, offering protection against heart failure with reduced and preserved ejection fraction largely due to ace2 deficiency, as well as preventing ang ii-induced myocardial hypertrophy, diastolic dysfunction, and myocardial fibrosis [74, 75] . earlier this year, monteil et. al did a similar study where they engineered human blood vessel organoids and human kidney organoids, both of which expressed a sufficient amount of ace2, to emulate sars-cov-2 infection in human tissues and organs. this idea was built on the knowledge that sars-cov-2 mainly uses ace2 as the entry point into cellular invasion. by introducing human recombinant soluble ace2 (hrsace2), the s-protein region of sars-cov-2 was competitively bound by the enzyme, which effectively limited the binding and entry of sars-cov-2 thereby preventing infection in a dose-dependent manner [76] . several more clinical trials emerged which suggested the importance of hace2 to sars-cov-2 infection, and which led hrsace2 to become one of most promising therapeutics for the early prevention of covid-19 to date. there were six amino acids identified in the rbd that is crucial for ace2 binding, and while five of these differed between sars-cov and sars-cov-2, the binding affinity of sars-cov-2 rbd to human ace2 receptors was found to be 10 times higher than its counterpart spike protein in sars-cov [12, 69] ). this ability of sars-cov-2 to bind the ace2 enzyme with a stronger affinity than sars-cov and hcov-nl63 under a non-ideal configuration strongly implies that prior mutations have been occurring as a form of natural selection. that is, sars-cov-2 may have been circulating the wet animal markets for several years before the outbreak and infecting individuals without triggering any symptoms [12, 13] . the downstream effect of the ace2 ligand-receptor complex is the degradation of angiotensin ii (ang ii) to produce angiotensin (1-7), initiating a negative feedback mechanism that inhibits further production of ang ii. decreased ang ii results in decreased salt and water reabsorption, which lowers blood pressure and vascular tone, and effectively prevents the excessive effects of ang ii [14, 15] . when the sars-cov-2 viral spike s protein binds the human ace2 enzyme, it fuses with the cell membrane to j o u r n a l p r e -p r o o f initiate viral entry by activating signal transduction pathways, resulting to cell uptake of the virusreceptor complex via a process called clathrin-mediated endocytosis. moreover, the s protein of sars-cov-2 possesses an s1/s2 site, which is cleaved by furin, to encourage entry of sars-cov-2 into the cell while evading the host immune response [16, 17, 18, 64] . in the process, ace2 is inhibited and internalized, and reduced membrane ace2 affects the homeostasis of raas due to decreased ang ii breakdown. ang ii is a direct vasoconstrictor known for its predominantly vasoactive properties, and its excessive production is associated with acute hemodynamic changes, such as hypertension and a drop in glomerular filtration rate (gfr). this triggers chronic inflammation if left unaddressed, and eventually leads to end-organ failure due to fibroid formation [70, 71, 72] . ace2 is an active homologue of ace seen in a diverse group of tissues including the oral and nasal mucosa, the lungs, the gastrointestinal tract, blood forming organs, liver, kidney, brain, and endothelial cells, with the majority seen localizing in the tongue and esophageal epithelial cells, enterocytes, male reproductive cells, type ii lung alveolar epithelial cells, renal tubules, ductal cells, bladder urothelial cells, endothelial cells, and cardiomyocytes [77, 78, 79, 80] . interestingly, the number of organs that express ace2 continues to increase as more data is published, which can be monitored using the covid-19 cell atlas database (https://www.covid19cellatlas.org/). the ramifications on the ability of the virion to provoke cellular or tissue damage remain intriguing, but for one, it is dependent not only on the presence or absence of ace2 but also on the amount of ace2 on a certain organ or tissue. one of the most commonly studied regions of sars-cov-2 entry is the respiratory system, for which it is thought to be the main route of infection, transmission, and pathophysiology. the specialized type ii alveolar cells express an abundant amount of ace2, and ace2 inactivity due to sars-cov-2-induced j o u r n a l p r e -p r o o f depletion early in the infection may disturb the ras homeostasis, leading to impaired tissue repair mechanisms, increased vascular permeability, fluid accumulation in extra-alveolar spaces, and oxidant/antioxidant imbalance [81] . in a study by ackerman et. al., which examined postmortem lungs of patients who died from covid-19 and compared them to 7 lungs obtained from patients who died from ards secondary to ah1n1 and to 10 age-matched healthy lungs, covid-19 patients were found to suffer more frequently from severe endothelial injury due to cell membrane damage by presence of sars-cov-2. widespread thrombosis with microangiopathy was seen, and patients were 9 times more likely to experience alveolar capillary microthrombi, and 2.7 times more likely to experience intussusceptive angiogenesis than with flu [82] . this phenomenon of thrombosis and other vascular events in covid-19 is likely the result of an abundance of ace2 in endothelial cells, which permits sars-cov-2 infection along the endothelium. this will be further discussed in later sections. sars-cov-2 has also been studied to disseminate into the cns, by initially invading the peripheral nerve terminals via the synapse-connected route, through the mechanoreceptors and chemoreceptors in the lungs, and into the medullary-cardiorespiratory center [83, 84] . however, ace2 is not particularly abundant in neurons, and other pathways of infection are being investigated. the oral and nasal mucosa also holds a significant distribution of ace2, especially on the tongue epithelial cells, which may explain its route of infection [80] . if and how the virus affects the activity of the tongue epithelial cells were investigated by a cross-sectional study in l. sacco hospital in milan, italy, where the prevalence of olfactory and taste disorders in hospitalized covid-19 patients were obtained. of the 88 hospitalized covid-19 patients, all 59 patients who were coherent and responsive reported the persistence of olfactory and taste disorders throughout the disease [85] , indirectly supporting the hypothesis of oral and mucosal routes of invasion to the nervous system. interestingly, ace2 population is also pervasively seen in pancreatic cells, which in fact shows a higher expression profile than pulmonary cells. this possibly makes the pancreas, by virtue of ace2-dependent viral entry, one of the major targets of sars-cov-2. complications may include acute pancreatitis resulting from direct injury to pancreatic acinar cells or uncontrollable systemic inflammatory response from cytokine storm syndrome, both of which have been documented in the disease [86] . acute pancreatitis due to sars-cov-2 infection could pose an additional threat to people afflicted with diabetes mellitus (dm) types 1 and 2 by promoting metabolic complications as the infection continues to alter pancreatic β-cell function, which may in fact explain the feared risks of severe covid-19 among patients with dm [87] . in the kidneys, the ace2 expression in the proximal tubules remains the highest in concentration, with markedly lower concentrations in the glomeruli [88] . stz-treated rats in previous experiments were found to have higher ace mrna/ace2 mrna ratio, a marker of kidney damage that resulted in renal injury similar to human diabetic nephropathy [89, 90] . due to a higher ace/ace2 ratio, there was less ang(1-7) synthesized in the kidney, which can effectively eliminate its renoprotective effects [88] . in an experimental study using c57bl/l mice models, investigators compared the physiology of normal 20-22 week-old c57bl/6 mice against ace2 -/y mice which lacked ace2 expression. the enos expression in both protein and mrna levels and no concentrations in the aortas of ace2 -/y mice were decreased, along with urine and plasma nitrite concentration. on the other hand, lipid peroxidation was markedly increased, in contrast to a decreased superoxide dismutase level in the aorta homogenates of ace2 -/y mice subjects -an indication of impaired antioxidant activity leading to the formation of reactive oxygen species (ros) [92] and observed vascular dysfunction in ace2-negative c57bl/6 mice [91] . disturbances in the oxidative activity of the vascular system could pose a risk for acquiring inflammation, vascular remodelling and vascular injury, eventually leading to activation of the coagulation cascade that could cause microthrombi formation [92, 115] . taken together, these correlate to the negative consequences impacting the vascular homeostasis of sars-cov-2-induced ace2 deficiency, with partial relations to the renin-angiotensin-aldosterone system (raas), which will be discussed. j o u r n a l p r e -p r o o f a correspondence by fang et al published at the lancet this march discussed that hypertensives and diabetics taking ace2 inhibitor (acei) and angiotensin receptor blocking (arb) drugs may be at an increased risk of infection and severity by sars-cov-2 and covid-19, respectively, citing three studies wherein diabetes and hypertension were major comorbidities of patients with severe covid-19 and of non-survivors [20] . several criticisms were received by this article, mainly due to the lack of clinical evidence for many of the author's claims at the time. a descriptive study on the pharmacologic characteristics of 96 covid-19 patients in the toulouse university hospital intensive care unit (icu) in france showed that many of the patients who were intubated had either taken paracetamol (33.8%), arbs (28.2), calcium channel blockers (15.5%), corticosteroids (15.5%), or metformin (14.1%). however, these patients also had pre-existing co-morbid conditions that warranted medication intake, such as obesity, arterial hypertension, and diabetes mellitus, which may regard the evidence as coincidental at best. since the study was also descriptive (case series), the results and their implications can only be suggestive [93] . however, a drug interaction cannot be ruled out. in terms of the raas, interaction between arb/acei drugs and viral entry is plausible, since these drugs either inhibit ace or the receptor of its enzymatic product angiotensin ii, and a known physiologic response of cells to inhibition or internalization is feedback upregulation [21] . since the known human receptor of sars-cov-2 is ace2, then it is possible for hypertensives chronically taking aceis to be susceptible to sars-cov-2 infection, if feedback upregulation of ace2 expression does occur, or due to the patients' impaired ability to decrease ang ii secondary to renin production, all of which may increase membrane ace2 levels [22, 23] (fig. 1) . a similar pathway is expected for arbs, since inhibition of angiotensin receptors can trigger a feedback mechanism to increase ang ii synthesis resulting to an upregulation of ace2 expression ( fig. 1 ), and this has been shown in previous studies [22, 24, 25, 26] . several other studies have since followed this hypothesis closely, and have in the process unraveled the role of the renin-angiotensin-j o u r n a l p r e -p r o o f aldosterone system (raas) on the observed clinical outcomes of some covid-19 patients, albeit with a surprisingly different mechanism. two experimental studies, one in silico and another in vitro, have clarified and suggested the role of the raas, as follows. an in silico approach, which utilized the coxpresdb v7, kyoto encyclopedia of genes and genomes (kegg) pathway and the human protein atlas databases to determine the co-expression, molecular interactions and organ-specific expression of ace2, neprilysin and carbonic anhydrase (ca), have shown that these three enzymes were highly coexpressed in the local ras, with important overlapping functions on the production of ang-(1-9) and ang-(1-7) for the regulation of systemic vascular resistance and the conversion of co 2 in times of hypoxia. further, the authors found that these enzymes were most likely co-expressed at organs of interest such as digestive, renal, respiratory, and reproductive systems, with the exception of ca which is ubiquitous [94] . likewise, an in vitro study showed that lung epithelial cells infected with sars-cov expressed 11 unique ras genes within a 48-hr period, which correlated well with the time of viral infection. these genes were divided into those that were significantly upregulated during first 24 hrs (ace, ace2, insulin-like growth factor 2 receptor (igf2r), angiotensinogen (agt), epidermal growth factor receptor (egfr), matrix metalloendopeptidase (mme), alanyl aminopeptidase (anpep)) and those that were significantly upregulated by the 48th hr (arginyl aminopeptidase (arpep), egfr, anpep, neurolysin, ace2, igfr, leucyl and cysteinyl aminopeptidase (lnpep) and cathepsin d), all of which has clearly shown the early response of the raas on sars infection [95] . studies have also recently reported that cleavage of ace2 due to the interactions with the viral s protein leads to inhibition of ace2 function through the ras ace/ang ii/at1a arm [61, 62] . another study has suggested that excessive activation of raas by sars-cov-2 plays a role in the onset of acute respiratory distress syndrome (ards), a severe form of acute lung injury [63] . in the presence of ards, fluid builds up in the alveoli, damaging the surfactant and promoting inflammation, eventually leaving fibrotic scars that prevents complete oxygen uptake. it is important to note that the binding of the sars-cov-2 spike rbd to ace2 effectively inhibits j o u r n a l p r e -p r o o f the enzyme receptor, leading to endocytosis and early ace2 depletion. this mechanism, which can interact with many other pathways, can also lead to the onset of ards, by virtue of feedback activation of the raas or by other mechanisms dependent solely on ace2. to date, there seems to be no sufficient clinical evidence that can confirm the biological relevance of these experimental results. in a retrospective multicenter cohort study of 154 covid-19-positive geriatric patients in belgian nursing homes, a non-significant association was observed between arb/acei use and severe or poor outcomes (or 0.48; ci 0.10-1.97) [96] . in another retrospective cohort study of 117 geriatric covid-19 patients with diabetes mellitus (dm) in daegun, south korea, use of acei/arb drugs did not significantly affect the severity of outcome of covid-19, apart from an incidental protective effect against acute cardiac injury (or 0.048), which was expected from the mechanism of action of the drugs [97] . another review article discussing the interaction between raas inhibitors and ace2 in the context of covid-19 claimed that although angiotensin-receptor blockers and mineralocorticoid blockers increased ace2 expression in a number of experimental and clinical models, ace2 inhibitors, while increasing cardiac ace2 mrna levels, had no effect on ace2 activity and expression. a direct inhibitor of renin, aliskiren, in contrast was associated with a decrease in ace2 expression but the mechanism of action remains to be studied further [98] . in contrast, wysocki and authors investigated the effects of aceis and arbs on membrane and total ace2 expression in lung and kidney cells and lysates, respectively, and found that use of these drugs do not significantly affect both the membrane and total ace2 protein levels in mouse lungs. likewise, acei (captopril) and arb (telmisartan) administration did not increase membrane ace2 levels in mouse idneys, but rather decreased them. although preliminary, this study provided direct evidence supporting continued use of aceis and arbs in covid-19, which also implied that feedback upregulation of ace2 does not occur even if the ras pathway is inhibited, at least for a span of 2 weeks [99] . it will also be interesting to j o u r n a l p r e -p r o o f determine similar responses to aceis and arbs in mouse models of hypertension, either genetic or nongenetic, to determine the consistency of these results in the setting of compensatory mechanisms. besides these studies, others that directly assessed the clinical effects of acei/arb drugs on covid-19 outcome severity were not found during the literature search. hence, the direct negative effects of acei/arb drugs on covid-19 outcome, as earlier hypothesized by clinicians and researchers, are at best experimental and, to the best of our knowledge, not yet supported by clinical evidence. a more in-depth discussion on the interactions between sars-cov-2 entry and acei/arb drugs can be found elsewhere, and readers are referred to those materials for more information. the renin-angiotensin-aldosterone system (raas), or simply the renin-angiotensin system, is a strong physiologic pathway that controls various processes relating to salt and water homeostasis (fig 1) . classically, the chronological order of the raas begins with the cleavage of angiotensinogen into ang i by renin, followed by the conversion of ang i into ang ii by ace which occurs extensively in the lungs. ace2 catalyzes both ang i and ang ii to produce ang (1-9) and ang (1-7), which provides negative feedback to ang ii thereby modulating its effects. prior to ace2 interaction, ang ii modulates the raas promotes vasopressin release from the posterior pituitary gland, and directs the adrenal gland to produce aldosterone, which then promotes na + and h 2 o retention in normal physiology. in patients with cardiovascular disease, available ang ii reacts with angiotensin 1 receptor (at 1 r) to stimulate pathologic behavior such as vasoconstriction, oxidative stress and profibrotic events. ang ii is then converted into ang (1-7) by ace2, followed by the activation of at 2 r and masr. elevation of ang ii may increase plasma aldosterone levels through the regulatory action of adrenocorticotropic hormone contrast, ang-(1-7) increases urinary na + excretion, endothelial oxide activity, insulin sensitivity [100] . this further shows that the ace 2/masr/at 2 r axis performs an opposite reaction with the ace/ang ii/at 1 r axis, offering protection against hypertension, fibrosis, and oxidative stress [65] . as mentioned above, sars-cov-2 exploits and depletes these ace2 receptors to facilitate viral host cell entry and increase its pathogenicity. the raas is dysregulated following ace2 depletion, promoting effects downstream of ang ii and therefore increasing aldosterone secretion. aldosterone acts by inhibiting sodium excretion in the distal convoluted tubule and collecting ducts via the mineralocorticoid receptors. while na + is reabsorbed and h 2 o is retained, potassium is consequently excreted, and an excess can lead to wastage and hypokalemia. this is consistent with the findings of chen et. al. from a cohort study of 175 covid-19-positive patients, wherein they classified patients as either being severely hypokalemic (17.71%), hypokalemic (36.57%), or normokalemic (45.71%). they found that patients with higher hypokalemia also had increased creatinine kinase (ck), lactate dehydrogenase (ld) and c-reactive protein (crp) levels, along with a higher body temperature, which were consistent with inflammatory and catabolic pathways [101] . some suggest that the observed hypokalemia among covid-19 patients is not isolated but rather is part of a general electrolyte imbalance. on the other hand, aldosterone in the setting of a dysregulated raas may or may not be the cause of hypokalemia in some covid-19 cases, but rather tubular dysfunction, such as renal tubular acidosis. however, studies on this aspect remain elusive. nonetheless, there is evidence that potassium levels, which are mainly regulated by aldosterone, are commonly deranged in covid-19 patients, and this could be due to interactions with the raas. in the same correspondence published at the lancet recommending the exercise of caution for covid-19 patients under acei and arb drugs, ibuprofen and thiazolidinediones were mentioned to potentially increase ace2 expression [20] . ibuprofen is under the class known as non-steroidal anti-inflammatory drugs (nsaids), and acts by non-selectively inhibiting cyclooxygenase enzymes (cox) 1 and 2, which confers its anti-inflammatory and antipyretic effects through the inhibition of prostaglandin synthesis from arachidonic acid [34, 35] . of the four prostaglandins synthesized by this pathway, pge 2 and pgi 2 , along with the prostanoid thromboxane txa 2 , are produced specifically in the kidneys, while pgd 2 is produced in the airways, and they act by interacting with rhodopsin-like g-protein coupled receptors or gpcrs [35] . interestingly, prostaglandins, particularly pge 2 and pgi 2 , have been well-studied and have been shown to affect kidneys by improving blood flow through vasodilation. under conditions of reduced renal flow, this mechanism leads to increased tubular flow, increased renal perfusion, and increased glomerular filtration rate, with a concomitant increase in the secretion of potassium ( fig. 1 and 2 ). this is supported by the fact that pge 2 regulates sodium and water reabsorption, while pgi 2 increases the production of renin --with the latter increasing salt and water retention and hence blood volume, while pge 2 counteracts this process via volume/pressure natriuresis [36]. since pge 2 is the main prostaglandin in the kidney, cox inhibition leads to an overall increase in sodium and water reabsorption. because ibuprofen and other nsaids decrease renal perfusion by increasing systemic vascular resistance, a vicious cycle can be formed by tricking the body to sense that there is reduced renal perfusion, leading to an inappropriate upregulation of ace to return kidney perfusion back to normal via ang ii (fig. 2) . concomitantly, an upregulation of ace2 is expected by virtue of its role as a physiologic antagonist of ang ii, by catabolizing ang ii to ang (1-7) to reduce vasoconstriction, reduce sodium and water retention, and provide reno-and cardio-protective effects [14, 19, 37, 38] . hence, the reduction in prostaglandin e 2 and i 2 syntheses is the main mechanism by which nsaids directly contribute to increased ace2 expression, which can be utilized by sars-cov-2 (fig. 2) . studies have shown that the raas is implicated in acute pulmonary injury, pulmonary hypertension, and pulmonary fibrosis [39] . likewise, raas is indeed activated in idiopathic pulmonary arterial hypertension [40] , and the expression of angiotensin receptor 1, ace, and ace2 is upregulated in the lungs as a response to lung pathology [39, 41, 42] . hence, ace2 may also be increased in pulmonary tissues secondary to ace-induced promotion of the raas. further, it has been shown that cox-2 and pge 2 mediate an antiviral response in lung epithelial cells infected with human coronavirus, and their expression results in decreased coronavirus replication [43] . similarly, pgd 2 , a prostaglandin known to be expressed in the brain and airways, mediate an anti-inflammatory response that may protect against acute respiratory distress and acute lung injury [44] . hence, the use of nsaids not only decreases pge 2and pgi 2 -mediated regulation of kidney function, but also pgd 2 -mediated protection against acute lung injury and pge 2 -mediated antiviral responses, which may lead to an enhanced coronavirus replication in the lung epithelia. a review of clinical evidence has shown that ibuprofen and naproxen improved the symptoms of influenza, and naproxen combined with clarithromycin and oseltamivir reduced both the rate and the duration of hospitalization of patients with influenza pneumonia. however, no evidence was provided for covid-19 [102] . the observed benefits in influenza infection may be due to the reduction in airway inflammation which is common in colds, and which is also observed in severe influenza. however, regarding viral entry and replication, this clinical evidence does not directly support nor reject any of the mechanisms discussed, especially with regards to sars-cov-2 infection. a similar systematic review of various nsaids, corticosteroids, and specific immunosuppressive or immunestimulating drugs have shown no strong evidence supporting the use of nsaids for covid-19; however, indomethacin was noted to have shown direct antiviral activity against sars-cov and canine coronavirus (ccov) in vitro [103] . the resulting benefit is not expected to contradict the mechanisms discussed; j o u r n a l p r e -p r o o f rather, it must have been a function of the structure of indomethacin, which is reflective of the authors' comment that the results were unexpected since indomethacin is a cox inhibitor [104] . a retrospective cohort study published in 2005 showed that patients in hong kong infected with sars-cov, which were given corticosteroids to manage acute respiratory distress, experienced higher rates of adverse outcomes despite favorable baseline characteristics. further, analysis showed that sars patients treated with corticosteroids (hydrocortisone, methylprednisolone) were at a 20.7-fold higher risk of icu admissions or mortality, independent of age and severity of disease [45] . this study alone, albeit based on sars-cov, showed that corticosteroid use may in fact increase the risk of unfavorable outcomes for coronavirus-infected patients. however, data supporting the validity of this conclusion on other coronavirus infections are indeed warranted. to note, a systematic review and meta-analysis of 15 studies on the management of coronavirus pneumonia showed that patients with severe coronavirus symptoms were more likely to require corticosteroid therapy (rr 1.56; ci 1.28-1.90), but that they were also twice as likely to contract bacterial infection (rr 2.08; ci 1.54-2.81) and were twice as likely to succumb to the disease (rr 2.11; ci 1.13-3.94) [105] . however, this systematic review and meta-analysis was not only limited to covid-19 (2 studies), but included other coronavirus infections such as those caused by sars-cov (11 studies) and mers-cov (2 studies), which may allow for differences in the prognosis and clinical outcomes of covid-19 patients. another systematic review and meta-analysis of 11 studies on corticosteroid use in coronavirus infections showed a statistically significant delay in virus clearing of 3.78 days (ci 1.16-6.41), but with no significant changes in hospitalization duration, use of mechanical ventilation, or survival [106] . in these two studies, the results may have differed mainly because the included studies were mostly retrospective in nature. hence, baseline characteristics and management may not have been the same within a singular study, and this may have affected the recommendations assessment, development, and evaluation) approach, which was mainly due to the nature of the studies included (mostly retrospective cohort studies). in addition, the inclusion of outcomes from non-coronavirus infections may have significantly confounded and affected the analysis of outcomes. on the other hand, a systematic review showed that corticosteroids may help reduce immunopathological damage and may improve outcomes when given high-dose with a quinolone early in the infection, but these results were specific for sars-cov and not for sars-cov-2 [103] . to resolve the contradicting reports, randomized controlled trials of corticosteroid use and its benefits or risks to covid-19 outcomes are needed, especially if more accurate results are to be generated. to date, there seems to be emerging clinical evidence that caution must be exercised in the use of corticosteroids for coronavirus-infected patients. however, due to the varying and conflicting results of their effects on clinical outcome, more specific and better designed studies are required for a better evaluation of current recommendations. notwithstanding, the observed risks are supported experimentally and pharmacologically as will be discussed. first, note that corticosteroids are divided into the mineralocorticoids and the j o u r n a l p r e -p r o o f glucocorticoids. mineralocorticoids comprise a group of proinflammatory hormones that primarily act on the kidney to modulate renal-sodium balance and blood pressure by activating mineralocorticoid receptors (mr) [46] (fig. 3) . it is thought to be proinflammatory, acting through the upregulation of inflammatory cytokines to influence t and b lymphocytes, monocytes, neutrophils, and dendritic cells to mount an immune response. this interaction subsequently promotes inflammation and increased levels of reactive oxygen species (ros) following the activation of hormonal response elements (hres) inside the nucleus (fig 4) [47] . in pathological conditions, the mineralocorticoid receptors (mrs) can remain activated regardless of ang ii levels, interfering with normal salt and water retention and electrolyte regulation. this eventually leads to the exacerbation of hypertension, which if not promptly addressed, can lead to progressive organ injury and finally organ failure. glucocorticoids on the other hand affect insulin metabolism, hepatic synthesis, and muscle protein catabolism by increasing amino acid production, and increase the availability of glycogen stores to buffer blood glucose concentration. insulin resistance triggers an increase in blood glucose, which causes plasma volume depletion secondary to osmotic diuresis (fig. 3) . in response to resulting plasma volume reduction, the juxtaglomerular cells of the kidneys will produce renin to act upon liver angiotensinogen to form ang i, followed by an ace-mediated conversion to ang ii in an effort to activate the pathway called the renin-angiotensin system to return plasma volume back to normal. subsequently, ace2 can increase proportionally with ang ii, acting to produce ang (1-7), which we hypothesize is the mechanism for glucocorticoid-enhanced sars-cov-2 virulence (fig. 3) . on the other hand, glucocorticoids suppress inflammation by binding to glucocorticoid response elements (gres) inside the nucleus to interact with genes and affect gene transcription in three basic ways (transactivation, trans-repression, and cis-repression) to suppress inflammation or to prevent an immune response from occurring altogether [47] (fig. 4) . j o u r n a l p r e -p r o o f recent guidelines released by the uk national health service (nhs) on the intake of ibuprofen for mild covid-19 symptoms has drawn flak from some health experts as it may pose risks on increased severity [48] . reduction of prostaglandin levels in the body, which is the main action of nsaids like ibuprofen, effectively reduces the immune response, and previous reports have shown no benefit of taking ibuprofen in symptom control and clinical outcome for respiratory infections, and have been shown to worsen the prognosis of community-acquired pneumonia [49, 50] . in a randomized, placebo-controlled study for ibuprofen use in managing respiratory syncytial virus (rsv) infection, ibuprofen was not able to prevent lung consolidation and was shown to increase viral shedding [51] . from these studies, we can infer the classical mechanism by which anti-inflammatory drugs aggravate infection, which is to reduce the degree of immune response that the body can mount against a virus and to allow uncontrolled progression. when a virus infects its host, it most often remains intracellular as viruses are obligate intracellular organisms, and extracellular viruses are most likely seen only during the lytic phase. when viruses infect a cell, antigen-presenting cells alert nearby immune cells through major histocompatibility complexes (mhcs) of the virus' intracellular presence. concomitantly, toll-like receptors (tlrs) recognize viral nucleic acids intracellularly and in effect promotes the cellular expression of interferons and inflammatory cytokines that act in an autocrine and paracrine fashion to induce an antiviral state in the host and in nearby, uninfected cells via the jak/stat pathway, and to alert the immune cells of an ongoing invasion [52, 53] . type i interferons have also been shown to promote prostaglandin synthesis [54] , the latter acting as a mediator between the thymus and the bone marrow to promote cellular and humoral types of immunity [55] . a previous study has shown that positive-strand rna viruses, to which the coronaviruses belong, induce the formation of the endoplasmic reticulum double membrane vesicles (er-dmvs) that promote viral replication and protect viruses from immune detection, and j o u r n a l p r e -p r o o f treatment with interferon-beta significantly reduces these viral-induced membrane structures [56] . hence, suppression of prostaglandin synthesis by the use of nsaids may effectively reduce cellmediated and humoral immunity against sars-cov-2, and may modulate immune cells towards the production of il-10 for t-cell suppression and suppression of phagocytosis, particularly by pge 2 [57, 58] . it is however noted that prostaglandins such as pge 2 act differently in different viral infections, and may promote viral replication and shedding in other viruses, such as herpes simplex virus (hsv), enterovirus 71, and cytomegalovirus, to name a few [59, 60] . hence, the effect of nsaids on different viral infections may differ, but for sars-cov-2 and for other viruses such as hepatitis b and human immunodeficiency virus (hiv), taking ibuprofen and other nsaids of similar action may aggravate viral replication. additional risks by nsaids however lie in its ability to increase ace2 expression by activating a gene called adam17, a metalloprotease that upregulates ace2 by enabling ace2 shedding, resulting in increased covid-19 infection [108] . from here, severity and susceptibility against covid-19 is expected to increase as viral load relatively increases. only recently, a prothrombotic state has been associated with covid-19 infection, and this has led to complicated clinical evaluations as covid-19 is slowly added to the ever increasing list of differentials for thromboembolism. studies included in a scoping review by al-ani et al showed that venous thromboembolism (vte) occurs in about 20% of covid-19 patients, with around 71.4% of those who died from the disease being classified as having disseminated intravascular coagulation (dic), according to one single-center study [109] . similarly, a systematic review and meta-analysis of 3 of the 44 included studies showed vte as a rather common complication of covid-19 in 15% of the 318 total patients from three different studies [110] . these suggest that covid-19, apart from being a mainly respiratory disease, is an ailment of the vasculature frequently complicating into vte, which is expected from its likely tropism for the endothelium. since nsaids, especially the selective cox-2 inhibitors, likely affect hemostatic balance by inhibiting prostacyclin synthesis without inhibiting cox-1-induced thromboxane j o u r n a l p r e -p r o o f production, a prothrombotic state is likely to occur especially among susceptible patients. this is the reason why rofecoxib was withdrawn from the market, after a clinical trial found an increased risk of myocardial infarction and stroke in participants after 18 months of use for colon cancer prevention, which is likely to be a class effect [111] . this points to a possible risk of vte or other thromboembolic events in covid-19 patients with multiple co-morbidities taking nsaids, although evidence is not yet clear. to the best of our knowledge, there is no direct evidence to date of nsaid use and induced thromboembolism among covid-19 patients, but serious caution is advised because associations between thromboembolism and nsaid use have been well established in previous years, albeit in different contexts of disease. the reader is referred to this article for an overview [112] . similarly, corticosteroids may enhance viral entry when provided for symptomatic treatment of sars infection. as aforementioned, a 20.7-fold increase in the risk for adverse outcomes (icu admission/mortality) was observed among patients in hong kong treated with corticosteroids for sars, which was independent of age and disease severity [45] . this may suggest the promotion of coronavirus replication secondary to immunosuppression, or of increased ace2 expression secondary to osmotic diuresis, which we hypothesized earlier above (fig. 4) . it is important to note, however, that the proposed effects of corticosteroids and nsaids on sars-cov2 infection is applicable only for the early stages of the disease. during the late stages, such as in acute respiratory distress syndrome (ards) or acute airway inflammation, treatment with corticosteroids or other more targeted therapies, such as monoclonal antibodies, may be lifesaving. recently, a large randomized clinical trial called the randomised evaluation of covid-19 therapy (recovery) trial conducted the world's largest clinical trial on covid-19 treatments. in this study they selected 2,104 covid-19 patients and subjected them to low to moderate dose (6mg/day) dexamethasone treatment, which then compared them to 4,321 controls who received the standard care for coronavirus infection. as a result, the investigators discovered a significant decrease in patient deaths. death among severely-ill covid-19 patients on ventilators were j o u r n a l p r e -p r o o f reduced by 33%, while death among patients requiring oxygen decreased by 20% [66] . in contrast, there was no clinical significance seen in patients with less severe cases, suggesting that the dexamethasone might be more useful in patients experiencing cytokine-mediated inflammation. this shows the potential of corticosteroids to provide benefit in severe covid-19; however, it is noted that great caution must be exercised when using dexamethasone, as it is neither indicated for mild cases nor with methylprednisolone [113] , the latter being consistent with our previous statement. however, it is not clear in the review if corticosteroids are indeed detrimental for covid-19, as the study they included, which showed a higher rate of icu admissions among covid-19 patients treated with corticosteroids, was only a case series and is at best suggestive [113] . further, the administration of corticosteroids among the studies varied in terms of chronology, with one study being given before ards and the other during ards. meanwhile, a rapid systematic review of 59 publications on inhaled corticosteroid (ics) use in covid-19 have found no sufficient evidence of benefit or harm on clinical outcomes [67] , which reflects the scarcity of covid-19 clinical studies to date. alternatively, paracetamol may be given as first-line for the management of fever without targeting inflammation, due to its effectiveness and safety profile. unlike the nsaids, paracetamol is a weak prostaglandin inhibitor, and in contrast to cox-1/cox-2 inhibition, it inhibits the cox-3 isoenzyme, which is a cox-1 splice variant abundant in cns rather than in the peripheral tissues. this provides paracetamol with analgesic and antipyretic properties, but with little to no peripheral anti-inflammatory properties [29, 30] . further, paracetamol employs the serotonergic pathway to provide analgesia. by stimulating the descending serotonergic pathway, mediated by the 5-ht1a/1b receptors in the cns and acting on afferent nerve fibers, paracetamol largely inhibits noxious stimuli [29, 31, 32] . in addition, paracetamol also demonstrates an opioid sparing effect of 20% that may increase analgesia in patients under opioid analgesics [33] . hence, by virtue of these pathways, paracetamol may be a safer and more effective alternative for early symptomatic management of headache, fever and pain in covid-19. j o u r n a l p r e -p r o o f covid-19 is a respiratory disease caused by the coronavirus sars-cov-2, and has caused a pandemic by infecting more than 31 million people in more than 190 countries to date, while claiming hundreds of thousands of lives as it impacts heavily on both economy and lifestyle. initial clinical reports have shown correlations between adverse outcomes for covid-19 and hypertension, which they attributed to increased expression of ace2. this was followed shortly by anecdotal reports of increased symptom severity among patients taking anti-inflammatory drugs like ibuprofen early in the disease, which was later hypothesized to be associated with ace2. in this paper, we provide concrete mechanisms of how nsaids and corticosteroids may aggravate sars-cov-2 infection in the early stages of disease, through their interaction with prostaglandin synthesis and the raas, apart from the conventional pathway of immunosuppression at a time when the body needs it the most. however, as evidence is often conflicting and scarce, more evidence is needed to gain a definitive understanding of covid-19. this research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. the authors do not have any conflicts of interest to disclose. we would like to thank the healthcare workers and scientists in this pandemic for continually providing care to covid-19 patients and for providing reports on the clinical and molecular aspects of this disease. dr. rafael a. manalo and emma manalo are acknowledged for providing great inspiration in this work. revised fig. 1: (arb-acei mismatch was now addressed) the raas is an important homeostatic mechanism of the body that involves the kidney, liver, lungs and adrenal glands, which responds mainly to electrolyte and volume changes as well as to renal blood flow. in times of reduced plasma volume, reduced perfusion to the kidneys and to a concomitant loss of na + , the juxtaglomerular cells of the kidney produce renin, which catalyzes liver angiotensinogen to produce angiotensin i (ang i). the lungs then upregulate angiotensinconverting enzyme (ace) that converts ang i to ang ii, which influences the adrenal glands to synthesize more aldosterone. in addition, ang ii has a direct vasoconstrictive effect. aldosterone (mimicked by other mineralocorticoids), which is released from the adrenal glands then influences the kidneys to increase na + and h2o reabsorption in the distal convoluted tubules and collecting ducts. under normal conditions, prostaglandins e2 and i2 are formed which, in the kidneys, generally vasodilate the afferent renal arterioles to increase the glomerular filtration rate (gfr) and renal perfusion. in addition, pge2 predominantly inhibits na + and h2o reabsorption in the thick ascending loop of henle (tal) to induce volume or pressure natriuresis, which harmonizes with the raas to maintain homeostasis. in hypertension and other conditions of reduced renal blood flow, the effects of pgi2 are marked and there is increased reabsorption of na + and h2o. chronic use of ace inhibitors (aceis) and angiotensin receptor type 1 blockers (arbs) can lead to a compensatory upregulation of ace and ace2 expression, which is the probable mechanism for the aggravation of sars-cov-2 infection among chronic hypertensives taking these drugs. arachidonic acid is converted to the prostaglandins and thromboxanes by the action of cyclooxygenases cox-1 and cox-2. of the four prostaglandins, pge2 and pgi2 promote optimal renal function by dilating afferent renal arterioles and promoting natriuresis in normal conditions, with pgi2-mediated na + and h2o reabsorption seen more evidently in unhealthy individuals with reduced renal perfusion. when nsaids like ibuprofen or naproxen are taken continuously, the net effects of cox inhibition are (1) increased na + and h2o reabsorption, (2) reduced renal afferent arteriolar dilatation, (3) reduced glomerular filtration rate (gfr), and (4) an increase in systemic vascular resistance. due to reduced gfr and renal perfusion, the body produces greater amounts of angiotensin ii (ang ii) to increase blood flow via increased plasma volume. however, increased ang ii levels leads to inappropriate vasoconstriction and increased systemic vascular resistance, which may ultimately result in a vicious cycle due to feedback compensation from ang ii in susceptible patients. subsequently, an upregulation of ace2 expression is expected to counteract ang ii providing its catabolic pathway to produce vasodilator and natriuretic angiotensins (1) (2) (3) (4) (5) (6) (7) . this compensatory increase in ace2 expression from nsaid intake, which is aggravated in unhealthy individuals such as those with hypertension, is the proposed mechanism by which sars-cov-2 can more aggressively infect the cell, by binding to greater amounts of ace2 for viral entry. the concomitant immunosuppression by nsaids also promotes viral replication and shedding of sars-cov2, which can complicate the early phases of covid-19. corticosteroids can be categorized into the mineralocorticoids and the glucocorticoids. the mineralocorticoids, which mimic the actions of aldosterone, affect kidney function by increasing the na + and h2o reabsorption. because this mechanism does not promote ang ii formation, it is not expected to contribute majorly to increased ace2 expression. on the other hand, corticosteroids affect the liver by increasing its capacity for gluconeogenesis. muscles enhance protein breakdown into 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medicine. glucocorticoids bind to their nuclear glucocorticoid receptors (gr), but also to mr, and affect gene transcription via two important mechanisms: (1) transactivation, which is the direct interaction of the glucocorticoid-gr complex with genetic material, or (2) trans-repression, in which the complex interacts with other proteins such as creb-binding protein (cbp) and nf-κb. trans-activation leads to an upregulation of anti-inflammatory proteins, while trans-repression leads to decreased expression of inflammatory proteins. together, these pathways converge to result in immunosuppression, which may aggravate covid-19 by promoting sars-cov-2 replication and shedding, especially in the early stages of the disease key: cord-290948-cuu78cvl authors: imbert, isabelle; snijder, eric j.; dimitrova, maria; guillemot, jean-claude; lécine, patrick; canard, bruno title: the sars-coronavirus plnc domain of nsp3 as a replication/transcription scaffolding protein date: 2008-02-05 journal: virus res doi: 10.1016/j.virusres.2007.11.017 sha: doc_id: 290948 cord_uid: cuu78cvl many genetic and mechanistic features distinguish the coronavirus replication machinery from that encoded by most other rna viruses. the coronavirus replication/transcription complex is an assembly of viral and, most probably, cellular proteins that mediate the synthesis of both the unusually large (∼30 kb) rna genome and an extensive set of subgenomic mrnas. the viral components of the complex are encoded by the giant replicase gene, which is expressed in the form of two polyproteins (pp1a and pp1ab) that are processed into 16 cleavage products (nonstructural proteins 1–16). using the combination of yeast two-hybrid screening and gst pull-down assays, we have now analyzed all potential interactions between sars-coronavirus nonstructural proteins, which may contribute to the structure and/or function of the viral replication/transcription complex. we demonstrate the existence of a complex network of interactions involving all 16 nonstructural proteins. our results both confirmed previously described associations and identified novel heterodimerizations. the interaction map thus provides a sum of the interactions that may occur at some point during coronavirus rna synthesis and provides a framework for future research. the family coronaviridae includes the genera coronavirus and torovirus that, together with the more distantly related arteriviridae and roniviridae families, form the order nidovirales (for reviews see gorbalenya et al., 2006) . in 2003, a novel coronavirus was identified as the etiological agent of the severe acute respiratory syndrome (sars; drosten et al., 2003; ksiazek et al., 2003; peiris et al., 2003) and is now known as the sars-coronavirus (sars-cov). coronaviruses are enveloped viruses with large positivesense single-stranded rna genomes ranging from 27 to 32 kb, the largest among the known rna viruses. like other nidoviruses, coronaviruses genomes are polycistronic with about two thirds being occupied by two large open reading frames (orfs 1a and 1b) that specify the viral nonstructural proteins (nsps) or "replicase". the coronavirus genome is expressed using a variety of regulatory mechanisms, including the synthesis and proteolytic processing of replicase polyproteins and the generation of an extensive nested set of subgenomic (sg) mrnas to express structural and accessory proteins from the 3 -proximal part of the genome (for recent reviews, see pasternak et al., 2006; sawicki et al., 2007) . genome expression starts with the translation of replicase orfs 1a and 1b, with expression of the latter involving a −1 ribosomal frameshift (brierley et al., 1989; brierley and dos ramos, 2006) occurring with an estimated efficiency of 20-40%. in the case of sars-cov, pp1a and pp1ab are 4382 and 7073 amino acids long, respectively (marra et al., 2003; rota et al., 2003; snijder et al., 2003; thiel et al., 2003) . these precursors are cleaved co-and post-translationally by two viral proteases, a papain-like protease (plp2) residing in nsp3 and the 3c-like protease (3clpro; nsp5), to produce the mature replicase subunits nsp1 to nsp16 (fig. 1) . most of these subunits are believed to associate with a poorly characterized membranebound complex (bost et al., 2001; gosert et al., 2002; shi et al., 1999; snijder et al., 2006; van der meer et al., 1999 ) that mediates the synthesis of genome rna (replication) and sg mrnas (transcription). the sg mrnas are produced by a unique mechanism involving discontinuous negative-strand rna synthesis to produce subgenome-length templates for sg mrna synthesis (sawicki and sawicki, 1995; reviewed in pasternak et al., 2006; sawicki et al., 2007) . knowledge related to the functions and interactions of coronavirus nsps has remained limited thus far. nsp1 and nsp2 are rather variable in sequence, and possibly subgroup-specific replicase subunits ziebuhr et al., 2001) . sars-cov nsp1 (20 kda) was implicated in suppression of cellular gene expression by promoting host cell mrna degradation (kamitani et al., 2006) and in chemokine dysregulation (law et al., 2007) . sars-cov nsp2 (70 kda) is dispensable for replication in cell culture, but its deletion attenuates viral replication (graham et al., 2005) . sars-cov nsp3 is a large multidomain protein of 1922 amino acids thiel et al., 2003) that is thought to contain at least seven domains: (1) an n-terminal glu-rich acidic domain (ad); (2) an x domain (xd) with poly(adp-ribose) binding properties saikatendu et al., 2005) ; (3) the sud domain (for sars-cov unique domain, an insertion not found in any other coronavirus thus far) with a specific affinity for oligo(g)-strings (tan et al., in press); (4) a papain-like protease (plp2), recently shown to exhibit deubiquitinating activity (barretto et al., 2005; harcourt et al., 2004; lindner et al., 2005; ratia et al., 2006) ; (5) an unknown domain possibly extending the papain-like protease domain, termed plnc for papain-like noncanonical (see below); (6) a transmembrane domain (kanjanahaluethai et al., 2007) corresponding to the n-terminal of the y domain; and (7) the remainder of the y domain, the abbreviation "y domain" will be used for this part in this study. the multi-spanning transmembrane domains in nsp3, nsp4 and nsp6 are presumed to serve as a scaffold for the assembly of the membrane-associated replication/transcription complex (rtc). nsp5 (34 kda) is a 3c-like cysteine protease (3clpro), also known as "main protease" (m pro ) (anand et al., 2003; gorbalenya et al., 1989) , cleaving the replicase polyproteins at 11 conserved sites containing a canonical leu-gln (ser, ala, gly) signature. nsp8 protein (22 kda) was recently postulated to be a processivity factor associated with the viral rna-dependent rna polymerase (rdrp) in nsp12. indeed, the crystal structure of a hexadecameric nsp7-nsp8 complex provided a first glimpse of the sophisticated architecture of the coronavirus replication and transcription machinery (zhai et al., 2005) . the properties of the central channel of this complex suggest that it may encircle double-stranded rna. this hypothesis was strengthened by the finding that nsp8 displays primase activity in an in vitro assay (imbert et al., 2006) and might thus catalyse the synthesis of rna primers for the primer-dependent rdrp. nsp9 (13 kda) is a ssrna-binding protein (egloff et al., 2004; sutton et al., 2004) postulated to stabilise and protect nascent and template rna strands during replication and/or transcription. much like nsp8 and nsp9, nsp10 (18 kda) is also a nucleic acid-binding protein with two zinc fingers (joseph et al., 2006; matthes et al., 2006) . the phenotype of a temperature-sensitive nsp10 mutant (q65e) of the coronavirus mouse hepatitis virus (mhv) implicated nsp10 in minus-strand rna synthesis (sawicki et al., 2005; siddell et al., 2001) . the c-terminal part of nsp12 (102 kda) contains the poorly characterized rdrp domain that is significantly divergent from both cellular and viral polymerases. only one study reported polymerase activity of a purified coronavirus rdrp (cheng et al., 2005) . nsp13 (67 kda) comprises a c-terminal helicase domain with a 5 -3 directionality and an n-terminal zinc-binding domain (ivanov et al., 2004b; seybert et al., 2000 seybert et al., , 2005 . finally, nsp14 (58 kda), nsp15 (38 kda) and nsp16 (33 kda) contain exoribonuclease (minskaia et al., 2006) , endoribonuclease (bhardwaj et al., 2006; ivanov et al., 2004a) and predicted 2 -o-ribose methyl transferase activities (von grotthuss et al., 2003) , respectively, which are all essential for efficient coronavirus rna synthesis (almazan et al., 2006) . in addition, as illustrated above, coronavirus replicases have evolved to include a number of nonessential functions that may provide a selective advantage in specific host cells only. most of the 16 coronavirus nsps are known or thought to be part of the rtc, which is formed following genome translation and replicase polyprotein processing. as for many plus-strand rna viruses (mackenzie, 2005; salonen et al., 2004; snijder et al., 2006) , it has been demonstrated that nidovirus rna synthesis is associated with cytoplasmic membranes, in this case double-membrane structures that likely originate from the endoplasmic reticulum. obvious benefits of complex formation are the possibility to couple functions residing in different subunits and the sequestration of viral components. the complex and sophisticated coronavirus replication/transcription strategy suggests that the composition of the complex may change during the life cycle of the virus or that -alternatively -partially different complexes dedicated to, e.g. either replication or transcription may co-exist, probably relying on the same central rna synthesizing catalytic subunit, the nsp12-rdrp. the network of interactions within the sars-cov rtc has only begun to emerge. dual-labelling studies in sars-covinfected cells have confirmed the colocalization of a variety of replicase subunits (harcourt et al., 2004; ivanov et al., 2004a; prentice et al., 2004; snijder et al., 2006) and newly synthesized viral rna (harcourt et al., 2004) . chemical cross-linking experiments demonstrated that sars-cov nsp10 could be cross-linked to nsp9 (su et al., 2006) , which in turn interacts with nsp8 (sutton et al., 2004) . very recently, a sars-cov genome-wide analysis for intraviral protein-protein interactions was performed (von brunn et al., 2007) . interactions were tested by yeast two-hybrid system and subsequently confirmed by coimmunoprecipitation experiments in mammalian cells. nsp2 and nsp8 showed a rather large number of interactions. in this study, the nsp3 protein was subdivided into 2 fragments by cutting to the sud domain. coronavirus nsp12-rdrp is believed to play a central role in both replication and transcription. here, we investigated interactions between all possible combinations of sars-cov nsps, including the nsp12-rdrp, which are potentially involved in the assembly and/or function of the rtc. the experiments were performed with in vitro translated nsps, as well as ex vivo, in saccharomyces cerevisiae expressing these nsps in the context of a two-hybrid system. we observed a complex network of interactions involving all 16 nsps, confirming previously documented associations and identifying numerous novel heterodimerization events, and thus providing a framework for future research. the frankfurt-1 isolate of sars-cov (genbank accession number ay291315; thiel et al., 2003) was amplified in vero-e6 cells and used in this study for the production of the expression vectors. all protocols used for the yeast two-hybrid system were described previously (walhout and vidal, 2001 ). mav103 and mav203 yeast strains (leu2-3112, tryp-901, his3 200, ade2-1, gal4a, gal80a, spal10::ura3, gal1::lacz, gal1::his3-@lys2, can1r, cyh2r), which are mata and matalpha, respectively, were used for the yeast two-hybrid matrix. rt-pcr products encoding different sars-cov proteins/domains, i.e. nsp1, nsp2, six nsp3 subdomains (ad, xd, sud, plp2, plnc, and y), nsp5 to nsp10, and nsp12 to nsp16 were first cloned into pdonr201 using the gateway system (invitrogen). all constructs were verified by sequence analysis. the boundaries of the domains used are detailed in table 1 . using the gateway system, each cdna was subcloned into pad and pdb vectors, which were used to transform mav203 and mav103 cells, respectively. diploid cells were generated by mating mav103 expressing genes fused to the dna-binding domain (db-orf) and mav203 expressing genes fused to the activation domain (ad-orf). selection was performed using selective complete (sc) medium lacking tryptophane and leucine. patches representing potential interaction pairs were then examined for two-hybrid phenotypes by replica-plating onto selective plates, i.e. ypd (yeast extract, peptone, d-glucose) medium plate with a nitrocellulose filter on to perform the ␤-galactosidase filter lift assay, as well as on sc-leu-trp-his + 25 mm 3-at and sc-leu-trp-ura plates to assess the three two-hybrid phenotypes (␤-galactosidase, his and ura) (walhout and vidal, 2001) . the matrix experiment was carried out three times, using clones derived from three different plasmid transformation and mating experiments. for gst fusion protein expression vectors, bamhi/ecori restriction fragments encoding the sars-cov plnc domain, nsp8 and nsp9, and a bamhi restriction fragment encoding nsp12 were inserted in-frame into pgex-4t-2 (ge healthcare), to generate gst-plnc, gst-nsp8, gst-nsp9 and gst-nsp12, respectively. gst-p55, encoding the hiv-1 gag precursor, was constructed as described in douaisi et al. (2004) . to create sars-cov protein expression vectors, ecori/kpni restriction fragments encoding nsp1, xd, sud, plp2, plnc, y, nsp5, nsp6, nsp7, nsp10, nsp16, ecori/xbai restriction fragments encoding nsp2, nsp8, nsp9, ad, a xbai/noti restriction fragment encoding nsp12, and kpni/noti restriction fragments encoding nsp13, nsp14, nsp15 were cloned into ptnt (promega). all constructs were sequenced, and the proteins expressed from these plasmids did not carry any foreign tags, except an additional n-terminal met residue. the gst-plnc (69 kda), gst-nsp8 (48 kda), gst-nsp9 (38.8 kda), gst-nsp12 (132 kda) and gst-p55 (80 kda) fusion proteins were expressed in e. coli strain c41(de3) (avidis sa, france), which contained the plyss plasmid (novagen). bacterial cultures were grown at 37 • c in lb medium supplemented with ampicillin (100 g ml −1 ) and chloramphenicol (17 g ml −1 ) until the od 600 reached 0.5. protein expression was induced by adding 500 m isopropyl 1-thio-␤-d-galactopyranoside (iptg) and cells were incubated for 4 h at 37 • c. in the case of gst-plnc and gst-nsp12 expression, however, induction was performed with 100 m of iptg and cells were incubated for 17 h at 17 • c. cell pellets were resuspended in pbs supplemented with 0.25 mg ml −1 , of lysozyme, 0.1 mg ml −1 dnase, 10 g ml −1 rnasea, 1 mm phenylmethylsulphonylfluoride (pmsf), protease inhibitor cocktail (sigma), and 1% triton x-100. the suspensions were sonicated and centrifuged at 25,000 × g for 30 min, after which 500 l of a 50% slurry of glutathione-sepharose 4b beads (ge healthcare) was added to the clarified supernatants. the beads were allowed to bind proteins for 1 h, washed three times with pbs, and finally resuspended in 500 l of pbs. rnas specifying sars-cov nsp1, nsp2, nsp3 domains (ad, xd, sud, plp2, plnc, y), nsp5 to nsp10, and nsp12 to nsp16 were in vitro transcribed from the t7 promoter present in the ptnt expression vector. transcripts were in vitro translated in the presence of [ 35 s]methionine using the tnt t7 coupled reticulocyte lysate system (promega) according to the manufacturer's instructions. in vitro binding analyses were performed as described previously (dimitrova et al., 2003) . each interaction was independently tested three times. protein samples were resolved by sds-page, transblotted onto hybond-p membranes (ge healthcare) and probed with an anti-gst antibody that was kindly provided by dr. teillaud (inserm unité 255, paris, france). the rtcs of mammalian positive-stranded rna viruses, including coronaviruses, are commonly associated with intracellular membranes (prentice et al., 2004; snijder et al., 2006) and consist of multiple virus-encoded protein subunits as well as host proteins. to further characterize the molecular interactions between sars-cov rtc subunits, we performed a yeast two-hybrid matrix screen with all nsps encoded by this virus. an interaction was considered valid when it reproducibly produced a positive result for one or more of the three different yeast phenotypes tested (fig. 2a) . subsequently, as a general strategy, gst pull-down assays were used to confirm interactions (see below). because of the large size of the membrane-associated nsp3 (213 kda), six nsp3 subdomains were defined (table 1) . the general bioinformatics approach that led to the design of nsp3 domains was described previously (ferron et al., 2005) . briefly, we performed a blast search against the vazymolo database with the full sequence corresponding to nsp3. the protein is predicted to be organized in 6 modules that are the f197 (ad) domain structurally confirmed by crystallography (serrano et al., 2007) , the f200 (xd) domain structurally confirmed by crystallography , the f209 (sud), the f203 (plp2) domain also structurally confirmed by crystallography (ratia et al., 2006) , the f250 (plnc) (tan et al., in press) , and a hydrophobic domain followed by the f202 (y domain). the hydrophobic cluster analysis of module f250 (plnc domain) can be interpreted as a single and globular domain flanked by the plp2 in n-terminal and a hydrophobic domain in c-terminal. a total of 19 proteins/domains were used in our screening matrix. out of the 361 protein pairs tested in the yeast two-hybrid analysis, 15 combinations produced a positive result, accounting for 4% of the possible protein interactions (fig. 2) . in comparison, about 6% of nonstructural protein-protein interactions were positive in the von brunn et al. (2007) y2h study. surprisingly, no interaction is common to both studies. this discrepancy could be explained by the fact that two-hybrid false positives may arise in two-hybrid arrays and by the difference between the y2h systems used (uetz, 2002) . with our y2h system, to eliminate potential false-positive interaction, three different phenotypes were assessed (␤-galactosidase, his and ura) and the transcription from each reporter gene (lacz, his3 and ura3) is driven by independent promoters. at last, we have considered an interaction as positive if at least one phenotype in 2 out of the 3 matrix experiments was positive. two sars-cov proteins (nsp3 and nsp14) were found to be involved in interactions with 4 or more partners. domains from nsp3 interacted with nsp5, nsp6, nsp12, nsp13, nsp14 and nsp16, and in particular for the plnc subdomain the unexpectedly large number of 5 interaction partners was identified. since this domain is comprised of only 386 amino acids, it is tempting to speculate that it might sequentially interact with these partners rather than with all of them simultaneously. consequently, nsp3 may serve as one of the rtc scaffolding proteins by bringing several nsps into close proximity of each other. the presence of a transmembrane domain within nsp3, and the observed interaction with the nsp6 transmembrane protein, reinforces the hypothesis of a membrane-associated scaffolding function for nsp3 in the infected cell. the nsp3 central role was not so clear in the von brunn et al. (2007) study because nsp3 protein was subdivided only into two fragments (nt 2719-4431 and nt 4885-8484), splitting the sud domain in two. the nsp14 protein is an exoribonuclease playing an elusive, yet essential, role in the sars-cov life cycle (minskaia et al., 2006) . in addition to its interaction with nsp3, nsp14 interacted strongly with both nsp10, a protein with two zinc-fingers potentially involved in nucleic acid recognition, and nsp16, the 2 -o-ribose methyltransferase. interestingly, the interactions between nsp10, nsp14 and nsp16 were the only reciprocal ones, meaning that they were also detected when the db and ad fusion partners were swapped. it is tempting to speculate that these three proteins form a complex that is associated with nsp3 and that the other interactions that were detected may reflect different and/or alternative complexes existing at different time points. the surface of interaction of nsp10 with nsp16 was mapped by reverse yeast two-hybrid system and in mammalian cells, using the bioluminescence resonance energy transfer technology (bret). point mutations that abrogate the nsp10-nsp16 interaction also inhibit the interaction between nsp10 and nsp14, albeit with subtle differences, suggesting that the interacting surface is overlapping (pl, unpublished data). two crystal structures were available for nsp10. one of them reveals a unique spherical dodecamer architecture (su et al., 2006) and the other shows a monomer (joseph et al., 2006) . nsp10 protein is able to bind single-and double-stranded rna and dna without apparent sequence specificity (joseph et al., 2006) and was suggested to be involved in sars-cov replication based on structural similarities with nuclear proteins (su et al., 2006) . the overall positive charge of the inner and outer surfaces of the dodecamer would be compatible with an rna-binding function. interestingly, nsp10 also interacts with nsp9, a single-stranded rna binding protein (egloff et al., 2004; su et al., 2006; sutton et al., 2004) that in turn interacts with nsp8, the putative rna primase (imbert et al., 2006) . in human coronavirus 229e, mutagenesis of the active site of the nsp14 exonuclease greatly reduced the accumulation of viral rna, suggesting an essential role for this enzyme in replication and transcription (minskaia et al., 2006) . this critical function of nsp14 was also supported by results obtained with a sars-cov replicon, in a study that also addressed the impact of mutations within or deletion of nsp15-and nsp16-coding sequences, clearly demonstrating that these proteins are required for efficient replication (almazan et al., 2006) . finally, by interacting with nsp3 through the plnc domain, and with nsp7, nsp9 and nsp10, the nsp14 exoribonuclease may also be a key rtc scaffolding component, irrespective of its nucleic acid binding activity. a number of nsps (nsp1, nsp13, and nsp15) interacted with a single partner and these interactions were only detected when they were used as prey (fig. 2) . sars-cov nsp1, a small protein , unique to sars-cov, believed to be involved in host cell rna degradation, may not directly participate in viral rna synthesis. consequently, it may be not too surprising that its only interaction partner was nsp9, a singlestranded rna binding protein that, in turn, was not densely connected to other nsps (fig. 2) . in the case of the large nsp13 and nsp15 subunits, it is impossible at this stage to assess whether their size (or any other factor) may have impeded their expression or correct folding in yeast, thus preventing detection of protein-protein interaction in the yeast two-hybrid system. alternatively, the toxicity of rna-modifying enzymes that is often observed when they are expressed as recombinant proteins may have played a role. nsp13 and nsp15 could also belong to larger hetero-multimers, like the nsp7-nsp8 complex. notably, and despite the documented interaction of nsp8 with nsp7, nsp15 and nsp8 formed the sole isolated protein pair. we failed to detect nsp10 dimerization in the yeast-two hybrid system, as in the von brunn et al. (2007) study, even though one of the two crystal structures reveals a dodecameric arrangement based on nsp10-nsp10 interactions (su et al., 2006) . the second crystal structure shows a monomer (joseph et al., 2006) . the dodecamer supercomplex may lack biological relevance, because the second zinc-finger lacks the last cysteine residue and the remainder of the cterminal tail is disordered. unexpectedly also, in particular since helicases are known to interact with rdrps in rtcs (dimitrova et al., 2003; piccininni et al., 2002) , the nsp13 helicase stood alone and interactions between nsp12 and nsp13 were not detected. in the yeast two-hybrid matrix screening, a number of proteins did not interact with any other nsp or subdomain. these were nsp2 and the xd, sud and y domains of nsp3. it is interesting to note that nsp2, which appears to be unique to sars-cov, is dispensable for replication and transcription (graham et al., 2005) . also, the adp-ribose-1 -monophosphatase (adrp) activity of the xd subdomain was found to be dispensable for fig. 4 . gst pull-down analysis to identify interaction partners of nsp12-rdrp. each panel shows an autoradiogram from a sds-page analysis of in vitro-translated nonstructural proteins pulled down by binding to either glutathione-sepharose-bound gst-p55 (negative control; lanes a) or gst-nsp12 (lanes b). equal amounts of [ 35 s]methionine-labelled target were used for lanes (a and b) and the input material was also run next to the pull-down samples, as indicated for each panel. coronavirus replication (putics et al., 2005) . the sud domain binds oligo(g) stretches and may be involved in the modulation of host-cell signalling pathways (tan et al., in press ). finally, the y domain of nsp3 has no obvious role in rna synthesis. therefore, it is logical to propose that they may rather play a role in virus-host interactions to, e.g. modulate immunity and/or pathogenesis. surprisingly, despite its presumed central role in rna synthesis, the nsp12-rdrp was found to interact with only two other nsps, namely nsp7 and nsp3. nsp7-nsp8 could be part of a larger complex including nsp12 on the one hand and nsp14, nsp9 and nsp10 on the other (fig. 2) . these results also reflected the potential limitations of the yeast-two-hybrid approach when analyzing large proteins like nsp12, prompting us to develop an alternative to confirm and/or extend our interaction map. it is well-known that -for technical reasons -yeast twohybrid screening may not reveal all interactions that normally occur in a cellular environment, owing, e.g. to steric hindrance of the fusion partners used in yeast two-hybrid assays or the inability of certain fusion proteins to be expressed and targeted to the yeast nucleus. therefore, as an additional approach to identify potential interactions of the plnc domain, nsp8, nsp9 and nsp12-rdrp with other sars-cov nsps, we carried out a set of pull-down assays using gst fusion proteins. this type of assay can be used to analyze the binding of a radiolabeled, in vitro-translated protein to an interaction partner presented in the form of a purified gst fusion protein bound to glutathione-sepharose beads. gst fusion proteins, as well as a hiv-1 gag fusion protein (gst-p55) that was used as negative control, were expressed in e. coli and immobilized on glutathione-sepharose beads (fig. 3a) . gst-p55, gst-plnc, gst-nsp8 and gst-nsp9 were much more soluble in bacteria fig. 6 . schematic summary of gst pull-down results. nodes in blue and green represent nsp3 domains and orf1b-encoded proteins, respectively. proteins that did not interact with the bait are depicted at the left side of each diagram. than gst-nsp12 and could be purified in considerably larger amounts (fig. 3a) . therefore, only 1/50 of a standard gst-p55, gst-nsp8 and gst-nsp9 preparation was used in the assays, whereas the complete yield of a gst-nsp12 purification was used (fig. 3a) . to obtain potential interaction partners, not containing any foreign tags, all sars-cov nsps and domains used before (table 1) were in vitro translated in the presence of [ 35 s]-labelled methionine, except for nsp4 (an integral transmembrane domain) and nsp11 (which is only 13 amino acids long) (fig. 3b) . despite repeated attempts, we could not achieve the expression of nsp6 and nsp9 in the tnt t7-coupled reticulocyte lysate system. the in vitro synthesis of full-length proteins was confirmed by sds-page analysis (fig. 3b) . a few truncated products were observed for the sud, plp2, nsp15 and nsp16 proteins. the ad protein corresponds to the lower band on the different gels, according to the protein molecular weight marker. we do not have an explanation regarding the other higher band observed for ad. nsp2 protein (70 kda) appears to migrate more slowly than nsp12 protein (102 kda) (fig. 3b, middle panel) . this discrepancy may be related to the amino-terminal part of nsp2 protein which appears to be disordered. it is documented that disordered protein might have an atypical migration pattern, which is variable according to the percentage of acrylamide sieving. gst pull-down assays were carried out with approximately equimolar levels of input material. pull-down assays with gst-nsp12 revealed that the sars-cov rdrp does not interact with nsp1, ad, nsp7, or nsp10 (figs. 4 and 6) . interactions between gst-nsp12 and nsp2, xd, sud, plnc, plp2, and nsp8 were detected, in the absence of a signal for the gst-p55 negative control (fig. 4, lanes b) . interactions were also observed with the nsp3-y domain, nsp5, nsp13, nsp14, nsp15 and nsp16. nsp3-y domain, nsp12, nsp14 and nsp16 also showed a weak interaction with gst-p55 (fig. 4 , lanes a) and gst alone (not shown), but the signal clearly increased when specific sars-cov gst-nsp fusion proteins were used. therefore, these background signals were attributed to the large amount of the control proteins used in comparison with the amount of gst-nsp12. in contrast to the yeast twohybrid analysis, gst-nsp12 failed to interact with nsp7. the same interactions were detected after rnase treatment of the in vitro translation products used for these binding studies, indicating that the observed interactions were not mediated by rna (data not shown). from these gst pull-down experiments with nsp12-rdrp, a clear pattern emerged: the rdrp-containing subunit is apparently able to built two interactomes: one with nsp3 domains and the other with products encoded by orf1b, which had been missed in the yeast two-hybrid experiments. notably, nsp12 is also able to dimerize, perhaps providing an explanation for the fact that many interactions involving nsp12 were missed using the yeast two hybrid system (see also below). the nsp12-nsp7 interaction could not be reproduced using the gst pull-down approach. the negative results for nsp1, ad, and nsp10 were consistent with the yeast two-hybrid data. the recently discovered primase activity of nsp8 made this protein a very interesting bait for an extended interaction analysis. the interaction pattern of gst-nsp8 was found to be related to that of gst-nsp12 (fig. 6 ). although interactions with mul-tiple nsp3 subdomains (xd, sud, plnc and y) were missing, nsp2, plp2, nsp5 and orf1b-encoded products, were identified as interactors, yielding the same interaction tree obtained for gst-nsp12. the bands corresponding to sud and y domains did not migrate to the expected distance but much lower further (fig. 5a, left panel) . for the sud domain, a strong tendency to proteolytic (self-)degradation was shown by others (tan et al., in press ). however, this discrepancy in size was not observed with the gst-plnc pull-down assay (see below). furthermore, these experiments confirmed the nsp8/nsp15 interaction previously identified by yeast two-hybrid screening (fig. 2) and revealed that nsp8 dimerized, in accordance with structural, biochemical, and enzymatic data (imbert et al., 2006; zhai et al., 2005) . the yeast two-hybrid screening (fig. 2) identified the nsp3 plnc domain as a central interactor in the sars-cov rtc. thus, plnc pull-down assays were performed in order to confirm and perhaps extend these findings. indeed, this alternative assay confirmed interactions of plnc with nsp5, nsp12, nsp13, nsp14 and nsp16 (figs. 5b and 6). moreover, additional interactions were revealed with nsp2, several nsp3 subdomains (ad, sud, plp2 and y), nsp7, nsp8 and nsp15. taken together, these data supported the previously proposed scaffolding role for nsp3, in which the plnc subdomain may fulfill a central organizing position. for unknown reasons, nsp9 expression in the tnt t7coupled reticulocyte lysate system remained unsuccessful. therefore, a gst-nsp9 fusion protein was produced and used to complete our interaction screening. gst-nsp9 interacted with nsp1, nsp2, some nsp3 domains (ad, sud, plp2, y), nsp5, nsp7, nsp8, nsp10, nsp12, nsp13, nsp14 and nsp15 (figs. 5c and 6). overall, like the nsp3 plnc domain, nsp9 seems to have many partners and interacts with almost all nsps except for nsp16. the gst-nsp9 pull-down analysis confirmed the interactions identified above in the yeast two-hybrid approach (nsp9 with nsp1 and nsp14) and also the previously documented nsp9-nsp10 interaction (su et al., 2006) . both are rna-binding proteins that may cooperate at some point in the virus life cycle. the nsp10/gst-nsp8 interaction and the nsp10/gst-plnc interaction are much weaker than nsp10/gst-nsp9 interaction. in this study, we investigated in vitro and in cellulo the network of interactions that must exist amongst the viral proteins that assemble into the sars-cov rtc. these data may enhance our understanding of coronavirus replication and transcription at the molecular level. we have not only confirmed interactions previously deduced from a variety of experimental approaches, but have also identified a significant number of previously unknown (potential) interactions between sars-cov replicase cleavage products or domains. in the first place, we have provided evi-dence for physical interactions that may be the basis for the previously described co-localization of various key enzymes of the sars-cov rtc in infected cells (nsp3, nsp5, nsp12, nsp13 and nsp15; snijder et al., 2006) . co-immunoprecipitation in mammalian 293 cells confirmed interactions of nsp8 with nsp2, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14 (von brunn et al., 2007) and highlighted its central role within the rtc machinery. secondly, the nsp3 plnc domain, nsp8 and nsp12 were able to form stable homodimers. thirdly, the nsp8/nsp15 interactions initially detected by yeast two-hybrid screening could be confirmed in gst pull-down assays. previously described nsp8/nsp9/nsp10 interactions (su et al., 2006; sutton et al., 2004) were also confirmed in the latter assay (fig. 5, panels a and c) . finally, three viral interaction partners for nsp2 (nsp8, nsp9 and nsp12) were identified in the pull-down analysis, although the yeast two-hybrid analysis had not revealed these interactions. the coronavirus rtc engages in a variety of rna synthesis processes with replication and sg mrna synthesis each proceeding through their specific minus-strand templates. how the rtc accomplishes and balances these various processes remains to be elucidated. it is unknown whether complexes with different composition exist that are specifically dedicated to, e.g. replication or transcription. alternatively, one type of complex may be converted into another by the binding or release of specific protein partners. in this manner, such protein factors may regulate the balance between replication and transcription, if carried out by the same complex, or direct the formation of specialized "replicase"and "transcriptase"complexes. the molecular mechanism and factors governing this switch in coronaviruses are unknown. in the related arterivirus system, nsp1 was identified as a clear example of a protein with a transcription-specific function. deletion of this replicase subunit did not affect replication, but reduced sg mrna synthesis to undetectable levels (tijms et al., 2001) . in alphaviruses, temporal regulation of replicase polyprotein cleavage by the nsp2 protease was found to be a key factor in regulating the switch from minus-to plusstrand rna synthesis (kaariainen and ahola, 2002; lemm et al., 1994) , illustrating another barely explored feature of the nidovirus/coronavirus system: replicase subunits may act either after complete maturation, or as part of larger processing intermediates, or both. thus, rtc function may be temporally and spatially coordinated by the activity of the protease domains embedded in replicase polyproteins. our interactome studies confirmed that the key enzymes encoded by replicase orf1b likely exist as a complex, which may be organized around the dimerizing nsp12-rdrp. it is unknown whether dimerization is essential for rdrp activity, as, e.g. in the case of hepatitis c virus (qin et al., 2002) . in general, rdrp error frequencies are estimated to be in the order of 10 −4 (huang et al., 2000) . for many years, this number, in combination with the unusual size of the coronavirus genome, has triggered speculations about the presence of proofreading or other repair mechanisms. the gst-nsp12 pull-down experiments identified nsp13 to nsp16 as strong interactors (fig. 4) , and the fact that this pattern overlaps with that of the nsp8primase is intriguing (fig. 5a) . the interaction of these proteins, either simultaneously or successively, with the helicase (nsp13), 3 → 5 exonuclease (nsp14), endonuclease (nsp15) and (putative) methytransferase (nsp16) supports the hypothesis that these subunits may mediate successive steps of the same pathway and provides a basis for further experiments aimed at, for example, exploring the existence of a proofreading mechanism. it is interesting to note that the nsp13 helicase, with a 5 -3 directionality, appears to lack interactions with nsp14, nsp15 and nsp16 (our study and von brunn et al., 2007) . in flaviviruses, ns3 helicase unwinds its substrates in a 3 -to-5 direction and physical mapping and structural studies indicate that the helicase most likely precedes the ns5 rdrp undergoing rna synthesis (malet et al., 2007) . upon isolation of an active coronavirus rdrp, it will be interesting to determine if this is also the case in coronaviruses, in view of the different rna unwinding polarities (patel and donmez, 2006) . in conclusion, our work confirms and defines several association groups among sars-cov nsps that will provide a basis to investigate cooperative enzymatic functions as well as currently unknown tertiary and quaternary structures. in this manner, these data may also contribute to the development of in vitro assays faithfully reproducing the different steps in coronavirus rna synthesis. furthermore, compounds affecting interactions between essential components of the 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protein nsp10 the nsp9 replicase protein of sars-coronavirus, structure and functional insights sars-unique domain" (sud) of sars coronavirus is an oligo(g)-binding protein mechanisms and enzymes involved in sars coronavirus genome expression a zinc finger-containing papain-like protease couples subgenomic mrna synthesis to genome translation in a positive-stranded rna virus localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication analysis of intraviral protein-protein interactions of the sars coronavirus orfeome high-throughput yeast two-hybrid assays for large-scale protein interaction mapping insights into sars-cov transcription and replication from the structure of the nsp7-nsp8 hexadecamer the autocatalytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond we thank andrew davidson and john ziebuhr for helpful discussion and sharing preliminary data. we acknowledge jessika zevenhoven-dobbe, françois ferron, christian cambillau, valérie campanacci and sonia longhi for material and their help in the initial phase of the project. we thank claire debarnot, etienne decroly and bruno coutard for excellent technical assistance. dr. j.p. borg is greatly acknowledged for lab space, equipment, and funding to p.l. this work was supported by the structural proteomics in europe (spine) project of the european union 5th framework research program (grant qlrt-2001-00988) and subsequently by the euro-asian sars-dtv network (sp22-ct-2004-511064) from the european commission specific research and technological development programme "integrating and strengthening the european research area" and partially by interdisciplinary cnrs 2007 program "infectious emerging diseases". key: cord-318319-efqf5e1i authors: yamasaki, yukitaka; ooka, seido; tsuchida, tomoya; nakamura, yuta; hagiwara, yuta; naitou, yoshiyuki; ishibashi, yuki; ikeda, hiroki; sakurada, tsutomu; handa, hiroshi; nishine, hiroki; takita, mumon; morikawa, daiki; yoshida, hideki; fujii, shuichi; morisawa, kenichiro; takemura, hiromu; fujitani, shigeki; kunishima, hiroyuki title: the peripheral lymphocyte count as a predictor of severe covid-19 and the effect of treatment with ciclesonide date: 2020-07-03 journal: virus res doi: 10.1016/j.virusres.2020.198089 sha: doc_id: 318319 cord_uid: efqf5e1i we investigated whether reduced lymphocyte count, could predict the development of severe covid-19. we also examined whether ciclesonide could prevent the development of severe covid-19 among patients with the predictors. this was a retrospective cohort study. of the 30 included patients, 12, 14, and 4 were allocated to severe pneumonia, non-severe pneumonia, and non-pneumonia groups, respectively. the group of the low level of lymphocyte counts of the sixth day after onset was significantly intubated approximately three days later. the incidence of the severe pneumoniae requiring intubation are significantly lower in the patients treated with ciclesonide than without it (11.18 % vs 83.33%, p = 0.0033). the lymphocyte count after ciclesonide treatment in the non-severe pneumonia group was significantly higher (p = 0. 0156) than before. the lymphocyte count could be used to identify patients that may develop severe covid-19. treatment with ciclesonide may prevent the development of severe covid-19. since first appearing in wuhan, hubei province, china, in december 2019, a novel coronavirus (sars-cov-2) has spread rapidly around the world. many patients with coronavirus infection disease 2019(covid-19) are subclinical, and it has been reported that people are j o u r n a l p r e -p r o o f contagious even when asymptomatic [1, 2] , which means preventing the spread of sars-cov-2 is challenging [3] . in addition, some patients have been reported to deteriorate rapidly in the early stages [4] . therefore, early detection and preventing cases from progressing to a severe stage is essential. about 20% of covid-19 cases progress to a severe stage, of which about 3% die. risk factors of severe pneumonia include age, comorbidities, smoking, reduced lymphocyte count, elevated ferritin levels, and elevated c-reactive protein (crp) levels [4] [5] [6] [7] [8] [9] . however, it is unclear which of these risk factors are predictors of progression to severe covid-19. as yet, no effective treatment has been found for covid-19. there have been many medications suggested, including remdesivir [10] [11] [12] , lopinavir and ritonavir [13] , and chloroquine [14] , but their efficaciousness have yet to be verified. ciclesonide is an inhaled corticosteroid that is approved to treat asthma. it has demonstrated antiviral effects in vitro [15] and has been reported to be effective in treating covid-19 [16] . according to a report by meehyun koa et al., the infection inhibitory effect of ciclesonide was confirmed in the mers-cov strain isolated in south korea [17] . furthermore, because ciclesonide is a local administration, there are few side effects, and administration is possible for a pregnant woman relatively safely. we believe that preventing the development of severe covid-19 will help to reduce the mortality rate. we investigated whether any of the factors that have been reported to correlate with severe pneumonia could predict the development of severe covid-19. in addition, we examined whether ciclesonide could prevent the development of severe covid-19 among patients with these predictors. this was a retrospective cohort study. all the patients were hospitalized at our institution between february 16 and april 14, 2020, and had tested positive for sars-cov-2 using polymerase chain reaction testing of pharyngeal or nasopharyngeal swabs taken. for all patients, the date of onset was the day clinical symptoms appeared, such as fever, cough, runny nose, and dysgeusia. the presence of pneumonia was confirmed by chest computed tomography (ct). patients who underwent intubation and respiratory management were defined as severe pneumonia group. written informed consent for this study was obtained. the study was conducted with the approval of our hospital's institutional review board (approval number: 4712). thirteen patients with covid-19, hospitalized between february 16 and march 18, 2020, before treatment with ciclesonide starts, were enrolled in this study. blood tests performed less than 14 days from the date of onset and before intubation were examined. if multiple blood tests were performed during the evaluation period, the minimum and maximum values were examined. the leukocyte count, lymphocyte count, platelet count, crp, ferritin, d-dimer, and kl-6 were examined. patients were divided into three groups: severe pneumonia, non-severe pneumonia, and non-pneumonia. for the lymphocyte count, the mean+1sd was used as the cutoff value of severe covid-19 pneumonia. the cases at or below this cutoff value were evaluated, and patients who started ciclesonide after intubation were excluded. the treatment group received 2 inhalations of 400 µg ciclesonide once a day, for a daily total of 800 µg. the relationship between ciclesonide use and severe pneumonia were examined. in addition, the lymphocyte count prior to and approximately j o u r n a l p r e -p r o o f 7 days after starting treatment were compared. data were analyzed with the mann-whitney u , fisher's exact and wilcoxon matchedpairs signed rank tests using graphpad prism ver.6.00 for windows, graphpad software, san diego california usa.. of the 31 patients who were hospitalized during the observation period, 1 was excluded due to a lack of data before intubation. of the 30 included patients, 12 were allocated to the severe pneumonia group, 14 to the non-severe pneumonia group, and 4 to the non-pneumonia group. the study design of this study was shown in figure1. baseline characteristics table 1 details the patients' demographic information. the mean age was 54.5 years, and 83.3% were male. of the total and those with pneumonia, 53.3% and 57.7% had j o u r n a l p r e -p r o o f comorbidities, respectively. blood tests were on average performed 5.8 days after onset (sd 2.72) and 12 days after treatment (sd 3.58). on average, patients developed severe covid-19 and underwent intubation and respiratory management 9 days after onset (sd 2.43). of the 13 patients with covid-19 hospitalized between february 16 and march 18, 2020, before the start of ciclesonide therapy, there were 5 in the severe pneumonia group, 4 in the non-severe pneumonia group, and 4 in the non-pneumonia group ( figure 2 ). lymphocyte counts of approximately sixth days after onset were significantly lower in the severe pneumonia group compared to both the non-severe pneumonia group and the nonpneumonia group (p = 0.0159, 0.0016, respectively) ( figure 2a) . the severe pneumonia group had a low mean lymphocyte count at 659 cells/mm 3 (sd 318.9). patients in the severe pneumonia group were significantly older than those in the non-severe pneumonia group (p=0.0079), but not significantly different from those in the non-pneumonia group (figure 2b ). significant differences were not observed between the severe and non-severe pneumonia groups in relation to ferritin, crp, and d-dimer. regarding sex differences, there tended to be more males in the severe and non-severe pneumonia groups. however, there was no significant difference in sex for the pneumonia cases. while 57.7% of patients in the pneumonia groups had a comorbidity, the difference between the severe and non-severe pneumonia groups was not significant. (table 2) , 12 patients had severe pneumonia, and 11 had non-severe pneumonia. eleven patients from 23 with a lymphocyte count at or below the cutoff value could be treated with ciclesonide. of these, 2 had severe covid-19 pneumonia, and the incidence of the severe pneumoniae requiring intubation are significantly lower in the patients treated with ciclesonide than without it (11.18 % vs 83.33%, p=0.0033). thus ciclesonide therapy is suspected to exhibit a significant correlation with the non-severe pneumonia group. moreover, the lymphocyte count after ciclesonide therapy in the non-severe pneumonia group was significantly higher (p=0.0156) compared to before treatment (mean 6.14 days, sd 2.17) (figure 3b ). five patients with pneumonia were subsequently transferred to other hospitals, so their lymphocyte counts after treatment are unknown. there is currently no therapy that has been proven to be efficacious in treating covid-19. as many covid-19 cases are subclinical, it is challenging to track infected individuals, making it hard to prevent infections from occurring [1] [2] [3] . however, some patients with covid-in the present study, 2 patients who received ciclesonide developed severe pneumonia. patients with covid-19 are known to deteriorate rapidly. both of these cases began treatment with ciclesonide 2 days before intubation, which suggests that the drug may have been introduced too late. previous research has found that age, comorbidities, lymphocyte count, ferritin, crp, and d-dimer are associated with severe pneumonia. of these, only the preintubation lymphocyte count appeared to be a possible predictor with counts in the nonpneumonia and non-severe pneumonia groups being significantly different from the severe pneumonia group. regarding age, there was a significant difference between the severe and nonsevere pneumonia groups, but not with the non-pneumonia group. the 73 year old case in the non-pneumonia group accounts for the lack of significant difference. we believe that this case did not cause pneumonia because there is no underlying disease and there are no risk factors other than age. significant differences were not observed for ferritin or d-dimer. there were cases with high ferritin and d-dimer levels in both the severe and non-severe pneumonia groups. in addition, there was a lot of missing data, making it difficult to accurately assess whether ferritin or d-dimer could be a predictor of severe covid-19. while the presence of subclinical covid-19 cases makes controlling infections difficult, death occurs suddenly in some cases [4] . covid-19 is known to be contagious 2 days before onset, with increased viral loads in the respiratory tract. it has been suggested that cytokine storms are associated with the development of severe covid-19. therefore, the early administration of antivirals could be efficacious, similar to influenza. it is important to identify patients with covid-19 as soon as possible and prevent it from progressing to a severe stage. the lymphocyte count could be used as an indicator for identifying patients that may develop severe covid-19. our results suggest that treatment with ciclesonide may prevent the j o u r n a l p r e -p r o o f development of severe covid-19 in these circumstances. it is best to introduce the drug as soon as possible in patients with reduced lymphocyte counts and other predictors of severe covid-19 [18] . this study had several limitations. this was a retrospective study with a small sample size. therefore, the results need to be confirmed in a larger, prospective study. the viral load determination could not be mentioned because it was not measured in all cases. we showed treatment with ciclesonide as the candidate of the factor which inhibited severe covid-19 in this study. we urgently need to establish a testing system that includes the antigen-antibody method, develop a vaccine, and find treatments that can prevent the development of and 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covid-19 coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio arguments in favour of remdesivir for treating sars-cov-2 infections broadspectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 the inhaled corticosteroid ciclesonide blocks coronavirus rna replication by targeting viral nsp15 therapeutic potential of ciclesonide inahalation for covid-19 pneumonia: report of three cases screening of fda-approved drugs using a mers-cov clinical isolate from south korea identifies potential therapeutic options for a mouse model for mers coronavirus-induced acute respiratory distress syndrome we thank all medical staff who treated covid19 with us.the authors have no conflicts of interest directly relevant to the content of this article.j o u r n a l p r e -p r o o f key: cord-279969-5nlmljcw authors: bastien, nathalie; normand, susan; taylor, tracy; ward, diane; peret, teresa c.t; boivin, guy; anderson, larry j; li, yan title: sequence analysis of the n, p, m and f genes of canadian human metapneumovirus strains() date: 2003-04-03 journal: virus res doi: 10.1016/s0168-1702(03)00065-0 sha: doc_id: 279969 cord_uid: 5nlmljcw the complete nucleotide sequences of the nucleoprotein (n), phosphoprotein (p), matrix protein (m), and fusion protein (f) genes of 15 canadian human metapneumovirus (hmpv) isolates were determined. phylogenetic analysis revealed two distinct genetic clusters, or groups for each gene with additional sequence variability within the individual groups. comparison of the deduced amino acid sequences for the n, m and f genes of the different isolates revealed that all three genes were well conserved with 94.1–97.6% identity between the two distinct clusters the p gene showed more diversity with 81.6–85.7% amino acid identity for isolates between the two clusters, and 94.6–100% for isolates within the same cluster. respiratory virus infections account for significant morbidity and mortality. viruses most frequently associated with respiratory tract infections include rhinoviruses, coronaviruses, influenza viruses, parainfluenza viruses, respiratory syncytial viruses and adenoviruses. however, the causative agents for a significant portion of respiratory infections are still unknown. recently, van den hoogen et al. reported the isolation of a new paramyxovirus in the netherlands that was tentatively assigned to the metapneumovirus genus of the pneumovirinae subfamily (van den hoogen et al., 2001) . this hmpv has been associated with respiratory illnesses ranging from upper respiratory tract disease to severe bronchiolitis and pneumonia similar to those caused by human respiratory syncytial virus (hrsv) infection. hmpv is currently being isolated from patients with respiratory disease in several countries suggesting that hmpv may be present throughout the world (pelletier et al., 2002; peret et al., 2002; stockon et al., 2002) . partial sequence analysis demonstrated sequence similarity between the dutch and canadian isolates and the presence of two potential genetic clusters. phylogenetic analysis of hmpv and the other members of the pneumovirinae revealed that hmpv is more closely related to avian pneumovirus (van den hoogen et al., 2001) . to gain a better understanding of the molecular biology of hmpv, we report the complete sequence of n, p, m and f genes and intervening regions of multiple recent isolates of this virus from canada. fifteen specimens were collected from patients with acute respiratory illnesses from 1997 to 2000 in quebec city, quebec, canada. the specimens were inoculated onto llc-mk2 cells and serially blind passaged (peret et al., 2002) . following viral isolation, the virus was cultured at 37 8c in eagle's mem supplemented with penicillin (100 u/ml), streptomycin (100 mg/ml), 30 mg/ ml l-glutamine (life technologies) and 0.02% tpkc trypsin (sigma). the virus isolates were cultured for a maximum of 21 days with weekly media change. the cells usually displayed cytopathic effects at 8 á/14 post inoculation, characterized by focal rounding and cell destruction without apparent syncytia formation. the specimens were negative when tested by indirect immunofluorescent assays for influenza virus a and b, parainfluenza viruses 1, 2 and 3, adenovirus and respiratory syncytial virus (bartels; chemicon). these isolates were also negative for influenza a and b, parainfluenza 1, 2, 3, 4 and hrsv by an in-house rt-pcr. primers used for amplification and sequencing were based on the published n (af371365), p (af371366), m and f (af371367) hmpv sequences (van den hoogen et al., 2001 hoogen et al., , 2002 . the primer pairs used to amplify each gene and gene junction are described in table 1 . viral rna was extracted from 100 ml of tissue culture fluid using the rneasy mini kit (qiagen). viral rna was amplified in a one step rt-pcr reaction (qiagen) following the manufacturer's recommendations. briefly, 2á/10 ml of rna was added to the rt-pcr mixture containing 2 ml qiagen onestep rt-pcr enzyme mix, 10 ml of 5x qiagen onestep rt-pcr buffer, 400 mm dntp, 0.6 mm of each primer and 10 ml of q-solution in a final volume of 50 ml. the thermocycler conditions used were: 50 8c for 30 min for reverse transcription, 95 8c for 15 min for the activation of the hotstart dna polymerase; then 35 cycles of 94 8c for 1 min, 50 8c for 1 min, 72 8c for 2 min, followed by an extension of 7 min at 72 8c. the pcr products were purified using qiaquick pcr purification kit (qiagen) and cloned in vector pcr2.1 (invitrogen) according to the manufacturer's instructions. two independent purified pcr amplicons for each gene were either sequenced directly, or sequenced using m13-specific primers after cloning in pcr2.1, on an abi 377 sequencer, using a fluorescent dye-terminator kit (applied biosystems). the dna sequences were assembled and analyzed with seqman, edit-seq and megalign programs in lasergene (dnas-tar, madison, wi). phylogenetic trees were generated by neighbor-joining method using the mega program (kumar et al., 2001) . the nucleotide sequence database accession numbers of the pneumovirus strains used in this paper are: hrsv-a (all orfs:m11486); hrsv-b (all orfnucleotide sequences and positions were derived from the ndl00-1 isolates (af371337). added restriction sites and unspecific sequences are underlined. s:af013254); brsv (all orfs:af092942); apv-a (n:u39295, p:u22110, m:x58639, f:y14294); apv-b (n:u39296, p:af325443, m:u37586, f:y14293); apv-c (n:af176590, p:af176591, m: af072682; f:af187152); pvm (n:d10331, p:u092942, m:u66893; f:d11128); hmpv netherlands isolate 00-1 (nld00-1) (n: af371365, p: af371366, m and f:af371367). the hmpv sequences described in this paper have been deposited in genebank under accession numbers ay145242á/ay145301. fifteen isolates obtained from patients suffering from respiratory illnesses were used for the current study. initial testing of the samples by rt-pcr analysis using primer sets specific for hrsv, parainfluenza virus 1, 2, 3, and 4 as well as influenza a and b were all negative. subsequently, the 15 isolates were identified as hmpv using a rt-pcr test based on the fusion (f) protein gene (peret et al., 2002) . phylogenetic trees were constructed based on the n, p, m and f orf sequences of 15 hmpv canadian isolates, the published full-length hmpv sequence and the a, b and c apv sequences. as shown in fig. 1 , for each of the four orfs analyzed, all fifteen isolates fell into two distinct genetic clusters or groups that were tentatively named 1 and 2 ( fig. 1) . additional variability was also observed within the individual groups. these results confirm data previously reported for the partial sequences of the n, m, f and l genes of hmpv isolates from the netherlands and canada (peret et al., 2002; van den hoogen et al., 2001) . partial sequences of the n and f genes of hmpv isolated in australia have also been reported (nissen et al., 2002) . phylogenetic analysis of the n and f genes of australian, canadian and dutch isolates also shows the same two distinct genetic clusters (data not shown). representatives of both clusters 1 and 2 were present in hmpv isolates from the netherlands and canada, whereas the australian isolates all belonged to cluster 1. the hmpv dutch isolates (ndl00-1) characterized by van den hoogen et al. clustered with group 1 isolates (van den hoogen et al., 2002) . following the phylogenetic analysis, isolates can99-81 (cluster 1) and can98-76 (cluster 2) were selected as representatives of each hmpv cluster. their genomes were sequenced from the gene start codon of the n gene to the one of the m2 gene. the putative gene start signals for the n, p, m and f genes were identified by comparison with those of the hmpv dutch isolate (ndl00-1) and other pneumoviruses: human and bovine respiratory virus (hrsv, brsv), avian pneumovirus (apv) and pneumonia virus of the mice (pvm). nucleotide sequence analysis revealed the presence of a well conserved region located at the beginning of each hmpv gene that potentially serves as gene-start signal (table 2) . however, since these signals were identified from the viral rna, they remain tentative and should be confirmed by sequencing of the viral mrna. the same is true for the gene end sequences described below. no sequence variation was observed between the two genetic clusters or the ndl00-1 isolate for n and p putative gene start signals (van den hoogen et al., 2002) . the putative gene start signal of the m gene had one a/c nucleotide change and the f gene one a/g substitution between the two clusters. the can99-81 (cluster 1) putative gene start sequence was identical to the one of ndl00-1 (cluster 1). all gene start sequences contained a (gggacaag/au) motif that is identical to the gene start signal characterized for the matching genes of apv subgroups a, b and c (dar et al., 2001; li et al., 1996; ling et al., 1995; naylor et al., 1998; randhawa et al., 1996; seal, 1998; seal et al., 2000; yu et al., 1992) . in contrast, no consensus sequences could be identified as a common gene termination signal. comparison with ndl00-1 sequences revealed the presence of a putative gene end signal for each of the genes analyzed (bolded in table 2 ). each putative gene end signal was conserved between the two hmpv genetic clusters and ndl00-1. further comparison with apv sequences showed that the putative gene end signal for the hmpv n gene (aguaauua 6 ) was identical to the one characterized for apv-c (dar et al., 2001; van den hoogen et al., 2002) . similar analysis revealed that the putative gene end sequence for the p gene (uaguuuaaua 5 ) contained the same (aguu) element of transcription as those of apv p genes (dar et al., 2001; ling et al., 1995) . the putative gene end signals identified for the m (aguuauaua 5 ) and f (uaguuaguuaauua 5 ) genes of hmpv possess a similar (aguua) element of transcription stop signal found for the f genes of apv-a and c, but varies from those found for apv m gene (aguca/apv-a, aguuu/apv-b, uauua/apv-c) (naylor et al., 1998; randhawa et al., 1996; seal, 1998; seal et al., 2000; yu et al., 1992) . similarly to other pneumoviruses, the putative gene end and intergenic sequences of the hmpv gene varied in length (table 2 ). that region of the p gene was strictly conserved between the two clusters and ndl00-1, whereas those of the n, m and f genes varied. in particular, that region of the m gene showed only 37% identity between the cluster 1 and 2, and cluster 1 sequence had three additional nucleotides. it was suggested that this region contained part of a potential secondary orf of 33 aa residues that starts at nt 2281 (van den hoogen et al., 2002) . the corresponding region of the f gene varied greatly in length (20 á/80 nucleotides long) between clusters. the ndl00-1 putative gene end and intergenic sequences were more closely related with can99-81, but were not identical. four nucleotide changes were found in those m noncoding sequences (nt: 2947-g/a, 2976-u/c, 2990-u/c, 3053-u/c) of ndl00-1. the f putative gene end and intergenic sequences of ndl00-1 contained two nucleotides substitution (4712-a/t, 4717-t/a) and had a 21 nucleotides , m (c) and f (d) orfs were amplified by pcr and sequenced. the corresponding gene sequences from apv subgroups and hmpv from the netherlands (nld00-1) were also analyzed. bootstrap proportions were plotted at the main internal branches of the phylogram to show support values. phylogenetic analysis was performed using the neighbor-joining method of the mega program. the year in which the isolate was collected is indicated by the first two numbers in the isolate name. deletion (4724-4744) in comparison to can99-81 sequences. sequence alignment with other members of the pneumovirus genus revealed that the n gene coding region was 1185 bases in length. the deduced n protein was 394 aa in length with an estimated molecular mass of 43.5 kda (fig. 2) . the n protein of hmpv is similar in size to the ones of other pneumoviruses (391 á/394 residues), which is relatively smaller than those reported for the other members of the paramyxoviridae family (489 á/583 residues) (barr et al., 1991; collins et al., 1985; curran and kolakofsky, 1999; dar et al., 2001; spriggs and collins, 1986) . the n gene of isolates within the same cluster were highly conserved with nucleotide sequence identity of 93.8 á/100%, whereas sequence identity between the two different groups was 85.3 á/ 86.1% (table 3 ). the aa identity was higher both within and between groups, 98.3 á/100% and 94.2 á/95.9%, respectively. the n protein of ndl00-1 was identical to the one of can99-81 and therefore showed similar level of aa identity with the respective hmpv cluster. the levels of aa identity observed between the two hmpv clusters are comparable to the ones observed between the two subgroups of hrsv (96%) (johnson and collins, 1989) . the n proteins of apvs are not highly conserved, with 69á/89% aa identity between the different subgroups (dar et al., 2001; li et al., 1996) . when compared to the other pneumovirus members, the hmpv n gene showed 69 á/89% aa identity with apv members, 42 á/44% with rsv members and 45% with pvm ( barr et al., 1991; buchholz et al., 1999; dar et al., 2001; karron et al., 1997; li et al., 1996; venkatesan et al., 1983) (table 4 ). the n genes of all non-segmented, negative-stranded rna viruses have three relatively conserved domains with homology in sequence and predicted secondary structure (barr et al., 1991; li et al., 1996; dar et al., 2001) . sequence comparison with apv n genes allowed us to identify the position of these domains at residues 161 á/173, 251 á/263 and 279 á/326 (li et al., 1996) (fig. 2) . these three domains were highly conserved between the two hmpv clusters and only one conservative aa substitution (aa 164) was found with their closest pneumovirus relative (apv-c). most of the aa changes (66.6%) in the n genes of the two hmpv clusters were located within the first 136 aa of the protein. however the majority of these (62.5%) were conservative substitutions. two of the remaining changes were found in the central part of the protein, and the last two in the last 38 aa. the coding region of the p gene was 885 bases in length, encoding a predicted protein of 294 aa residues with a molecular mass of 32.5 kda. the size of the hmpv p protein was comparable to those of apvs and pvm (279 á/295 aa), but was larger than the non-avian pneumovirus (241 aa) (barr et al., 1994; buchholz et al., 1999; dar et al., 2001; karron et al., 1997; ling et al., 1995; satake et al., 1984) (fig. 3) . within each cluster the nucleotide sequence identity was 91á/100% and the identity for the predicted aa sequence was 94.6 á/100% (table 3 ). the p gene was less conserved between clusters with 78.0 á/79.4% and 81.6 á/85.7% identity levels for the nucleotide and aa sequences, respectively. the majority of the aa differences (34) between the two clusters were observed at the amino-proximal end of the protein (19 á/130) (fig. 3) . the central portion of the proteins (aa 131 á/278) was well conserved, containing only five aa changes, while the remaining changes (five) were found in the last 16 aa of the protein. the majority (65.9%) of the aa substitutions were non-conservative. six aa changes were found between ndl00-1 and can99-81 (s/g-31, l/p-64, i/t-78, a/t-201, t/a-202, i/t280). none of these aa changes were unique to the ndl00-1 sequence. the 201 and 280 threonine residues present in the ndl00-1 sequence were also found in other cluster 1 and 2 canadian hmpv isolates and the four remainder aa substitutions were identical to cluster 2 sequences. the p protein of hmpv showed a significant level of divergence with those of other members of the pneumoviruses (barr et al., 1994; buchholz et al., 1999; dar et al., 2001; karron et al., 1997; ling et al., 1995; satake et al., 1984) (table 4 ). the highest levels of identity were observed with the p protein of apvs (50 á/68%), with the greatest identity shared with apv-c (dar et al., 2001; ling et al., 1995) . the identity level was only 23á/28% with mammalian pneumoviruses (barr et al., 1994; buchholz et al., 1999; karron et al., 1997; satake et al., 1984) . the aa region encompassing residues 171 á/226 represents a highly conserved domain in pneumovirus p proteins (ling et al., 1995) (fig. 3) . the amino-terminal portion of this region has a heptad repeat sequence that is frequently implicated in the formation of a-helices and coiled coils (ling et al., 1995; mclachlan and karn, 1983) . this region of the hmpv p proteins had greater aa identity with both avian and mammalian pneumovirus 80.0 á/87.5% and 62.5 á/67.9%, respectively (fig. 4) . similar to the p proteins of other pneumovirus, the majority (82 á/85%) of proline residues present in the hmpv p protein were found in the n-terminal half of the protein (dar et al., 2001; ling et al., 1995) . the large number of charged residues found at the c-terminal portion of hmpv p is another common feature shared in all pneumovirus. as observed for apv-c, hrsv and brsv, the p protein of hmpv did not contain any cysteine residues. in contrast, the p proteins of apv-a and b have two cysteine residues, and five cysteine residues are found in the pvm protein (barr et al., 1994; ling et al., 1995) . sequence alignment analysis showed that the m gene of hmpv was 765 nucleotides in length, encoding a predicted protein of 254 aa residues with a predicted molecular mass of 27.6 kda (fig. 4) . the m genes of hmpv isolates were highly conserved both within (94.1á/ 100% identity) and between (83.5 á/85.6% identity) clusters (table 3 ). these differences were reflected at the aa level with identity of 96.9 á/97.6% for members of different clusters and 98.8 á/100% for isolates belonging to the same cluster. the aa sequences of the m protein of ndl00-1 was identical to the one of can99-81 and showed similar levels of aa identity with the two hmpv clusters. the majority of aa substitutions between the two clusters were conservative and distributed throughout the entire protein (fig. 4) . the m protein of hmpv was most closely related to the m proteins of apvs with aa identity ranging from 76 á/88%, compared to the reduced aa identity 37 á/40% observed with the remaining pneumoviruses (table 4 ) (buchholz et al., 1999; easton and chambers, 1997; karron et al., 1997; randhawa et al., 1996; satake et al., 1984; seal, 1998; yu et al., 1992) . a short region located at the nterminal end of the protein (aa 14 á/19) was conserved for all pneumovirus m proteins, although the biological significance of this region is unknown. the f gene was 1620 bases in length, encoding a protein of 539 aa residues with a predicted molecular mass of 58.4 kda (fig. 5) . the f gene was well conserved within each group with 94.3 á/100% and 98.3% á/100% for the nucleotide and aa sequences identity, respectively (table 3) . between the two clusters the identity was 83.0 á/83.6% and 94.1 á/95.4% for the nucleotide and aa sequences, respectively. the aa sequence of the f protein of ndl00-1 had 100% identity with the one of can99-81. the majority (75.0%) of aa substitutions between the two clusters were conservative. sequence comparison with avian pneumoviruses showed identity levels of 67 á/68% with apv subgroup a and b and 81 á/82% with subgroup c (table 4 ) (naylor et al., 1998; seal et al., 2000) . the f gene of hmpv was less conserved with the mammalian pneumoviruses and showed 30á/43% aa identity (buchholz et al., 1999; chambers et al., 1992; collins et al., 1984; karron et al., 1997; seal, 1998) . the f proteins of pneumoviruses are synthesized as a precursor f0 that is cleaved by cellular protease into two subunits, f1 and f2. most pneumoviruses cleavage peptide sequences contain the cleavage motif for the the results represent the range of identity (%) obtained for the comparison of the most divergent to the most conserved sequences within and between each genetic clusters. cellular furin protease (r/k-x-r/k-r) (collins et al., 2001) . however, the cleavage peptide sequence of apv-c (rkar) varies from the consensus sequence (seal et al., 2000) . similarly, sequence analyses revealed that the cleavage peptide sequence of hmpv f protein (rqsr) differs from that of the furin protease motif (fig. 5) . cleavage of the f0 precursor releases the hydrophobic fusion peptide at the n-terminus of the f1 subunit. the f1 and f2 subunits of the two hmpv lineages showed a similar level of conservation with aa identities of 94% and 97%, respectively. these identity levels were similar to those observed within hrsv subtypes (f1, 98% and f2, 95%) (naylor et al., 1998) . in contrast, greater divergence (83% identity) was observed between subtypes of hrsv and apv (naylor et al., 1998) . similar to apvs, the carboxy-terminal transmembrane domain and cytoplasmic tail of the two hmpv clusters diverge significantly (16%) (naylor et al., 1998; seal et al., 2000) . however, this region of the hmpv f protein showed greater aa identity between clusters (84%) than the one observed between the different apv subgroups (32% á/ 39%) (naylor et al., 1998; seal et al., 2000) . the remaining portion of the hmpv f1 subunit was more conserved with only 4.2% aa divergence between the two clusters. the two heptad repeats domains (hra and hrb) necessary for viral fusion of paramyxoviruses (chambers et al., 1990; buckland and wild, 1989; lamb, 1993; russell et al., 2001) are also present in the hmpv f gene. there were three potential nglycosylation sites at aa 57, 172 and 353, two of which (57 and 172) were also present among the f proteins of all apv subgroups (naylor et al., 1998; seal et al., 2000) . the aa residues at position 102 (arg), 103 (phe), 106 (gly) and 111 (gly) are structurally important to paramyxovirus f proteins (horvath and lamb, 1992) and are present in all pneumoviruses. these aa residues were conserved for the two clusters of hmpv f proteins. the phylogenetic analysis of the complete n, p, m and f genes of the canadian isolates was consistent with previous reports (peret et al., 2002; van den hoogen et al., 2001) , and showed the presence of two genetic clusters. comparison of these sequences with the ones reported from the netherlands and australia showed that all hmpv isolates reported to date cluster within the same two genetic groups, suggesting that a relatively homogeneous population of hmpv is circulating worldwide (nissen et al., 2002; van den hoogen et al., 2001) . in fact, hmpv canadian cluster 1 isolates showed over 96% amino acid identity with the ndl00-1 dutch isolates for all the viral protein analyzed (van den fig. 4 . alignment of the predicted amino acid sequences of the hmpv m protein of hmpv can99-81 (cluster 1) with those of can98-76 (cluster 2), apv-a, b and c, hrsv a and b, brsv and pvm. only residues that differ from isolate can98-76 are shown, identical amino acids are represented by periods, gaps are represented by dashes. hoogen et al., 2002) . our results also show that both hmpv clusters were co-circulating in canada during 1997 canada during , 1999 canada during and 2000 . this is comparable to what is observed for the two subgroups of hrsv, which have been co-circulating in most yearly epidemics (collins et al., 2001) . similar to other pneumoviruses, the genetic diversity observed for the hmpv genes may lead to antigenic variability and these two hmpv genetic clusters may also represent two different antigenic groups. however, distinct lineages of hrsv have been identified within each subgroup, and shown to cocirculate during epidemics and to have broad geographic distribution pringle, 1991, 1995; coggins et al., 1998; garcia et al., 1994; martinez et al., 1999) . the nucleotide identities observed between the two hmpv clusters for each of the genes analyzed are comparable to the ones obtained between members of the two hrsv subgroups (naylor et al., 1998) . however, a greater proportion of silent nucleic acid substitutions are found in the f genes of hmpv clusters leading to an aa identity that is closer to the one observed within hrsv subgroups. at this point, it is still unclear if the differences observed between the hmpv isolates are representative of two distinct hmpv antigenic subgroups. the characterization of the hmpv g genes for which the antigenic diversity is most evident for rsv and apv, as well as serologic studies, are needed to determine the antigenic significance of the genetic diversity observed for hmpv. molecular analysis of the n, p, m and f genes provided further support for the assignment of hmpv to the metapneumovirus genus of the paramyxovirinae subfamily. analysis of the genomic organization of hmpv revealed several similar features to apvs, especially to the subgroup c (van den hoogen et al., 2001) . they share the same putative gene start sequence (gggacaagugaaa), as well as the same gene end sequences for the n gene (aguaauua 6 ). although not identical, the gene end sequences of the other genes also show some similarity to apvs. the 3? noncoding region of the m and f genes varies significantly between the two hmpv clusters. this finding is comparable to the low conservation of some 3? noncoding sequences of hrsv and apv isolates belonging to different subgroups (bayon-auboyer et al., 2000; seal et al., 2000) . sequence alignment studies show that the hmpv proteins are more closely related to the subgroup c of apv, with aa identities for the different proteins ranging from 66 to 89% (van den hoogen et al., 2002) . a greater divergence is observed with the non-avian pneumovirus members and the aa identity drops to 23á/45%. the n, m and f genes were the most conserved whereas the p genes show greater divergence. comparison of the predicted aa sequences revealed that the n, m, p and f proteins share most of the common features characterized for these proteins in other pneumoviruses. however, the cleavage peptide sequence for the hmpv f protein (rqsr) varies from the one described for other pneumoviruses, and differs from the furin protease motif (r/k-x-r/k-r) present in all pneumovirus except for apv-c. when compared to hmpv, the cleavage sites of other pneumoviruses contain additional arg-lys residues. the substitution of these basic aa in the hmpv cleavage site may play a role in viral pathogenesis, since it may modify the efficiency of cleavage by the host protease. the virulence of newcastle disease virus (ndv) a member of the rubulavirus genus of the paramyxovirinae subfamily has been linked to the number of basic residues present at the f protein cleavage site. ndv virulent strains have two pairs of basic residues at the cleavage site, whereas avirulent strains have two basic residues (collins et al., 1993) . the close relationship observed between hmpv and apv-c suggests that hmpv originated from birds. it is also possible that apv-c could have evolved from human precursor strains of hmpv that infected birds. apv-c has only recently been described infecting commercial poultry (mid-1990s) (dar et al., 2001; seal, 1998; seal et al., 2000) . it was speculated that hmpv is an avian virus that is also able to infect humans (van den hoogen et al., 2001) . however, data obtained by van den hoogen et al., indicate that the human virus is unable to infect turkeys and chickens, but can infect monkeys experimentally (van den hoogen et al., 2001) . the relative importance of hmpv on viral respiratory tract illnesses is still not known, but serological studies demonstrated that by the age 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of a subgroup b avian pneumovirus membrane fusion machines of paramyxoviruses: capture of intermediates of fusion sequence analysis of the respiratory syncytial virus phosphoprotein gene matrix protein gene nucleotide and predicted amino acid sequence demonstrate that the first us avian pneumovirus isolate is distinct from european strains fusion protein predicted amino acid sequence of the first us avian pneumovirus isolate and lack of heterogeneity among other us isolates sequence analysis of the p and c protein genes of human parainfluenza virus type 3: patterns of amino acid sequence homology among paramyxovirus proteins a newly discovered human pneumovirus isolated from young children with respiratory tract disease construction and characterization of cdna clones for four respiratory syncytial viral genes cloning and sequencing of the matrix protein (m) gene of turkey rhinotracheitis virus reveal a gene order different from that of respiratory syncytial virus we thank dr albert d.m.e. osterhaus (erasmus medical center rotterdam, the netherlands) for providing sequence information for primer synthesis. we are also grateful to dr dean d. erdman (cdc, atlanta) for critical reading of the manuscript. key: cord-286416-8eu6wp9b authors: valiente-echeverría, fernando; melnychuk, luca; mouland, andrew j. title: viral modulation of stress granules date: 2012-06-14 journal: virus res doi: 10.1016/j.virusres.2012.06.004 sha: doc_id: 286416 cord_uid: 8eu6wp9b following viral infection, the host responds by mounting a robust anti-viral response with the aim of creating an unfavorable environment for viral replication. as a countermeasure, viruses have elaborated mechanisms to subvert the host response in order to maintain viral protein synthesis and production. in the last decade, several reports have shown that viruses modulate the assembly of stress granules (sgs), which are translationally silent ribonucleoproteins (rnps) and sites of rna triage. this review discusses recent advances in our understanding of the interactions between viruses and the host response and how virus-induced modulations in sg abundance play fundamental roles in dictating the success of viral replication. exposure of cells to environmental stress (e.g., heat shock, uv irradiation, hypoxia, endoplasmic reticulum (er) stress and viral infection) trigger a rapid translational arrest generating polysome disassembly . this event triggers a molecular triage, where the affected cell must make a decision on the fate of mrna that is released from polysomes: decay or silencing (anderson and kedersha, 2008) . for these events, cells have elaborated different classes of rna granules named processing p-bodies (pbs) or stress granules (sgs) that contribute to the regulation and lifecycle of mrnas. both pbs and sgs contain share proteins and are assembled in cells subjected to stress, but differ in: (i) only pbs are observed in unstressed cells, (ii) sg assembly typically requires phosphorylation of translation initiation factor eif2␣, but not pb assembly (fig. 1) , and (iii) pbs contain proteins involved in mrna decay, whereas sgs contain proteins of translation initiation complex (eulalio et al., 2007) . pbs are cytoplasmic structures that, unlike sgs, are responsible for mrna decay, rna-mediated gene silencing (microrna and sirna-based gene silencing) and mrna surveillance (or rna quality control) (beckham and parker, 2008) . pbs were discovered by bashkirov et al. (1997) and they showed that xrn1, a 5 -3 exoribonuclease, was localized in small granular structures within the cytoplasm. other proteins related to mrna degradation were also found to localize to this granules, such as a deadenylase (ccr4), decapping enzymes dcp1 and dcp2 as well as the activators of decapping dhh1/p54/rck/ddx6, pat1, scd6/rap55, edc3, hedls and lsm1-7 complex (eulalio et al., 2007; ingelfinger et al., 2002; van dijk et al., 2002) . moreover, pbs can contain mrnas and proteins involved in nonsense-mediated decay (nmd) (e.g., smg5, smg7, and upf1) (fukuhara et al., 2005; unterholzner and izaurralde, 2004) and components of the rna-induced silencing complexes (risc) (e.g., argonaute, microrna and gw182) (liu et al., 2005; rehwinkel et al., 2005) (fig. 2) . on the other hand, sgs were first observed in the cytoplasm of plant cells exposed to heat shock (nover et al., 1983) . sgs are translationally silent ribonucleoproteins and serve as storage sites of mrnas and proteins (fig. 2) , while other functions also have been discussed . sgs typically contain poly(a) + mrna, 40s ribosomal subunits, eif4e, eif4g, eif4a, eif4b, poly(a)-binding protein (pabp1), eif3, eif2, p54/rck/ddx6, and many other rna-binding proteins that regulate mrna structure and function, including human antigen r (hur), staufen 1, polysomal ribonuclease 1 (pmr-1), smaug, tristetraprolin (ttp), t-cell restricted intracellular antigen 1 (tia-1) and tia-1-related protein (tiar), fragile x mental retardation protein (fxmr/fxr1), ras-gap sh3-binding protein (g3bp-1), cytoplasmic polyadenylation binding protein (cpeb) and survival of motor neurons (smn) protein, although the composition can vary (listed in table 1) . during a stress response, cells induce a shut-off of cellular protein synthesis and subsequently promote sg assembly (anderson and kedersha, 2009) . different pathways in sg assembly have control of translation by eukaryotic initiation factor 2 (eif2). eif2 bound to gdp (eif2-gdp) is recycled to the active eif2-gtp by a reaction catalyzed by eif2b. once recycled, eif2-gtp forms a ternary complex with initiator-methionine trna (met-trnai) and 40s ribosome resulting in 43s pre-initiation complex. four kinases activated by hemin deficiency/oxidative stress (hri), viral infection (pkr), endoplasmic reticulum stress/hypoxia (perk/pek) and amino acid starvation/uv irradiation (gcn2); can phosphorylate eif2 subunit ␣, stabilize eif2-gdp-eif2b complex (inactive) and prevents eif2 recycling. these events result in a shut-off of the host protein synthesis and subsequently sg assembly (fig. 2 , i). been described. the most popular pathway is the phosphorylation of the critical translation initiation factor, eif2␣ by a family of four serine/threonine kinases hri, pkr, perk/pek and gcn2. hri (eif2␣k1) is activated in heme deprivation and oxidative stress (han et al., 2001) ; pkr (eif2␣k2) is activated by viral infection (williams, 2001) ; perk/pek (eif2␣k3) is activated in the presence of unfolded proteins in the endoplasmic reticulum (er) and during hypoxia (harding et al., 2000) ; and gcn2 (eif2␣k4) is activated during amino acid starvation and uv irradiation (jiang and wek, 2005) . each kinase causes the phosphorylation of the ␣-subunit of eif2 at ser52, which implies the tight binding with eif2b, inhibiting the exchange of gdp for gtp ( fig. 1) . therefore, there is a decrease in translation tertiary complex assembly (eif2/gtp/met-trna) which suppresses the initiation of translation and promotes sg assembly (fig. 2 , step i) . other mechanisms independent of the phosphorylation of eif2␣ have also been explored. hippuristanol and pateamine a, drugs that inhibit the helicase activity of eif4a, are able to induce the assembly of sgs (fig. 2 , step ii) (dang et al., 2006; mazroui et al., 2006) . as well, the overexpression of sg markers (anderson and kedersha, 2008) , such as tia1 (kedersha et al., 1999) or g3bp-1 (tourriere et al., 2003) , can trigger the assembly of sgs (fig. 2 , step iii). the activation of eif2␣ kinases by viral infection may result in the inhibition of cellular protein synthesis (walsh and mohr, 2011) and/or promotion of autophagy, process involving lysosomaldependent recycling of intracellular components (talloczy et al., 2002) . moreover, some viral proteins can bind eif4a (aoyagi et al., 2010; page and read, 2010) . all of these mechanisms induce sg assembly (i.e., shut-off of cellular protein synthesis), but the viruses have found ways to bypass the hostile environment generated by the cell to ensure their survival. in the last decade, several studies have also demonstrated that the assembly of sgs can be dramatically influenced by viruses: the induction and blockage of sg assembly mediated by viral infections have both been described as means to promote virus replication (beckham and parker, 2008; montero and trujillo-alonso, 2011; white and lloyd, 2012) . in this review we will summarize the current understanding that exists between different virus families and the regulation of stress granules. fig. 2 . sg assembly pathways. polysomes disassembly can lead to the assembly of cytoplasmic granules know as processing p-bodies (pbs) or stress granules (sgs). if deadenylation (e.g., ccr4/not1), destabilization (e.g., ttp/xrn1) and decapping (e.g., dcp1/dcp2) complex; and even risc (ago) complex are recruited to mrna, these will be targeted to pbs. conversely, if tia-1/tiar or proteins such as g3bp/usp10 are recruited to the stalled initiation complexes, these will be directed to sgs. different pathways in sg assembly are described (in red): (i) phosphorylation of eif2␣ induced by the exposure to different stress inducers (e.g., arsenite and thapsigargin) (fig. 1) ; (ii) hippuristanol and pateamine a, drugs that inhibit the helicase activity of eif4a altering atp binding or atpase activity; and (iii) the overexpression of sg markers, such as g3bp or tia-1. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) in 2002, the first evidence was reported showing an interaction between viruses and what we understand to be protein components of sgs. li et al. showed that the negative strand 3 terminal stem-loop structure present in the genome of west-nile virus (wnv) interacts with two sg markers, tia-1 and tiar . in support of the necessity for these virus-host interactions, wnv replication was reduced when tiar −/− cells were infected . wnv is a neurotropic flavivirus responsible for viral meningoencephalitis which has an enzootic cycle between mosquitoes and birds, but can infect amphibians, reptiles, horses and humans (dauphin et al., 2004) . moreover, emara et al. expanded these observations to other members of the same viral flaviviridae family, where tia-1/tiar were shown to co-localize with viral replication complexes (dsrna and ns3) in both wnvand dengue virus-infected cells (emara and brinton, 2007) . sgs can be induced in mammalian cells by several drugs (kedersha and anderson, 2007) , apparently as a consequence of the phosphorylation of eif2␣. in order to determine if viral infection would have any effect on sg assembly, baby hamster kidney (bhk) cells were infected with wild-type wnv and subjected to arsenitemediated oxidative stress. infected cells were found to be resistant to sg induction (emara and brinton, 2007) . however, recent studies showed that chimeric wnv produces high levels of an early viral rna (w956ic), allowing pkr activation and subsequent induction of sg, likely due to translational arrest (courtney et al., 2012) . another flavivirus, hepatitis c virus (hcv), the major etiologic agent of hepatitis c in humans, is able to disrupt pb assembly but at the same time, promote sg assembly during the course of viral infection (ariumi et al., 2011) . however, late in hcv infection corresponding to 48 h post-infection, g3bp-1 and ddx6, both components of sg (table 1) , are found to co-localize with the hcv core, resulting in the suppression of sg assembly. this blockade to sg assembly was found to be due to an interaction between g3bp-1 and the hcv non-structural protein, (ns)5b and the 5 end of the hcv minus-strand rna (yi et al., 2011) . thus, as shown in the examples above, through sequestration of factors essential for the assembly of sgs, several viruses have elaborated mechanisms to impose a blockade to sg assembly. some viruses inhibit cap-dependent translation (hence host cell mrna translation) to ensure the synthesis of their own proteins. pelletier et al. discovered that the translation of the uncapped picornaviral mrna is mediated by an rna structure known as the internal ribosome entry site, ires, at the 5 end of the viral rna (pelletier et al., 1988) . infection by poliovirus (pv), the etiologic agent of paralytic disease known as poliomyelitis, induces the inhibition of cap-dependent translation initiation by the cleavage of the translation initiation factors eif4gi, eif4gii, and pabp mediated by viral proteinases (gradi et al., 1998; kuyumcu-martinez et al., 2002) . sg assembly is induced at a very early time post-pv infection (at approximately 2-4 h), but later, sgs disappear because the same viral 3c proteinase (3cpro) cleaves g3bp-1, but not tia-1 or tiar, and thereby prevents sg assembly (white et al., 2007) . the sgs found in pv-infected cells contain viral rna and tia-1, but are compositionally distinct since they exclude well-described sg components such as g3bp-1, pabp, and eif4g, all of which are eventually cleaved by 3cpro (piotrowska et al., 2010; white and lloyd, 2011) . furthermore, pv infection also disrupts the assembly of pbs. also during pv infection, xrn1, dcp1a and pan3, three factors involved in mrna decapping, degradation and deadenylation, respectively, undergo degradation or cleavage by the viral 3cpro (dougherty et al., 2011) . likewise, cricket paralysis virus (crpv) infection in drosophila cells leads to a rapid shut-off of host protein synthesis concomitant with phosphorylation of eif2␣ (wilson et al., 2000) . because these characteristics are common to the induction of sgs, khong et al. investigated the assembly of sg after crpv infection. through an immunofluorescence assay, the authors showed that rox8 and rin, drosophila sg marker homologs of tia-1 and g3bp-1, respectively, do not aggregate in crpv infected cells, even in the presence of sg inducers such as heat shock, oxidative stress and pateamine a. it was also demonstrated that crpv viral 3c proteinase is sequestered to sgs under cellular stress but not during virus infection (khong and jan, 2011) . another picornavirus, theiler's murine encephalomyelitis virus (tmev) which causes a demyelinating disease similar to multiple sclerosis in the central nervous system, also inhibits sg assembly. borghese et al. showed that tmev infection induces sg assembly, but the expression of the leader (l) protein during infection was sufficient to inhibit sg assembly induced by arsenite-mediated oxidative stress or by thapsigargin-mediated er stress. unlike the effects induced by pv 3c proteinase, g3bp-1 was not cleaved by tmev and was in fact found in sgs post-tmev infection (borghese and michiels, 2011) . for efficient protein synthesis, mrna circularization is required during translation. pabp, that is bound to poly (a) 3 tail, interacts with eif4gi at the 5 , causing circularization of the mrna by linking the 5 and 3 mrna ends, increasing the binding of eif4e to the cap (lopez-lastra et al., 2010) . rotavirus, the causative agent of a common infantile gastroenteritis, subverts the host translation machinery at this step. because rotavirus mrnas are capped but lack poly(a) tails, the virus-encoded protein, non-structural (ns) p3, binds to a consensus rna sequence in the 3 end of viral mrna, enabling mrna circularization by interaction with eif4gi (piron et al., 1998) . as a consequence, a shut-off of host protein synthesis ensues and thereby provides an advantage for viral protein synthesis. in infected cells, montero et al. found that eif2␣ is phosphorylated during the entire virus replication cycle but this does not have an impact in the formation of viroplasms (cytoplasmic viral factories found in rotavirus-infected cells) or viral replication and surprisingly, sg assembly was not induced. one possibility for explain this observation may be due to pabp, a component of sg (table 1) , is able to translocate from the cytoplasm to the nucleus in rotavirus infected cells in a nsp3-dependent manner (montero et al., 2008) . instead, junin virus (junv), that is responsible for argentine hemorrhagic fever, is able to impair the phosphorylation of eif2␣. linero et al. showed that in junv-infected vero cells exposed to arsenite-mediated oxidative stress, eif2␣ phosphorylation was impaired but this did not lead to the induction of sg assembly (linero et al., 2011) . furthermore, the junv nucleoprotein (n) and/or the glycoprotein precursor (gpc) was responsible for this virus-induced blockade to sg assembly. rather, when junv-infected cells were treated with hippuristanol, an eif4a-helicase activity inhibitor that induces sgs in an eif2␣-independent manner (mazroui et al., 2006) , sg assembly was observed in 100% of cells indicating that junv affects an unidentified event downstream of eif2␣ phosphorylation or the integrity of viral mrnas on polysomes (linero et al., 2011) . another virus that efficiently shuts off host protein synthesis is influenza a virus (iav) (kash et al., 2006) . iav is an animal pathogen that causes severe respiratory disease and pandemics in humans around the world. viral transcription involves a cap-snatching mechanism during which a nucleotide sequence between 10 and 20 nt, including the 5 cap structure, is cleaved from the 5 end of cellular mrnas. this sequence is used to prime transcription on the viral genome and is ultimately used during translation initiation of viral mrnas (lopez-lastra et al., 2010) . additionally, iav encodes capbinding proteins that are able to preferentially recognize capped viral mrnas. the influenza non-structural protein 1 (ns1) binds eif4gi and pabp-1, thus stimulating the assembly of the translation initiation complex on capped iav mrnas (lopez-lastra et al., 2010) . iav actively suppresses sg assembly during viral infection, thereby allowing translation of viral mrnas. complete inhibition of sg assembly is dependent on the function of ns1 and its ability to inhibit pkr, the double-stranded rna-activated protein kinase (khaperskyy et al., 2011) . recently, retroviruses such as the human immunodeficiency virus type-1 (hiv-1) and human t-cell lymphotropic virus type-1 (htlv-1) were shown to impose a blockade to sg assembly in infected cells. recent work from the authors' laboratory showed that hiv-1 preferably assembles ribonucleoprotein complexes to which staufen1, the viral genomic rna and the structural protein gag are recruited, called staufen1 hiv-1-dependent rnps (shrnps). these were compositionally different than sgs since they did not contain many of the classical sg marker proteins g3bp-1, eif3, tia-1, tiar, hur, pabp-1, but contained staufen1. the assembly shrnps during the late stages of viral replication is believed to impose a blockade to the assembly of sgs but to favor the encapsidation of hiv-1 genomic rna into assembling virus (abrahamyan et al., 2010; white and lloyd, 2012) . follow-up work, reported at the last international nucleocapsid (nc) meeting in barcelona, spain in september 2011, now demonstrates that the viral gag protein controls the kinetics of sg assembly and interferes with the cellular stress response pathway (valiente-echeverría et al., unpublished) . the oncoretrovirus, htlv-1 elicits a blockade to sg assembly in a different manner and this was found to be mediated by the viral regulatory protein, tax. legros et al. observed that tax relocated from the nucleus to the cytoplasm in response to environmental stress. while tax is present in the cytoplasm, it interacts with histone deacetylase 6 (hdac6), a critical component of sgs (kwon et al., 2007) , and thereby impairs sg assembly (legros et al., 2011) . while the details on the mechanisms by which viruses elicit favorable environments in which to replicate will require further work, the sequestration of critical factors for the induction of sgs by viral proteins appears to be an increasingly studied area of research and should yield important new information on how viruses gain control over host cell biology. while all of the examples described above belong to rna viruses, herpes simplex virus (hsv) and cytomegalovirus (hcmv) are the only members of the dna virus family that have been shown to regulate sg assembly. hsv-1 causes a shut-off of host cell protein synthesis by the virion host shutoff (vhs) protein and subsequently induces degradation of cellular rnas (kwong and frenkel, 1987) . several adenosine-uracil (au)-rich binding proteins that promote mrna stability, such as tia-1/tiar, and ttp (bevilacqua et al., 2003) , were upregulated in hsv-1 infected cells (esclatine et al., 2004) . ttp and tia-1/tiar were activated during the infection and accumulated in the cytoplasm, but only ttp was able to interact with vhs. as a consequence, sgs were not observed after infection (esclatine et al., 2004) . more recently, finnen et al. have shown that hsv-2 infection blocks sg accumulation in cells exposed to arsenite-mediated oxidative stress, but not in cells exposed to pateamine a, a drug that induces sg assembly in an eif2␣-independent manner (finnen et al., 2012) . these results were similar to those found in junv infected cells described above (linero et al., 2011) . on the other hand, hcmv infection induces an unfolded protein response (upr), activates perk, but eif2␣ phosphorylation levels were limited and viral rna translation was maintained (isler et al., 2005b) . likewise, the same group showed that sg assembly was suppressed in hcmv infected cells treated with the er stressor, thapsigargin (isler et al., 2005a) . as discussed in the previous section, viruses have chosen different mechanisms to inhibit the sg assembly to ensure efficient and unmitigated replication. some studies have demonstrated that the sg assembly is not always correlated with a shut-off of host protein synthesis (kimball et al., 2003; loschi et al., 2009) . moreover, other authors have showed that sgs could sequester apoptotic molecules favoring cell survival upon exposure to certain types of stress such as heat shock (kim et al., 2005; tsai and wei, 2010) . thus, a virus-mediated induction of sg assembly also represents a strategy employed by some viruses to ensure replication. respiratory syncytial virus (rsv), which is responsible for lower respiratory tract illnesses in both infants and the elderly, induces sgs during the course of infection (lindquist et al., 2010) . lindquist et al. showed the correlation between higher viral protein levels and the presence of sgs in infected cells. in addition, g3bp −/− cells, that are unable to generate sgs because of a disrupted g3bp gene locus, exhibited diminished rsv replication (lindquist et al., 2010) . however, a later study by the same group concluded that the stress response may not play an important role in viral replication. they did not see a difference in viral replication in cells that were not able to elicit a stress response because pkr was depleted by sirna (lindquist et al., 2011) . this later study also noted that rsv infection does cause eif2␣ phosphorylation and pkr is needed to induce sgs during viral infection. these results indicate that the assembly of sg neither aids nor interferes with the replication of this virus. the role of the stress response involving sgs in the reoviridiae family of viruses has been shown to be implicated in viral replication. mammalian orthoreovirus (mrv) infection in humans is usually asymptomatic or associated with symptoms of a common cold. during the early stages of infection, mrv induces sg assembly and the expression of atf4, a transcription factor, through eif2␣ phosphorylation (smith et al., 2006) . the assembly of sgs creates a competitive advantage for the viral mrna to be translated because cellular mrnas are sequestered in sg. when atf4 is expressed in mrv infected cells, viral production increases by up to 100-fold (smith et al., 2006) . a later study implicated a role for sg assembly in viral replication since sg formation occurs after viral uncoating but before viral mrna transcription (qin et al., 2009) . qin et al. (2011) found that viral mrnas escape translational inhibition when sgs are disrupted and viral translation occurs in the presence of high levels of phosphorylated eif2␣ in a manner that is independent of pkr inhibition. this study also mentions that mrv-infected cos7 cells are able to block the assembly of sgs induced by arsenitemediated oxidative stress later in infection (qin et al., 2011) . the implication of these findings is that the stress response and the resulting assembly of sgs must be involved in the early stages of the viral replication cycle but is ultimately detrimental to the virus if it is not able to disassemble sg during later stages of infection. semliki forest virus (sfv), which causes lethal encephalitis in rodents, seems to modulate the cellular stress response in a similar fashion than mrv. upon infection, sfv is able to induce the phosphorylation of eif2␣ and promote sg assembly in mouse embryo fibroblasts (mef) (mcinerney et al., 2005) . despite a shut-off of host protein synthesis during these events, sfv is still able to translate its mrna due to a translational enhancer element present in the viral genome. this study also indicated that areas around viral rna in the cytoplasm were devoid of sgs. this observation likely indicates that viral proteins or viral rna could locally disassemble sg to favor viral translation and this was shown to correlate with increased vrna levels (mcinerney et al., 2005) . the theme of utilizing the stress response to shut-off of host protein synthesis appears once again in coronaviridae. the mouse hepatitis coronavirus (mhv), which is closely related to the sars coronavirus, has been shown to subvert the host translation machinery through eif2␣ phosphorylation (raaben et al., 2007) . eif2␣ phosphorylation also leads to the assembly of sg and pb. a genome wide microarray analysis of regulated mrnas in mhvinfected lr7 cells revealed the decrease in the expression of many cellular mrnas, which may be due to an increase in pbs activity and function (raaben et al., 2007) . likewise, viral rnas transcripts make up 40% of total rna in the cell, so the virus may be overloading the host cell cytoplasm to ensure that its transcripts will be translated (raaben et al., 2007) . however, the authors come to the conclusion that the inhibition of cellular translation is not beneficial to the virus since in systems lacking the ability to inhibit cellular translation, viral production did not change and thus, the assembly of sg in mhv-infected cells does not appear to dramatically favor viral replication (raaben et al., 2007) . finally, rubella virus (rubv) infection generates aggregates of g3bp-1 in the cytoplasm (matthews and frey, 2012) . these aggregates differ from typical sg because they do not contain proteins such as pabp and tia-1 (table 1) . rubv is a positive strand rna where viral replication is mediated for intermediary double stranded rna (dsrna). matthews et al. found that g3bp-1 does not overlap with dsrna, but rather colocalizes with viral ssrna in perinuclear clusters (matthews and frey, 2012) , suggesting that these may represent sites of encapsidation (beatch and hobman, 2000) . despite an intensifying research focus to understand the relationships between the cytoplasmic rnps called sg and virus replication (refer to table 2), many questions remain to be answered in this growing field of virology. the roles for many sg components (table 1) that have been found to participate in viral replication either by inclusion or exclusion still remain incompletely defined in host cell biology. as well, the literature has only touched the surface as to how viruses hijack and commandeer sg components. in several cases in which sg assembly is shown to be inhibited, it remains unclear if viruses block the assembly or induce the disassembly of sg. there is also a need to determine at what level viruses are hijacking or co-opting the host cell stress responses that exhibit sg. there is also a need to understand how sg may lead to deleterious effects if they remain present during viral infection. indeed, further characterization of a virus' ability to overcome the inhibition of sg assembly or induce their assembly to prevent translation of host mrnas may be beneficial in developing new anti-viral drugs that could be useful against multiple viruses. anti-cancer drugs such as etoposide, bortezomib and doxorubicin, do induce sg assembly, however their roles as anti-virals are not known (arimoto et al., 2008; fournier et al., 2010; morita et al., 2012) . the many mechanisms by which viruses inhibit or induce sg may pose a problem to developing a broad anti-viral drug targeting sg. viruses such as pv, which inhibit sg formation through cleavage, would likely be unaffected by drugs that activate the stress response upstream of these cleaved factors. another caveat to the potential use of these drugs is that sg formation may help the replication of certain viruses which induce sg to create a better environment for viral replication. the knowledge gained on the biology of sg and how it is influenced by viral infections will play a role in further characterizing innate responses to infection and how this system can be taken advantage of to curb viral infections. novel staufen1 ribonucleoproteins prevent formation of stress granules but favour encapsidation of hiv-1 genomic rna stressful initiations rna granules stress granules: the tao of rna triage stress granules human cytomegalovirus ul69 protein facilitates translation by associating with the mrna cap-binding complex and excluding 4ebp1 formation of stress granules inhibits apoptosis by suppressing stress-responsive mapk pathways hepatitis c virus hijacks p-body and stress granule components around lipid droplets mammalian smaug is a translational repressor that forms cytoplasmic foci similar to stress granules a mouse cytoplasmic exoribonuclease (mxrn1p) with preference for g4 tetraplex substrates rubella virus capsid associates with host cell protein p32 and localizes to mitochondria p bodies, stress granules, and viral life cycles post-transcriptional regulation of gene expression by degradation of messenger rnas the leader protein of cardioviruses inhibits stress granule assembly west nile virus infections suppress early viral rna synthesis and avoid inducing the cell stress granule response eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine a west nile: worldwide current situation in animals and humans poliovirus-mediated disruption of cytoplasmic processing bodies codependent functions of rsk2 and the apoptosis-promoting factor tia-1 in stress granule assembly and cell survival interaction of tia-1/tiar with west nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly herpes simplex virus 1 induces cytoplasmic accumulation of tia-1/tiar and both synthesis and cytoplasmic accumulation of tristetraprolin, two cellular proteins that bind and destabilize au-rich rnas p bodies: at the crossroads of post-transcriptional pathways herpes simplex virus type 2 infection impacts stress granule accumulation the chemotherapeutic agent bortezomib induces the formation of stress granules smg7 is a 14-3-3-like adaptor in the nonsense-mediated mrna decay pathway stress granule assembly is mediated by prion-like aggregation of tia-1 a novel functional human eukaryotic translation initiation factor 4g heme-regulated eif2alpha kinase (hri) is required for translational regulation and survival of erythroid precursors in iron deficiency perk is essential for translational regulation and cell survival during the unfolded protein response rpp20 interacts with smn and is re-distributed into smn granules in response to stress the human lsm1-7 proteins colocalize with the mrna-degrading enzymes dcp1/2 and xrnl in distinct cytoplasmic foci production of infectious human cytomegalovirus virions is inhibited by drugs that disrupt calcium homeostasis in the endoplasmic reticulum human cytomegalovirus infection activates and regulates the unfolded protein response gcn2 phosphorylation of eif2alpha activates nf-kappab in response to uv irradiation hijacking of the hostcell response and translational control during influenza virus infection mammalian stress granules and processing bodies evidence that ternary complex (eif2-gtp-trna(i)(met))-deficient preinitiation complexes are core constituents of mammalian stress granules rna-binding proteins tia-1 and tiar link the phosphorylation of eif-2 alpha to the assembly of mammalian stress granules stress granules and processing bodies are dynamically linked sites of mrnp remodeling influenza a virus inhibits cytoplasmic stress granule formation modulation of stress granules and p bodies during dicistrovirus infection sequestration of traf2 into stress granules interrupts tumor necrosis factor signaling under stress conditions mammalian stress granules represent sites of accumulation of stalled translation initiation complexes efficient cleavage of ribosome-associated poly(a)-binding protein by enterovirus 3c protease the deacetylase hdac6 is a novel critical component of stress granules involved in the stress response herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mrnas the htlv-1 tax protein inhibits formation of stress granules by interacting with histone deacetylase 6 cell proteins tia-1 and tiar interact with the 3' stem-loop of the west nile virus complementary minus-strand rna and facilitate virus replication respiratory syncytial virus induces host rna stress granules to facilitate viral replication activation of protein kinase r is required for induction of stress granules by respiratory syncytial virus but dispensable for viral replication junin virus infection impairs stress-granule formation in vero cells treated with arsenite via inhibition of eif2alpha phosphorylation microrna-dependent localization of targeted mrnas to mammalian p-bodies translation initiation of viral mrnas dynein and kinesin regulate stress-granule and p-body dynamics analysis of subcellular g3bp redistribution during rubella virus infection inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation importance of eif2alpha phosphorylation and stress granule assembly in alphavirus translation regulation rotavirus infection induces the phosphorylation of eif2alpha but prevents the formation of stress granules stress granules in the viral replication cycle the stress granule protein vgl1 and poly(a)-binding protein pab1 are required for doxorubicin resistance in the fission yeast schizosaccharomyces pombe formation of cytoplasmic heat shock granules in tomato cell cultures and leaves the virion host shutoff endonuclease (ul41) of herpes simplex virus interacts with the cellular cap-binding complex eif4f translational efficiency of poliovirus mrna: mapping inhibitory cis-acting elements within the 5 noncoding region stable formation of compositionally unique stress granules in virus-infected cells rotavirus rna-binding protein nsp3 interacts with eif4gi and evicts the poly(a) binding protein from eif4f mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eif2{alpha} phosphorylation and pkr mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies a crucial role for gw182 and the dcp1:dcp2 decapping complex in mirna-mediated gene silencing reovirus induces and benefits from an integrated cellular stress response mk2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and are-mrna decay regulation of starvation-and virus-induced autophagy by the eif2alpha kinase signaling pathway rna granules: the good, the bad and the ugly mammalian staufen 1 is recruited to stress granules and impairs their assembly the rasgap-associated endoribonuclease g3bp assembles stress granules rhoa/rock1 signaling regulates stress granule formation and apoptosis smg7 acts as a molecular link between mrna surveillance and mrna decay human dcp2: a catalytically active mrna decapping enzyme located in specific cytoplasmic structures viral subversion of the host protein synthesis machinery inhibition of cytoplasmic mrna stress granule formation by a viral proteinase poliovirus unlinks tia1 aggregation and mrna stress granule formation regulation of stress granules in virus systems naturally occurring dicistronic cricket paralysis virus rna is regulated by two internal ribosome entry sites hepatitis c virus co-opts ras-gtpase-activating protein-binding protein 1 for its genome replication we thank marie-joelle miron for critical reading of the manuscript. this work is supported by grants from the cihr (mop-38111) to a.j.m.; f.v.e. is supported by a postdoctoral fellowship from becas chile-conicyt. key: cord-304137-vxqkztio authors: bueno, carlos a.; barquero, andrea a.; di cónsoli, hernán; maier, marta s.; alché, laura e. title: a natural tetranortriterpenoid with immunomodulating properties as a potential anti-hsv agent date: 2009-01-20 journal: virus res doi: 10.1016/j.virusres.2008.12.013 sha: doc_id: 304137 cord_uid: vxqkztio meliacine (ma), an antiviral principle present in partially purified leaf extracts of melia azedarach l., prevents the development of herpetic stromal keratitis (hsk) in mice by diminishing the viral load in the eye and the severity of lesions caused by a virus-induced immunopathological reaction. the tetranortriterpenoid 1-cinnamoyl-3,11-dihydroxymeliacarpin (cdm), obtained from ma purification, displays anti-herpetic activity and impedes nuclear factor κb (nf-κb) activation in hsv-1 infected conjunctival cells. to extend our understanding about cdm biological properties, we investigated its anti-hsv-1 activity as well as the effect on nf-κb activation and cytokine secretion induced by viral (hsv-1) and no-viral (lps) stimuli, in corneal cells and macrophages. cdm exerted a potent anti-hsv-1 effect on corneal cells and inhibited nf-κb translocation to the nucleus, leading to a decrease in il-6 production. besides, cdm seemed to modulate il-6 and tnf-α responses in macrophages, whether they were infected with hsv-1 or stimulated with lps. however, cdm did not affect nf-κb activation in these cells, suggesting that an alternative nf-κb cell signaling pathway would be involved in the modulation of cytokine production. we conclude that, in addition to its antiviral effect, cdm would be acting as an immunomodulating compound which would be responsible for the improvement of murine hsk already reported. a great variety of ethnomedicinal plants are being studied as a source of natural products useful in the development of novel drugs. it has been established that many of them inhibit several steps of the viral replication cycle of many dna and/or rna viruses (chattopadhyay and naik, 2007) . we have reported that meliacine (ma), an antiviral principle present in partially purified leaf extracts of melia azedarach l., exerts an antiviral action on the development of herpetic stromal keratitis (hsk) in mice by causing a significant decrease in the viral load in the eye of herpes simplex virus type 1 (hsv-1) infected animals, as well as in the incidence and severity of lesions due to a virus-induced immunopathological reaction (pifarré et al., 2002) . bioassay guided purification of ma led to the isolation of the limonoid 1-cinnamoyl-3,11-dihydroxymeliacarpin (cdm) that reduces both vesicular stomatitis virus (vsv) and hsv-1 multiplication in vero cells (alché et al., 2003) . we have found that a late step in vsv and hsv-1 multiplication cycles is hindered by cdm since glycoproteins (g) b, gc and gd of hsv-1, as well as gg of vsv, are confined to the golgi apparatus when cdm is added after infection (barquero et al., 2004 (barquero et al., , 2006 . hsv-1-induced ocular disease occurs as a result of a primary infection in the corneal epithelium and then, cells like macrophages intervene in clearing the virus from the infected eye and in the development of the immunologically driven stromal keratitis (bauer et al., 2002; biswas and rouse, 2005) . besides, conjunctival cells are also involved in amplifying the inflammatory processes in the eye (kase et al., 2004) . it has been shown that activation of nuclear factor b (nf-b) plays a pivotal role in triggering an immune inflammatory response to a range of stimuli, including viral infections, such as hiv-1, human t cell leukemia virus type 1, hepatitis b and influenza viruses. it has been reported that hsv-1 induces a strong nuclear translocation of nf-b in human cell lines that 0168-1702/$ -see front matter © 2008 elsevier b.v. all rights reserved. doi:10.1016/j.virusres.2008.12.013 could have several functions: to promote viral replication, prevent virus-induced apoptosis, and mediate the immune response to the invading pathogen (gregory et al., 2004; hiscott et al., 2001) . a great number of plant-derived substances, such as sesquiterpenes, di-and triterpenes, that prevent nf-b activation, have been described (nam, 2006) . we have found that cdm is able to block hsv-1 induced activation of nf-b by inhibiting its translocation to the nucleus of infected human conjunctival cells (nhc), and postulated that cdm would be able to abolish murine hsk by controlling viral spread and the associated immunopathology as well (barquero et al., 2006) . the aim of the present study was to determine whether cdm displays an antiviral activity in infected corneal cells, the target of hsv-1 multiplication in vivo, as well as its effect on the translocation of nf-b to the nucleus. since nf-b plays a particularly important role as far as expression of cytokines is concerned (santoro et al., 2003) , we also evaluated the modulating effect of cdm on the production of different cytokines in corneal cells and macrophages. human corneal-limbal epithelial (hcle) cells were kindly provided by dr ilene k. gipson and dr. pablo argüeso (the schepens eye research institute, harvard medical school, boston, usa) and grown in gibco keratinocyte serum free medium, supplemented with 25 g/ml bovine pituitary extract (bpe), 0.2 ng/ml epidermal growth factor (egf), and 0.4 mm cacl 2 , and maintained in low calcium dmem/f12. murine macrophage cell line j774a.1 was kindly provided by dr. osvaldo zabal (inta-castelar, buenos aires) and grown in rpmi 1640 medium supplemented with 10% inactivated fetal bovine serum (fbs) (rpmi 10%) and maintained in rpmi supplemented with 2% inactivated fbs (rpmi 2%). murine l929 cells and vero cells were grown in eagle's minimal essential medium supplemented with 10% inactivated fbs (mem 10%), and maintained in mem supplemented with 1.5% inactivated fbs (mem 1.5%). the hsv-1 kos strain was propagated at low multiplicity and used for in vitro experiments. lps from e. coli serotype 055: b5 was obtained from sigma. the rabbit polyclonal anti-p65 and anti-ib␣ antibodies, and the mouse monoclonal antibody anti-gd of hsv-1 were obtained from santa cruz biotechnology, usa. the monoclonal anti-calnexin antibody was obtained from chemicon. the anti-actin (merck) antibody was kindly provided by dr. viviana castilla, laboratory of virology, school of sciences, university of buenos aires. secondary goat anti-rabbit fluorolink tm cy tm 2 and anti-mouse fluorolink tm cy tm 3 antibodies were purchased from ge healthcare bio-sciences, argentina. peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies were obtained from icn immunobiological. cdm was purified from leaves of m. azedarach l., as described by alché et al. (2003) , solubilized in mem 1.5% to a final concentration of 1 mg/ml (1.5 mm), and stored at −20 • c. cell viability in the presence of the compound was determined using the cleavage of the tetrazolium salt mtt (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (sigma) by the mitochondrial enzyme succinate dehydrogenase to give a blue product (formazan) (denizot and lang, 1986) . hcle cells were seeded at a concentration of 10 4 cells/well in 96-well plates and grown at 37 • c for 24 h. the culture medium was replaced by dmem/f12 medium containing cdm at various concentrations by triplicate, and cells were further grown for 24 h. after that, we added 0.01 ml mtt (5 mg/ml in distilled water) to cells in culture medium. after mtt cleavage (2 h at 37 • c), formazan product was solubilized by the addition of 0.2 ml of ethanol. the absorbance of each well was measured on an eurogenetics mpr-a 4i microplate reader using a test wavelength of 570 nm and a reference wavelength of 630 nm. results were expressed as a percentage of absorbance of treated cell cultures with respect to untreated ones. hcle and j774a.1 cells grown in 96-well tissue culture plates were infected with hsv-1 at a multiplicity of infection (m.o.i.) of 0.067 pfu/cell. after 1 h adsorption at 37 • c, the inoculum was removed and medium containing different concentrations of cdm was added, by triplicate. the plates were incubated at 37 • c in 4% co 2 atmosphere until 100% cell death was observed microscopically in untreated infected control cells, approximately at 24 h post-infection (p.i.). after cell disruption by three cycles of freezing and thawing, supernatants were harvested and pooled. virus yields were titrated by plaque assay in vero cells and the effective concentration (ec 50 ) values were calculated as the concentration of cdm required to reduce the yield of infectious virus by 50% relative to the untreated virus control. hcle cells grown on coverslips were treated with 40 m cdm for 2 h at 37 • c and stained with acridine orange (1 g/ml) for 15 min at 37 • c. then, cells were washed twice with cold pbs, mounted on pbs and visualized on an olympus bx51 with epifluorescence optics. subconfluent cells grown on glass coverslips in 24-well plates were fixed with methanol for 10 min at −20 • c. after three washes with pbs, the coverslips were inverted on a drop of diluted primary antibody for 30 min at 37 • c, and then returned to culture dishes and subjected to three additional washes with pbs. afterwards, cells were incubated with diluted secondary antibody for 30 min at 37 • c. finally, coverslips were rinsed, mounted and photographed with an olympus fb300 confocal microscope or an olympus bx51 with epifluorescence optics. whole extracts from cells grown in 24-well plates for 24 h were loaded on 10% sodium dodecyl sulphate-polyacrilamide gel electrophoresis (sds-page) and transferred onto polyvinylidene fluoride (pvdf) membranes for 60 min at 75 ma. membranes were blocked in pbs containing 5% unfitted milk overnight and then incubated with diluted primary antibodies for 2 h at 37 • c. after washing, membranes were incubated with diluted peroxidaseconjugated antibodies for 1.5 h at 37 • c. the immunoreactive bands were visualized using an enhanced chemiluminesence system (ecl, perkinelmer). calnexin and actin were used as loading controls. cells were frozen and thawed, and then, supernatants were harvested, centrifuged at 1000 rpm for 10 min, and cytokines were quantified by elisa, or in a biological assay by triplicate. human tnf-␣ and il-6, and mouse il-6 were quantified by commercial elisa sets (bd opteiatm, becton dickinson, usa) according to manufacturer instructions. measurement of mouse tnf-␣ bioactivity was performed with the l929 cell-based bioassay (decker et al., 1987) , with minor modifications. l929 cells were grown in 96-well culture plates (2 × 10 4 cells/well) for 24 h at 37 • c. supernatants were removed and substituted with the samples to be assayed for tnf-␣ content in successive twofold dilutions and incubated at 38.5 • c with 5 g/ml of actinomycin d (acd) (sidus, argentina), for 22 h. cells were fixed in 10% formaldehyde and stained with crystal violet 0.05%. to assess tnf-␣ activity, light absorbance at 580 nm of the eluted crystal violet from the samples was measured and compared to a mouse recombinant tnf-␣ standard dilution series (sigma). the bioassay was specific for tnf-␣ since the activity was neutralized by an antibody against tnf-␣. student's t-test was used for statistical analysis of all data. it is well known that hsv-1 establishes infection usually in the epithelial layer of the cornea (deshpande et al., 2004) . considering that cdm suppressed hsv-1 replication in vero and human conjunctival cells, we decided to investigate the anti-hsv-1 effect of cdm in hcle cells where hsv-1 multiplied reaching a maximum viral titer of 5.3 × 10 6 at 24 h p.i. (alché et al., 2003; barquero et al., 2006) . when hcle cells infected with hsv-1 were treated with different concentrations of cdm, a dose-dependent inhibition of viral yields was observed, and an ec 50 value of 0.78 m was calculated. besides, cdm proved to have no cytotoxic effect at all the concentrations tested (fig. 1a) . we have previously reported that cdm displays its antiviral action by affecting the trafficking of gb, gc and gd of hsv-1 in vero and nhc cells when supplied after infection (barquero et al., 2006) . in order to study the effect of cdm on the transport of viral glycoproteins in corneal cells, hcle cells were infected with hsv-1 (m.o.i. = 2) and treated with cdm (40 m). when a total ifi staining was performed using an anti-gd monoclonal antibody, we found that gd of hsv-1 exhibited a perinuclear localization associated with the golgi apparatus (fig. 1b) . it has been already shown that the inhibition of viral glycoprotein transport may be related to the perturbation of the acidic ph of intracellular organelles (sidhu et al., 1999) . although the mechanism by which cdm affects the exocytic pathway is still unraveled, we have shown that cdm provokes the basification of the ph of the endosomal vesicles in vero cells (barquero et al., 2004) . now, we demonstrated that cdm also modified the endosomal ph of hcle cells. the vital fluorescence microscopic study revealed that untreated cells exhibited a bright orange punctate fluorescence concentrated in low-ph vesicles. nevertheless, the ph of acidic intracellular vesicles from cdm-treated cells was markedly affected since only a faint granular fluorescence was observed after 2 h of treatment (fig. 1c) . in summary, cdm induced hcle cytoplasmic alkalinization and exerted a potent anti-hsv-1 effect by confining hsv-1 glycoproteins to the golgi apparatus. we have previously shown that cdm blocks hsv-1 induced activation of nf-b by inhibiting its translocation to the nucleus of conjunctival cells (barquero et al., 2006) . hence, to examine the effect of cdm on nf-b intracellular localization, we infected hcle cells with hsv-1 (m.o.i. = 1) and, at 24 h p.i., cells were fixed and processed for an ifi staining using a rabbit polyclonal anti-p65 antibody. by visual inspection of the images, we observed that p65 remained in the cytoplasm of uninfected cells, whereas hsv-1 induced nf-b translocation to the nucleus of hcle cells. these qualitative observations were corroborated by calculating the percentage of translocation as the number of cells with fluorescence in the nucleus relative to the total cell number, in 50 cells per coverslip. nf-b nuclear translocation occurred in the majority of infected cells (92%), whereas viral-induced nf-b activation was efficiently impaired by cdm, since only 20% of hcle treatedinfected cells retained p65 in their nuclei ( fig. 2a) . together with nf-b, we also detected hsv-1 gd as a marker of viral infection, by a double ifi staining. we found that nuclear p65 fluorescence in the nuclei of infected cells was associated with gd expression. when hcle infected cells were treated with cdm, both p65 and gd were detected coincidently in a few and scattered foci of infection ( fig. 2a) . thus, the inhibition of nf-b translocation in treated-infected cells could be ascribed to the restraint of viral propagation caused by cdm rather than to an intrinsic effect on nf-b. since the translocation of nf-b to the nucleus is preceded by the proteolytic degradation of its inhibitor in the cytoplasm, ib␣, we determined whether the inhibition of nf-b activation by cdm was concomitant to ib␣ degradation. hcle cells infected with hsv-1 (m.o.i. = 1) were treated or not with 40 m of cdm, and, at 24 h p.i., cells were processed through western blot analysis by using a rabbit polyclonal anti ib␣ antibody. we found that ib␣ was almost completely degraded after infection with hsv-1; as expected, the addition of cdm hampered hsv-1 induced persistent ib␣ degradation. besides, we observed that cdm didn't induce ib␣ degradation in uninfected cells (fig. 2b) . it has been already reported that hsv-1 is able to activate nf-b in human telomerase-immortalized corneal epithelial cells (hucl) concomitant with a transcriptional expression of il-6 and tnf-␣, among other cytokines (li et al., 2005) . however, hcle cells failed to produce tnf-␣ after infection with hsv-1 (michelini et al., 2007) . to assess the biological relevance of the inhibition of nf-b translocation provoked by cdm on cytokine production, we measured its effect on il-6 secretion. supernatants harvested from hcle cells infected with hsv-1 (m.o.i. = 1) treated or not with cdm (40 m), were used to quantify il-6 by elisa, in triplicate. we found that the production of il-6 was ten fold higher than that of uninfected cells (p < 0.005). this increase was significantly reduced by half when cdm was added to hsv-1 infected cells (p < 0.005). in uninfected cells, no significant differences between il-6 release from non-treated and cdm-treated cells were detected (fig. 2c) . taken together, these results suggest that nf-b translocation prevented as a consequence of the anti-hsv-1 activity of cdm, would lead to a decrease in il-6 production. although corneal epithelial cells constitute the first line of defence after ocular hsv-1 infection, macrophages also play a crucial role as one of the dominant cell infiltrates in the infected cornea (mott et al., 2007) . thus, we decided to evaluate the effect of cdm in j774a.1 cells infected with hsv-1. consequently, we first investigated the antiviral action of cdm, finding that it inhibited hsv-1 multiplication with an ec 50 value of 12.05 m, without cytotoxic effect (data not shown). as it has been described in other cell lines, a similar pattern of nf-b translocation and ib␣ degradation was observed in j774a.1 cells after hsv-1 infection (gregory et al., 2004) . by means of an ifi staining, we found that p65 fluorescence remained in the cytoplasm of uninfected cells, whereas hsv-1 induces translocation to the nucleus in 82% of infected j774a.1 cells, which correlated with ib␣ degradation observed through an immunoblot assay. when cdm was added, the percentage of cells exhibiting nf-b translocation to the nucleus dropped to 17% and, as expected, ib␣ degradation was inhibited in cdm-treated infected cells ( fig. 3a and b) . it is well established that hsv-1 infection triggers proinflammatory cytokine production in macrophages (biswas and rouse, 2005; melchjorsen et al., 2006) . thus, we investigated the effect of cdm on il-6 and tnf-␣ secretion by elisa and a biological assay, respectively. in the case of uninfected cells, levels of il-6 and tnf-␣ accumulated in the culture medium for 24 h varied between control and cdm-treated j774a.1 cells. while the concentration of il-6 was significantly lowered in the supernatants from cdm-treated cells with respect to untreated j774a.1 cells (p < 0.01), the tnf-␣ yield belonging to cdm-treated cells was much higher than that accumulated in control cells (p < 0.05) (fig. 3c) . as shown in fig. 3c , hsv-1 infection induced macrophages to secrete increased amounts of il-6 and tnf-␣ in comparison with uninfected cells (p < 0.01 and p < 0.05, respectively). however, cdmtreated cells failed to produce il-6 in response to hsv-1 infection (p < 0.005), whereas a strong enhancement in tnf-␣ secretion was observed (p < 0.05). hence, cdm seemed to modulate the response of both cytokines in j774a.1 cells, whether infected or not. since it has been previously shown that the addition of increasing concentrations of ma to lps-stimulated mouse peritoneal macrophages synergized the amount of tnf-␣ released in a dosedependent manner, we used lps as a stimulus instead of hsv-1 to evaluate solely the immunomodulating effect of cdm (petrera and coto, 2003) . j774a.1 cells were stimulated with lps (10 g/ml) in the presence or absence of cdm (40 m) for 30 min at 37 • c. when cells were stained for nf-b p65 subunit detection, we observed that cdm did not inhibit lps induced-nf-b translocation (fig. 4a) . besides, cdm did not affect the kinetic of ib␣ degradation induced by lps. cdm alone neither activated nf-b nor led to ib␣ degradation ( fig. 4a and b) . taking into account that cdm did not affect lps-induced nf-b translocation, it was important to determine if cdm was able to affect il-6 and tnf-␣ yields. when j774a.1 cells were stimulated with lps during 8 h, the release of il-6 and tnf-␣ was enhanced significantly (p < 0.001 and p < 0.01, respectively) (fig. 4c) . nevertheless, cdm considerably reduced the release of il-6 (p < 0.001), and strongly enhanced tnf-␣ production (p < 0.01), after 8 h of treatment of lps-stimulated j774a.1 cells (fig. 4c) . in these cells, cdm inhibited nf-b activation and ib␣ degradation depending on the fact that macrophages were induced with lps or infected with hsv-1, while it modulated il-6 and tnf-␣ production regardless the stimuli used. since a long time, medicinal plants have been used to treat viral infections. the chemical diversity, structural complexity, lack of substantial toxic effects, and broad spectrum of antiviral activity of natural products, make them ideal candidates for new therapeutics (mukhtar et al., 2008) . in fact, terpenoids isolated from medicinal plants have attracted attention because many of them exhibit specific antiviral effect against hsv-1 and 2, and the coronavirus sars-cov, in vitro (wen et al., 2007) . triterpenoids and limonoids isolated from meliaceae plants proved to inhibit hsv-1, hiv-1 and rsv multiplication (chen et al., 2007; joycharat et al., 2008; manners, 2007) . the murine model of hsv-induced ocular disease provides valuable information related to the viral and immune stages of the hsk. we have previously reported that topical administration of ma in the corneas of hsv-1 infected mice exerts its therapeutic effect by reducing viral load as well as by abolishing the ocular inflammatory reaction and neovascularization, displaying, in addition to its antiviral activity, an eventual immunomodulatory effect (pifarré et al., 2002) . in another report, we described the chemical nature of the antiviral principle (cdm) and its mechanism of action in vero and nhc cells infected with hsv-1 ( barquero et al., 2004 barquero et al., , 2006 . in the present paper, we have shown that cdm displayed a potent antiviral effect in hcle cells (fig. 1a) , 25-fold higher in comparison to that obtained in vero cells (alché et al., 2003) . this is important because hsv-1 replication in the eye occurs almost exclusively in corneal epithelial cells (biswas and rouse, 2005) . the direct anti-hsv-1 activity of cdm achieved when the compound is supplied after infection has already been demonstrated: cdm affects gb, gc and gd glycoprotein exocytic pathway from nhc and vero cells, confining them to the golgi complex (barquero et al., 2006) . now, we demonstrate that cdm also affects the trafficking of, at least, gd glycoprotein of hsv-1 by exerting an analogous antiviral effect in hcle cells (fig. 1b) . furthermore, the hindrance of hsv-1 multiplication by cdm is responsible for the inhibition of nf-b translocation in corneal cells ( fig. 2a) . taken together, these results would explain the reduction of virus yield in the eyes of ma-treated infected mice (pifarré et al., 2002) . in recent past, a number of studies have explored immunomodulatory properties of plant extracts having antiviral properties (mukhtar et al., 2008) . a methanolic extract of capparis spinosa l. buds up-regulates the expression of proinflammatory cytokines, such as il-12, ifn-␥ and tnf-␣, besides its anti-hsv activity (arena et al., 2008) . likewise, pure compounds from plantago genus enhance the activity of lymphocyte proliferation and secretion of ifn-␥ (chiang et al., 2003) . the development of murine hsk involves production of proinflammatory cytokines, chemokines and neovascularization, and takes place between 5 and 7 days after infection, once the virus was cleared from the eye. interestingly, ma prevents ocular disease in hsv-1 infected mice when administered from 4 days p.i. onwards, suggesting the presence of a compound with immunomodulating activity (pifarré et al., 2002) . in this sense, now we have found that cdm modulates cytokine production in vitro (figs. 2b, 3c and 4c). il-6 and tnf-␣ are some of the numerous cytokines known to be upregulated following hsv-1 ocular infection (biswas and rouse, 2005) . the significant reduction in il-6 secretion detected in supernatants from cdm treated-infected epithelial cells and macrophages would be related to the inhibition of nf-b translocation by cdm, as a result of its antiviral activity (figs. 1, 2 and 3) . on the contrary, the strong increase of tnf-␣ secretion in j774a.1 cdmtreated infected cells observed in the absence of nf-b activation (fig. 3c ) would be exerted through an alternative nf-b cell signaling pathway, such as the activation of distinct mitogen-activated protein (map) kinase cascades (diao et al., 2005; zachos et al., 1999) . this possibility might be supported by the fact that cdm enhanced levels of tnf-␣ production, decreased il-6 secretion and did not impede nf-b translocation when j774a.1 cells were induced with lps rather than infected with hsv-1 (fig. 4) . it has been demonstrated that both cytokines play a different role in corneal angiogenesis: while il-6 induces the production of vascular endothelial growth factor (vegf), tnf-␣ may counter the induction effect of vegf on vascular endothelial cells, resulting in a blockade of neovascularization in injured corneas in vivo (biswas et al., 2006; fujita et al., 2007) . if this is the case, we would argue that the strong increase of tnf-␣ secretion together with il-6 reduction due to cdm could be responsible for the inhibition of neovascularization registered when mice were infected in the cornea with hsv-1 and treated with ma. we hypothesize that the improvement of hsk reported with ma may be a consequence of the antiviral and immunomodulating effects observed in vitro with cdm. further studies on the antiangiogenic and immunomodulating effects of cdm will be needed to decipher its mechanism of action, since the availability of a compound gathering together both biological properties will make it a viable candidate for a new and novel anti-hsv agent. an antiviral meliacarpin from leaves of melia azedarach l antiviral and immunomodulatory effect of a lyophilized extract of capparis spinosa l. buds block of vesicular stomatitis virus endocytic and exocytic pathways by 1-cinnamoyl-3, 11-dihydroxymeliacarpin, a tetranortriterpenoid of natural origin 1-cinnamoyl-3,11-dihydroxymeliacarpin is a natural compound with antiviral and nuclear factor-b modulating properties conjunctival macrophage-mediated influence of the local and systemic immune response after corneal herpes simplex virus-1 infection early events in hsv keratitis-setting the stage for a blinding disease involvement of il-6 in the paracrine production of vegf in ocular hsv-1 infection antivirals of ethnomedicinal origin: structureactivity relantionship and scope dysoxylins a-d, tetranortriterpenoids with potent anti-rsv activity from dysoxylum gaudichaudianum immunomodulatory activities of flavonoids, monoterpenoids, triterpenoids, iridoid glycosides and phenolic compounds of plantago species cell-associated tumor necrosis factor (tnf) as a killing mechanism of activated cytotoxic macrophages rapid colorimetric assay for cell growth and survival herpetic eye disease: immunopathogenesis and therapeutic measures activation of c-jun n-terminal kinase (jnk) pathway by hsv-1 immediate early protein icp0 endogenous tnf alpha suppression of neovascularization in corneal stroma in mice efficient replication by herpes simplex virus type 1 involves activation of the ikappab kinase-ikappab-p65 pathway hostile takeovers: viral appropriation of the nf-kappab pathway flavaglines and triterpenoids from the leaves of aglaia forbesii activation of nuclear factor-kappa b in conjunctiva with epithelial scraping of the mouse cornea and human epidemia keratoconjunctivitis herpes simplex virus 1 infection induces the expression of proinflammatory cytokines, interferons and tlr7 in human corneal epithelial cells citrus limonoids: analysis, bioactivity, and biomedical prospects induction of cytokine expression by herpes simplex virus in human monocyte-derived macrophages and dendritic cells is dependent on virus replication and is counteracted by icp27 targeting nf-kappab and irf-3 the in vitro immunomodulatory activity of a synthetic brassinosteroid analogue world account for the improvement of herpetic stromal keratitis in mice macrophages are important determinants of acute ocular hsv-1 infection in immunized mice antiviral potentials of medicinal plants naturally occurring nf-b inhibitors effect of meliacine, a plant derived antiviral, on tumor necrosis factor alpha therapeutic action of meliacine, a plant-derived antiviral, on hsv-induced ocular disease in mice nf-kappab and virus infection: who controls whom role of vacuolar h + -atpase in interferon-induced inhibition of viral glycoprotein transport specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus herpes simplex virus type 1 infection stimulates p38/c-jun n-terminal mitogen-activated protein kinase pathways and activates transcription factor ap-1 the authors wish to thank isabel paz and guillermo assad ferek for their technical assistance. this work was supported by grants from the agencia nacional de promoción científica y técnica (anpcyt) (pict 38260/05) and conicet (pip 6033). dr. a.a. barquero key: cord-309428-qkjjxr6p authors: li, liwei; wei, zuzhang; zhou, yanjun; gao, fei; jiang, yifeng; yu, lingxue; zheng, hao; tong, wu; yang, shen; zheng, haihong; shan, tongling; liu, fei; xia, tianqi; tong, guangzhi title: host mir-26a suppresses replication of porcine reproductive and respiratory syndrome virus by upregulating type i interferons date: 2015-01-02 journal: virus res doi: 10.1016/j.virusres.2014.08.012 sha: doc_id: 309428 cord_uid: qkjjxr6p micrornas (mirnas) play important roles in viral infections, especially by modulating the expression of cellular factors essential to viral replication or the host innate immune response to infection. to identify host mirnas important to controlling porcine reproductive and respiratory syndrome virus (prrsv) infection, we screened 15 mirnas that were previously implicated in innate immunity or antiviral functions. over-expression of the mir-26 family strongly inhibited prrsv replication in vitro, as shown by virus titer assays, western blotting, and qrt-pcr assays. mir-26a inhibited the replication of both type 1 and type 2 prrsv strains. mutating the seed region of mir-26 restored viral titers. luciferase reporters showed that mir-26a does not target the prrsv genome directly but instead affects the expression of type i interferon and the ifn-stimulated genes mx1 and isg15 during prrsv infection. these results demonstrate the important role of mir-26a in modulating prrsv infection and also support the possibility of using host mir-26a to achieve rnai-mediated antiviral therapeutic strategies. micrornas (mirnas) are small (∼22 nucleotides) non-coding rnas that bind to complementary sequences in the untranslated regions of target mrnas and contribute to gene regulation by reducing mrna translation or destabilizing transcripts (grassmann and jeang, 2008; skalsky and cullen, 2010) . the commonly accepted mechanism of mirna regulation is that the seed region (2 ∼ 8 nucleotides at the 5 end) of an mirna is complementary to the 5 or 3 untranslated region (5 -or 3 -utr) of an mrna, leading to mrna degradation or translational inhibition (bartel, 2009; gottwein and cullen, 2008) . recent work has shown the importance of mirnas in regulating host-pathogen interactions and innate immunity (lodish et al., 2008; scaria et al., 2007; tenoever, 2013) . host mirnas can affect viral replication by binding directly to viral rna (lecellier et al., 2005) or by indirectly modulating host factors to provide a less permissive environment for virus replication (triboulet et al., 2007) . as mirnas are small molecules without antigenic properties, they are considered to have potential efficacy in antiviral therapeutic applications. for example, human mir-122 is an essential component of the biology of hepatitis c virus replication (jopling, 2010; jopling et al., 2005) and therapeutic blocking of mir-122 suppresses hepatitis c viremia in non-human primates (lanford et al., 2010) . porcine reproductive and respiratory syndrome virus (prrsv), a member of the arterivirus family, is the causative agent of porcine reproductive and respiratory syndrome [prrs; (chand et al., 2012) ]. based on their genetic and antigenic differences, prrsv strains are classified into two distinct genotypes, north american (type 2) and european (type 1), represented by the vr-2332 (benfield et al., 1992) and lelystad virus (lv) (wensvoort et al., 1991) , respectively. these two genotypes on two different continents share only approximately 60% nucleotide sequence identity (forsberg, 2005; hanada et al., 2005) . many strategies for controlling prrsv transmission have been proposed but have generally shown little success, which has stimulated the search for new ways to control prrsv transmission. prrsv can escape from innate immunity and cause persistent infections (miller et al., 2004) . in mammalian cells, viral infection is a potent trigger of the interferon (ifn) response (sadler and williams, 2008; sen, 2001) . type i interferons can initiate the activation of jak/stat signaling to induce the expression of hundreds of ifn-stimulated genes (isgs), which play an important role in antiviral activities (albina et al., 1998; katze et al., 2002; overend et al., 2007) . however, in contrast to porcine respiratory coronavirus, prrsv is a poor ifn-inducer (buddaert et al., 1998) . many mirnas regulate ifn production pedersen et al., 2007) , maintain mrna stability (li et al., 2012) , and regulate signals downstream of ifn to modulate antiviral immunity (wang et al., 2010; yoshikawa et al., 2012) . while most mirnas characterized to date decrease the production of ifns (alam and o'neill, 2011) , a few mirnas that upregulate type i ifns have been reported. recent research has revealed that mir-23 may play a positive modulatory role in ifn production during prrsv infection (zhang et al., 2014) . given the breadth of mirna-mediated regulation of mammalian immunity (grassmann and jeang, 2008) , the role of host mirnas in prrsv infection is of significant interest. here, we found that mir-26a is an anti-prrsv host factor. over-expression of mir-26a inhibited infection by both of the major prrsv genotypes in a dosedependent manner. we found that mir-26a does not target the prrsv genome directly, but rather affects the expression of type i interferon and the ifn-stimulated genes mx1 and isg15 during prrsv infection. our study reveals an example of a mirna that affects viral propagation and highlights a host factor that may be important for future control measures against prrs. marc-145 cells were grown in mem (invitrogen) with 10% fetal bovine serum (fbs, gibco-brl, gaithersburg, md, usa) and were maintained with 2% fbs at 37 • c in a humidified 5% co 2 atmosphere as described previously (yuan and wei, 2008) . baby hamster kidney (bhk-21, atcc ccl10) cells were cultured in emem (atcc, manassas, va, usa) supplemented with 10% fbs. porcine alveolar macrophages (pams) were harvested from the lungs of 6-week-old prrsv-negative piglets as described previously (wensvoort et al., 1991) and maintained at 37 • c in rpmi 1640 (gibco) supplemented with 10% fetal bovine serum (fbs). vaprrs (genbank accession no. gq330474) (yuan and wei, 2008) and vshe (genbank accession no. gq461593) (tian et al., 2011) were rescued from paprrs and pshe, respectively. vjx143 (at passage 3) was isolated from the serum of a dying piglet displaying the clinical sings of porcine high fever disease (phfd) in 2006. vjxm100 (genbank accession no. gq475526) was obtained through 100 serial passages of the highly pathogenic prrsv vjx143 strain (eu708726) in marc-145 cells (wang et al., 2013) . the infectious cdna clone pjx143 was derived from vjx143 (lv et al., 2008) . high-titer virus stocks were obtained by infecting marc-145 cells at low multiplicities of infection (mois). infected cell supernatants were harvested after an 80% cytopathic effect (cpe) appeared, then the viruses were stored at −80 • c as stocks for further use. virus titer was determined by standard tcid 50 assay using marc-145 cells. mirna mimics (table 1) , which are double-stranded 2 -omethyl-modified rna oligonucleotides with sequence complementarity to mature mirnas were synthesized by genepharma (shanghai, china). the sense sequences of the mir-26 mimics were: mir-26a-5 -uucaaguaauccaggauaggcu-3 ; mir-26b-5 -uucaaguaauucaggauaggu-3 ; corresponding non-seed-mutated mir-26 mimics (26-1a, 26-9u, and 26-1a9u) or seed-mutated mir-26 mimics (26a-m, 26b-m, c-m) are listed in table 1 (underlined letters are mutated bases). the negative-control (nc) mimic sequence was 5 -uucuccgaacgugucacgutt-3 . 2.3. transfection of mirna mimic and viral multi-step growth kinetics mirna or nc mimics were transfected into pams or marc-145 cells at a concentration of 80 nm (except for dosedependence experiments) using x-tremegene sirna transfection reagent (roche). twenty-four hours after transfection, cells were infected with prrsv. for analysis of prrsv growth, supernatants (0.2 ml/well) from cell cultures were collected at different time points post-infection and frozen at −80 • c. for virus quantification at each time point, a viral titer was measured in marc-145 cells by standard tcid 50 assay using the method of reed and muench (reed and muench, 1938) . indirect immunofluorescence assays (ifa) were performed as described previously (tian et al., 2011) for the detection of nucleocapsid (n) protein in prrsv vjx143 infected marc-145 cells or pams pre-transfected with mir-26 family or mutant mimics. cells were fixed with cold methanol followed by blocking with 1% bovine serum albumin (bsa) and then incubated for 2 h with a monoclonal antibody (sr30a, rural technologies) that specifically recognizes type 2 prrsv n proteins. after washing with phosphate-buffered saline (pbs), the cells were incubated for 1 h with alexa fluor 568-labeled goat anti-mouse secondary antibody (invitrogen). cell nuclei were counterstained with 1 g/ml of 4 , 6 -diamidino-2phenylindole (dapi) for 5 min. after a final pbs wash step, cells were visually analyzed using an olympus inverted fluorescence microscope. marc-145 cells were transfected with mir-26a or nc mimics prior to prrsv infection. cells were washed twice with pbs at 48 h post-infection and lysed with lysis buffer in the presence of 1 mm n-ethylmaleimide (nem). after incubation for 10 min on ice, cell lysates were centrifuged at 12,000 × g for 15 min at 4 • c and the supernatants were collected. protein samples were prepared in reducing buffer (50 mm tris, ph 6.8, 10% glycerol, 2% sds, 0.02% [wt./vol.] bromophenol blue, 100 mm dtt). samples then were heated at 95 • c for 5 min, resolved on 15% sds polyacrylamide gels, and transferred to hybond-pmembranes (amersham biosciences). membranes were blocked with 5% nonfat dry milk in tbst (100 mm nacl, 10 mm tris, ph 7.6, 0.1% tween 20) for 2 h at room temperature. membranes were incubated overnight at 4 • c with primary antibody (1ag11) that specifically recognizes both type 2 and type 1 prrsv n proteins (kindly provided by ingenasa co., madrid, spain). after washing with tbst, blots were incubated with horseradish peroxidase (hrp)conjugated goat anti-mouse secondary antibody (santa cruz) for 1 h at room temperature, washed again with tbst, and developed using supersignal west pico or femto chemiluminescent substrate according to the manufacturer's instructions (thermo fisher scientific). total rna and mirna were extracted with trizol (invitrogen) following the manufacturer's instructions. primescript tm 1st strand cdna synthesis kit (takara) was used for reverse transcription. quantitative rt-pcr (qpcr) analysis was performed using a step-one plus real-time pcr system (applied biosystems) and a sybr premix ex taq tm (takara). for detection of endogenous mirnas, a commercial mircute mirna first-strand cdna synthesis was purchased from tiangen biotech (beijing, china) and used for polyadenylation and reverse transcription. a commercial mircute mirna qpcr detection kit was purchased from tiangen biotech (beijing, china) for measuring mirna abundance. marc-145 cells infected with prrsv at a moi of 0.01 were collected at the indicated time points and total rna was extracted using trizol reagent (invitrogen). one g of this total rna was then used for reverse transcription with an rt-primer. the abundance of the mirna of interest in the resulting cdna was determined by qpcr using a universal reverse primer and a mirna-specific forward primer. the pcr procedure comprised pre-denaturation at 94 • c for 2 min, and 40 cycles of 94 • c for 20 s, 60 • c for 15 s. the ubiquitously expressed u6 small nuclear rna (tiangen) was used for normalization purpose. all primers used for mirna qpcr were included in the commercial kit. the levels of orf7 rna, ifn-␣/␤, mx1, isg15 mrna were quantified using a sybr premix ex taq tm (takara). relative expression levels were analyzed using the ct method (bookout et al., 2006) , and glyceraldehyde-3-phosphate dehydrogenase (gapdh) mrna was used as an endogenous control. universal type i interferon was purchased from pbl interferonsource. all pcr experiments were performed in triplicate. other primers are listed in table 2 . the pgl3-control luciferase reporter vector (promega) was used as the cloning vector for luciferase assays to analyze potential mir-26a target regions in the prrsv genome. twenty cdna fragments encompassing the prrsv genome were amplified by pcr from prrsv pjx143 and subcloned into the pgl3-control vector downstream of the luciferase orf. the primers used are listed table 2 sequence of oligonucleotide primers used in this study. sequence table 2 . all cdna constructs were verified by dna sequencing. plasmids and mirna mimics were transfected into cells using lipofectamine 2000 (invitrogen) according to the manufacturer s protocol. for luciferase reporter assays, subconfluent bhk-21 cells cultured in 12-well plates were co-transfected with 500 ng/well of the indicated reporter plasmid and 100 ng/well of prl-cmv (as an internal control to normalize transfection efficiency, promega) along with the indicated amount of mir-26a mimic. cells were lysed 24 h later for determination of firefly luciferase activities using the luciferase assay system (promega). data are presented as the relative luciferase activities in mir-26a mimic-transfected cells relative to nc mimic-transfected controls and are representative of three independent experiments. to screen potential mirnas for their ability to inhibit prrsv replication, mimics of 15 mirnas that are well-conserved among different species and have been previously implicated in innate immunity and/or antiviral functions (banerjee et al., 2013; foley and o'neill, 2012; huang et al., 2014; yoo and liu, 2013; pauley and chan, 2008; schulte et al., 2013; selvamani et al., 2014) were synthesized (table 1) . marc-145 cells were transfected with individual mirna mimics (80 nm) and then infected with prrsv (vaprrs) at an moi of 0.01. supernatants from infected cells were collected at 24 and 48 h post-infection to determine viral titers. among the mirnas tested, over-expression of the mir-26a mimic strongly reduced prrsv titers (fig. 1a) . transfection of mir-26a/26b inhibitors demonstrated the opposite effects (fig. 1b) , indicating that mir-26 has antiviral activity against prrsv replication and that mir-26a is a more efficient suppressor than mir-26b. all the other mirna mimics tested had no demonstrable impact on prrsv titers in marc-145 cells (fig. 1a) . furthermore, immunofluorescence assays using a fitc-conjugated monoclonal antibody against the prrsv n protein were consistent with viral titer data (fig. 1c) . to rule out the possibility that this antiviral effect of mir-26a was specific to an individual prrsv strain, we analyzed the viral growth curves of two type 2 prrsv strains (vjx143, vjxm100) and a type 1 prrsv strain (vshe) in marc-145 cells transfected with nc or mir-26a mimics. over-expression of the mir-26a mimic, but not the nc mimic, reduced prrsv replication in multiple prrsv strains of differing genotypes ( fig. 2a) . to corroborate our findings with mir-26a further, marc-145 cells were transfected with increasing concentrations of mir-26a mimic (5, 20, 40, 80 nm) and then infected with vaprrs. both prrsv growth and the amount of orf7 mrna level were inhibited as a function of the dose of mir-26a mimic ( fig. 2b and c) . consistent with this, transfecting the mir-26a mimic also reduced the accumulation of the prrsv nucleocapsid (n) protein in a dose-dependent manner (fig. 2d) . to exclude the possibility that reduced prrsv replication was due to potential toxicity of the mir-26a mimic, marc-145 cells were transfected with the mir-26a mimic at different doses (40 nm, 80 nm, and 160 nm). no appreciable effect of the mir-26a mimic (at up to 160 nm) on cellular viability and morphology was observed (data not shown). collectively, these data show that mir-26a reduces prrsv replication in multiple prrsv genotypes in a dose-dependent manner. we next investigated the effect of other mir-26 mirna and mutants on prrsv replication. because mir-26a is highly conserved between monkeys and pigs, we conducted the subsequent investigations in pams, which are the target cells of prrsv infection in vivo. as previously reported, mirna-mrna interactions may require seed-matched sites at nucleotides 2-8 (bartel, 2009 ). thus, we mutated mir-26a mimic at non-seed nucleotides 1, 9, or 1 and 9 and mir-26 at seed nucleotides 2-6 (26a-m and 26bm). both mir-26a and mir-26b had anti-prrsv activity in pams. pams transfected with mir-26a or mir-26b mimics yielded significantly lower prrsv titers and orf7 gene expression compared with cells transfected with the nc mimic ( fig. 3a and b) . three mir-26a mutants with non-seed mutations retained their ability to inhibit prrsv progeny production and gene expression ( fig. 3a and b). by contrast, seed mutations at nts 2-6 abrogated the ability of mir-26 family members to repress prrsv replication and gene expression (fig. 3a and b) , showing that the seed region was essential for inhibiting prrsv replication. to investigate further inhibition effect of mir-26 family and mutants on prrsv infection, we used an immunofluorescence assay to detect the prrsv n protein in pams. n protein expression in pams was suppressed by both mir-26a and mir-26b (fig. 3c , top) and by mir-26 non-seed mutants (fig. 3c, middle) , but was not affected by mir-26 seed mutants (fig. 3c, bottom) . we then analyzed the growth dynamics of hp-prrsv isolate vjx143 in pams transfected with mir-26 family or nc mimics. viral growth was suppressed about 1000-fold in pams transfected with mir-26a and about 100-fold in pams transfected with mir-26b at 24 h postinfection (fig. 3d) . notably, mir-26a was more efficient suppressing viral growth than mir-26b. these results indicated that mir-26 family members, especially mir-26a, can inhibit vjx143 replication in pams. we then analyzed the kinetics of mir-26 expression in prrsv infected pam cells. the relative expression of mir-26 was upregulated as a function of prrsv infection time (fig. 3e) . targeting a specific viral sequence represents an efficient strategy by which mirnas can inhibit viral replication (jopling, 2010; lecellier et al., 2005) . in recent studies, mir-181 and mir-23 were confirmed to reduce viral gene expression and viral growth due to direct targeting of prrsv genomic rna (guo et al., 2013; zhang et al., 2014) . we determined whether mir-26a specifically targets the prrsv genome to exert its antiviral effect by constructing a range of firefly luciferase reporter pgl3-control based plasmids, which contained the cdna fragments representing the 5 utr, nsp1-nsp12, orf2-orf7, and the 3 utr of the prrsv genome statistical significance was analyzed using t-tests; *, p < 0.05; **, p < 0.01; ***, p < 0.001. c. pams were transfected with the indicated mirna mimics and then infected with prrsv vjx143 (moi = 0.01) for 24 h. cells were fixed and immunostained with the mouse monoclonal sr30a antibody against the viral n protein and fitc-conjugated goat anti mouse igg. cellular nuclei were counterstained with dapi (1 mg/ml). d. prrsv growth in pams transfected with mir-26 family mimics. pams were transfected with mir-26 family or nc mimics for 24 h and then infected with prrsv vjx143 at an moi of 0.01. culture supernatants were collected at the indicated times and titrated. e. time-course of mir-26a/26b expression after prrsv infection. pam cells infected with vjx143 at a moi of 0.01 were collected at the indicated times and qrt-pcr analysis was performed to detect mir-26a/26b expression. relative mir-26a/b expression refers to the change in mir-26a/b expression levels in prrsv-infected pams relative to mock pams. downstream of the firefly luciferase gene (fig. 4a ). if the prrsv cdna insert contains a mir-26a target sequence, luciferase reporter expression is expected to be subjected to mir-26a-regulation. mir-26a or nc mimics were co-transfected with the individual reporter vectors into bhk-21 cells, along with an internal control vector prl-cmv. relative luciferase activities were quantified 24 h post-transfection. the relative luciferase activities for different vectors containing various prrsv cdna fragments were not significantly different between cells transfected with mir-26a mimic as compared with cells transfected with the nc mimic. thus, mir-26a does not appear to target directly the prrsv genome. we found that over-expression of mir-26a increased ifn-␣/␤ expression during vjx143 infection (moi = 0.01) at 36 h in pams as compared with over-expression of nc (fig. 5a ). the ifn-stimulated genes mx1 and isg15 were also significantly upregulated (fig. 5b) . transfecting the mir-26a mimic into pams in the absence of prrsv infection also enhanced type i ifn expression ( fig. 5a and b) . ifn-␣ and ifn-␤ were induced about 1.6-fold in un-infected pams, and about 4.9-and 2.8-fold in prrsv infected pams, respectively. isg15 and mx1 were increased about 3.5-and 2.2-fold in un-infected pams, and about 3.6-and 2.4-fold in prrsv infected pams, respectively. in marc-145 cells, over-expression of mir-26a up-regulated ifn-␣ and isg15 more strongly (fig. 5c ). ifn-␣ and isg15 were induced about 4.4-and 9.8-fold in un-infected marc-145 cells, and about 9.1-and 20.4-fold in prrsv infected marc-145 cells, respectively. transfection of mir-26a inhibitors did not increase the expression of ifn-␣ or isg15 (fig. 5c) , confirming that the induction of the innate immune response is specifically mediated by mir-26a. there is a growing body of evidence that cellular mirnas are important regulators of innate and adaptive immune responses and the intricate networks of host-pathogen interactions. herein, we identified mir-26a as an inhibitor of prrsv replication that does not directly target the prrsv genome ( figs. 1 and 4) . overexpressed mir-26a reduced prrsv replication and viral gene expression (fig. 2) , in not only marc-145 cells, but also in pams (fig. 3) , the main target cell for prrsv replication in vivo, confirming the biological relevance of this finding. mir-26a belongs to a broadly conserved mirna family with perfectly identical sequences among vertebrates (griffiths-jones et al., 2006) . previous studies of mir-26a have shown that this mirna is an important regulator of cell proliferation and differentiation that targets the smad1 transcription factor (ezh2), a suppressor of skeletal muscle cell differentiation (lu et al., 2011; luzi et al., 2008; sander et al., 2008; zhang et al., 2011) . by infecting pams with prrsv strain vr-2332, liu et al. generated small rna expression profiles at 12, 24 and 48 h post-infection to identify alterations in mirna expression associated with prrsv (yoo and liu, 2013) . overall, 40 cellular mirnas were differentially expressed during at least one time point in prrsv-infected pams. however, in this study, mir-26a was not mentioned (yoo and liu, 2013) . contrary to the previous study, we found that the expression of mir-26a was up-regulated about 2-fold at 48 h post-infection (fig. 3e) . one mechanism by which host mirnas regulate viral replication is the direct targeting of viral sequences (jopling, 2010; lecellier et al., 2005) . however, prrsv is a fast-evolving rna virus (prieto et al., 2009) and the relatively high mutation rate may limit the application of this kind of rnai-mediated antiviral therapeutic. cellular mirnas can also indirectly modulate cellular pathways that perturb the viral life cycle. in particular, the activation or enhancement of innate antiviral immune pathways has been suggested to be responsible for the antiviral effect of certain mirnas (lecellier et al., 2005; pedersen et al., 2007) . in the current study, the reduction of prrsv replication by mir-26a did not appear to involve direct targeting of the prrsv genomic rna (fig. 4) . moreover, this reduction occurred in both type 1 and type 2 prrsv strains ( fig. 2a) although these two genotypes share only approximately 60% nucleotide sequence identity. these data led us to hypothesize that mir-26a might act on a cellular factor to reduce prrsv replication. the results presented here support a link between prrsv replication and the altered expression of mir-26a in targeting host innate immune responses (fig. 5) . type i interferons (ifns) are potent antiviral cytokines whose expression is triggered through recognition of viral components by pattern recognition receptors via a cascade of signaling molecules . pams are the main target cells for prrsv infection, and many gene expression studies have explored the immune response of pams to prrsv. such studies have shown that the expression levels of mx1, usp, ifn-␤, il-10, and tnf-␣ are affected by prrsv infection (albina et al., 1998; luo et al., 2008; van reeth et al., 1999) . overall, these analyses suggest that prrsv subverts host defenses by inhibiting the expression of pro-inflammatory cytokines (van reeth et al., 1999) and stimulating weak production of ifn-␣ . our results showed that over-expression of mir-26a was capable of inducing expression of ifn-␣/␤ and the ifn-stimulated genes isg15 and mx1, which might result in activation of the ifn response and further lead to the inhibition of virus infection. the restoration of innate immune responses to produce type i ifns in pams seems to be mirna specific, because another mirna (mir-181b) had no such effect (data not shown). thus, it is possible that mir-26a-induced type i ifn expression can overcome prrsv interference, contributing to viral clearance. this mechanism provides a higher genetic barrier to the emergence of viral escape mutants, so the identification and characterization of mir-26a as an inhibitor of prrsv replication may open new ways to control fig. 5 . mir-26a increases type i ifn expression during prrsv infection. qrt-pcr analysis of (a. type i ifn ␣/␤ and b. mx1/isg15) expression in pams transfected with nc or mir-26a mimics or left untreated (mock) for 24 h, and then infected with vjx143 for 36 h at an moi of 0.01, or left untreated. data were normalized to gapdh expression and are the mean ± standard deviation of three independent experiments. c. qrt-pcr analysis of ifn-␣ and isg15 expression in marc-145 cells transfected with nc, mir-26a mimics or inhibitors, and then infected with vjx143 for 36 h at an moi of 0.01. data were normalized to ␤-actin expression. statistical significance was analyzed using t-tests; *, p < 0.05; **, p < 0.01; ***, p < 0.001. future prrs outbreaks, for which effective control measures remain scant. our results showed that mir-26a also can mediate the activation of ifns in the absence of prrsv infection (fig. 5 ). the possible causes may relate to recent studies about a new function of mirnas, which is independent of their conventional role in post-transcriptional gene regulation fabbri et al., 2012; lehmann et al., 2012) . mir-21, mir-29a, and let-7b have dual functions; on one hand, they bind to argonaute proteins and guide the silencing of target genes, and on the other hand, they act independently of argonaute proteins by interacting directly with tlrs. although there is no current evidence, mir-26a may also serve as ligands for tlrs and activate ifns. future studies will be necessary to unravel the diverse functions of mir-26a. overall, we demonstrated that over-expression of mir-26a inhibits prrsv replication. although clearly defining the target and physiological role of mir-26a remains an unfinished task, our study provided evidence that over-expression of mir-26a enhances ifn-␣/␤ expression during prrsv infection, suggesting that mir-26a could be used as a potential target for antiviral development. micrornas and the resolution phase of inflammation in macrophages immune responses in pigs infected with porcine reproductive and respiratory 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upregulating type i interferons the study was supported by the grants from the national basic research program (973 plan) (no. 2014cb542700), the national natural science foundation of china (no. 31100121, no. 31302098, no. 31300140), and the natural science foundation of shanghai (no. 11jc1415200). key: cord-326349-59566vqe authors: ding, li; huang, yong; dai, meiling; zhao, xiaomin; du, qian; dong, feng; wang, lili; huo, ruichao; zhang, wenlong; xu, xingang; tong, dewen title: transmissible gastroenteritis virus infection induces cell cycle arrest at s and g2/m phases via p53-dependent pathway date: 2013-12-26 journal: virus res doi: 10.1016/j.virusres.2013.09.036 sha: doc_id: 326349 cord_uid: 59566vqe p53 signaling pathway plays an important role in the regulation of cell cycle. our previous studies have demonstrated that tgev infection induces the activation of p53 signaling pathway. in this study we investigated the effects of tgev infection on the cell cycle of host cells and the roles of p53 activation in this process. the results showed that tgev infection induced cell cycle arrest at s and g2/m phases in both asynchronous and synchronized pk-15 and st cells, while uv-inactivated tgev lost the ability of induction of cell cycle arrest. tgev infection promoted p21 accumulation, down-regulated cell cycle-regulatory proteins cyclins b1, cdc2, cdk2 and pcna. further studies showed that inhibition of p53 signaling could attenuate the tgev-induced sand g2/m-phase arrest by reversing the expression of p21 and corresponding cyclin/cdk. in addition, tgev infection of the cells synchronized in various stages of cell cycle showed that viral genomic rna and subgenomic rna, and virus titer were higher in the cells released from s-phaseor g2/m phase-synchronized cells than that in the cells released from the g0/g1 phase-synchronized or asynchronous cells after 18 h p.i. taken together, our data suggested that tgev infection induced s and g2/m phase arrest in host cells, which might provide a favorable condition for viral replication. viral infection could activate a variety of signal transduction pathways to induce subversion of the host cell cycle, which plays important roles in the viral life cycle by facilitating the replication of progeny virus after viral infection (davy and doorbar, 2007) . a variety of rna viruses have been shown to induce g0/g1, s or g2/m arrest in infected cells. for example, human immunodeficiency virus (hiv)-infected t lymphocytes isolated from patients are arrested in g2/m (zimmerman et al., 2006) ; influenza a virus a/wsn/33 (h1n1) infection results in g0/g1-phase accumulation of infected cells, which increase viral protein expression and progeny virus production (he et al., 2010) ; hepatitis c virus (hcv) ns2 protein induces cell cycle arrest in the s-phase in mammalian cells to facilitate hcv viral replication (yang et al., 2006) . in term of coronaviruses, murine coronavirus mouse hepatitis virus (mhv) has been shown to induce g0/g1 arrest (chen and makino, 2004) ; sars-cov nucleocapsid (n) protein can disrupt cytokinesis and block s-phase progression in mammalian cells (surjit et al., 2006) ; bronchitis virus (ibv) infection can induce g2/m phase arrest to facilitate viral replication (dove et al., 2006) . transmissible gastroenteritis virus (tgev) infection has been shown to alter some cell signaling pathways implicated in cell cycle regulation in our previous study (huang et al., 2013) . however, the effects of tgev infection on the cell cycle of host cells and the significance of cell cycle regulation in tgev replication need to be further investigated. the cell-cycle progression is tightly regulated through a complex network of cell-cycle regulatory molecules. cyclind-cdk2 complex and cycline-cdk4 complex regulate cell cycle progression in the g0/g1 phase. cyclina-cdk2 complex regulates cell cycle progression in the s phase. cyclinb-cdc2 complex is a crucial regulator for progression through late g2 and early m (malumbres and barbacid, 2009 ). these cell-cycle regulatory molecules have been shown to be regulated by some upstream pathways like p53 signaling. p53 signaling can control the expression of p21, which directly bind to some cdk-cyclin complexes to inhibit their kinases activity (he et al., 2005) . our previous studies have demonstrated that tgev infection induced the activation of p53 signaling pathway to regulate cell growth and apoptosis (huang et al., 2013) . in this study, we further investigated the effects of tgev infection on the cell cycle of host cells, the roles of p53 signaling activation in regulation of cell cycle progression in tgev-infected cells, and the significance of cell cycle regulation in tgev replication. results showed that that tgev infection could perturb the progression of cell cycle and facilitate virus gene replication. pk-15 cells (atcc, ccl-33) and st cells (atcc, crl-1746) were grown in dulbecco minimal essential medium (d-mem) (gibco brl, md, us) supplemented with 10% heat-inactivated fetal bovine serum (gibco), 100 iu of penicillin and 100 g of streptomycin per ml, at 37 • c in a 5% co 2 atmosphere incubator. the tgev shaanxi strain was isolated from intestinal tract contents of tgev-infected piglets in shaanxi province of china and propagated in pk-15 cells and st cells (ding et al., 2011) . virus titers determined by 50% tissue culture infective doses (tcid50) as described previously (reed and muench, 1938) . cell cycle analysis was measured by propidium iodide staining. briefly, cells were fixed in 70% ethanol for 30 min at 4 • c. after several washes with phosphate-buffered saline (pbs), the cell pellets were resuspended in 0.5 ml pbs containing 0.1% triton x-100 (sigma-aldrich, us), 20 g /ml rnase a (sigma) and 10 g/ml propidium iodide (sigma) for 30 min, prior to facs analysis (beckman the results are shown as mean ± sem of three independent experiments. *p < 0.05, **p < 0.01 versus mock infection. (b) serum-starved pk-15 cells were mock infected or infected with 0.5 moi of tgev. cells were co-stained with brdu and pi and analyzed using flow cytometry. the histograms were analyzed to determine the percentage of cells in s phase of the cell cycle. s phase, upper gate; g1, lower left; g2, lower right. the results are shown as mean ± sem of three independent experiments. **p < 0.01 versus mock infection. coulter, inc. fullerton, ca, us). at least 15 000 nuclei were counted for each sample. the thymidine analog bromodeoxyuridine (brdu) (sigma) is incorporated into actively replicating dna and thus accurately determines the proportion of cells in s phase. briefly, 10 m brdu was added to cell medium and incubated at 37 • c for 30 min to allow brdu incorporation. cells were collected and then fixed in 70% ethanol. then cells were pelleted and incubated in 2 m hcl in pbs at 37 • c for 30 min. after incubating in wash buffer and pelleting, the cell pellet was resuspended in 0.1 m sodium borate. samples were then pelleted before addition of 100 l of anti-brdu antibody (cell signalling technology) and incubated for 60 min at room temperature. samples were then incubated by fluorescein isothiocyanate (fitc)-labeled antibody for 30 min in the dark. samples were stained by pi before analyzed using a facs calibur analyzer. pk-15 and st cells were synchronized at g0/g1 phase using serum deprivation by maintenance of cells in dmem containing no fbs supplementation for 48 h. synchronized cells were mock infected or infected with 0.5 moi of tgev. after 1 h of virus adsorption, cells were treated with medium containing 10% fbs and harvested at various times post infection (p.i.) for cell cycle analysis. pk-15 and st cells were synchronized at the g1/s phase border using double-thymidine treatment by incubation for 12 h in maintenance media supplemented with 2 mm thymidine (sigma). cells were then washed three times with pbs and incubated for 10 h in maintenance media, followed by additional 12 h incubation in maintenance media supplemented with 2 mm thymidine. then, synchronized cells were mock infected or infected with 0.5 moi of tgev. pk-15 and st cells were synchronized at g2/m phase using nocodazole (sigma) treatment by incubation of cells in . at the indicated times, cells were lysed and equal amounts of proteins from the samples were tested by western blot analysis. the same membranes were also probed with ␤-actin as a loading control. the numbers below the proteins indicated the relative folds of mock infection after normalized to ␤-actin. maintenance media supplemented with 100 ng/ml nocodazole for 16 h. cells were washed three times with pbs, and then infected with 0.5 moi of tgev. at indicated times p.i., cells were processed for rt-pcr and flow cytometric analysis. cell extracts were prepared as described previously (ding et al., 2012) . protein concentrations were measured using bca protein assay reagent (pierce, rockford, il, us). equivalent amounts of proteins were loaded and electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gel (sds-page). subsequently, proteins were transferred to polyvinylidene difluoride (pvdf) membranes (millipore corp, atlanta, ga, us). the membranes were blocked with 5% nonfat dry milk at room temperature for 1 h, and then incubated with indicated primary antibodies over night at 4 • c, followed by hrp-conjugated secondary antibodies at room temperature for 1 h. the signal was detected using ecl reagent (pierce, rockford, il, us). total rna extraction and reverse transcription were performed as described (ding et al., 2012) . quantitative analysis of genomic rna (grna) and subgenomic mrnas (sgrnas) from tgev-derived replicons was performed by real-time rt-pcr using bio-rad iq5 real time pcr system. the primers for qrt-pcr in this study were described in previous study (dufour et al., 2011) . reactions were carried out in 25 l volume containing 1× sybr premix ex taqtm ii (takara, dalian, china), sense and anti-sense primers and target cdna. the relative quantification of gene expression was analyzed by the two-ddct method. pifithrin-␣ (ptf-␣) was purchased from sigma and stored as a 50 mm stock solution in dmso. to reduce the activation of p53, ptf-␣ (20 m) was diluted in cell culture medium without serum and added to cultures 1 h prior to infection. inhibitor was not included in the virus inoculum. after 1 h of tgev adsorption, the virus inoculum was removed and fresh basal medium containing fresh inhibitor was added to the culture. at indicated times, cells were harvested and correlative indicators were detected. data are mean ± sem of three independent experiments. results were analyzed by one-way analysis of variance (anova). for each assay, student's t-test was used for statistical comparison. a value of p < 0.05 was considered significant. to investigate the influence of tgev infection on cell cycle progression, mock infected or tgev-infected asynchronously growing pk-15 and st cells were harvested at different times p.i., and cell cycle profiles were detected by flow cytometry. representative cell cycle histograms and profiles in pk-15 and st cells were presented in fig. 1a and b, respectively. in tgev-infected pk-15 cells, there was a significant increase in the proportion of cells in the s phases and g2/m phases of the cell cycle from 6 h p.i. and 12 h p.i., respectively, and continued to increase with infection time, when compared to mock-infected cells (fig. 1a) . in tgev-infected st cells, the proportion of cells in the g2/m phases and s phases significantly increased from 12 h p.i. and 18 h p.i., respectively, when compared to mock-infected cells (fig. 1b) . these results suggest that tgev infection induced the arrest of the cell cycle in the s and g2/m phase. in tgev-infected cells, tgev infectious levels were evaluated by detection of the grna and sgrnas (n sgrna, m sgrna and orf 7 sgrna) of tgev using qrt-pcr. results showed that the levels of tgev grna increased significantly from 12 h p.i. in pk-15 ( fig. 2a , left panel) and st cells (fig. 2b, left panel) . the sgrna levels of n, m, orf 7 could not been detected at 0 h p.i., detected at 6 h p.i., significantly increased at 12 h p.i., and continued to increase with following infectious time in tgev-infected cells ( fig. 2a and b, right panel) . moreover, the titers of tgev were determined at various times postinoculation. results showed that little infectious virus was detected at 0 and 6 h p.i., thereafter the virus titer showed a rapid increase between 12 and 18 h p.i. and then reached a plateau (fig. 2c) . to further determine whether the tgev-induced s and g2/m arrest requires virus replication, uv-inactivated tgev was inoculated in pk-15 and st cells, and cell cycle profiles were measured at 18 h p.i. by flow cytometry. results showed that cell cycle arrest were not observed in cells infected with uv-inactivated tgev, while there was no significant difference between mock-infected cells and cells treated with uv-inactivated tgev (fig. 2d) , suggesting that viral replication was required for induction of cell cycle arrest in tgev-infected cells. to further confirm that tgev replication caused s and g2/m cell cycle arrest, we infected serum-starved quiescent cells with tgev and examined cell cycle progression after serum stimulation. as shown in fig. 3a , prior to infection, approximately 85% of serum-starved cells were arrested at the g0/g1 phase in pk-15 cells. at 18, 24 and 30 h p.i., the cells population at g0/g1-phase significantly decreased, while the cells population at s-phase dramatically increased in tgev-infected cells compared to that in mock-infected cells, and apparent increase of g2/m-phase cells was observed at 30 h p.i. (fig. 3a) . similar feature were also seen in quiescent st cells after infection (fig. 3b) . these results suggest that tgev-infected cells exhibited s-phase delay and g2/m-phase arrest. to determine whether tgev replication may induce the release of some soluble factors to affect the cell cycle progression of uninfected cells, we used high mois of tgev to infect serumstarved quiescent pk-15 cells and detect cell cycle profiles. the results showed that s-phase delay induced by 1 and 5 moi of tgev occurred at 18 h p.i., and the number of s-phase arrested cells induced by 1 moi and 5 moi of tgev were approximately two and four folds as many as that by 0.5 moi tgev, respectively (fig. 4a) . however, the s-phase and g2/m-phase arrested cells number induced by 1 moi and 5 moi of tgev were not significantly higher than that by 0.5 moi tgev at 24 h p.i. (fig. 4a) . these results suggested that tgev replication just affected the cell cycle progression of infected cells, and that over 1 moi of tgev infection would interfere the progression of cell cycle due to the involvement of cell apoptosis. to further confirm the changes of tgev-infected cells in the s phase of cell cycle, we used brdu incorporation assay to accurately determine the cell number in s phase. results showed that the proportion of cells in s phases significantly increased at 18 h p.i., and further increased with infection time, when compared to mockinfected cells (fig. 4b) , which were in consistent with the results of pi staining. to determine the molecular mechanism underlying the tgevinduced cell cycle arrest, we detected the levels of associated cell cycle regulatory factors at 0, 4, 8, 12, 18 and 24 h p.i. in tgev-infected cells. results showed that cdc2, acting as a main coordinator complexed with cyclin a and cyclin b to advance the cell cycle into m phase, began to decrease at 8 h p.i., and continued to decrease with infection time in infected cells compared to that in mock-infected cells (fig. 5a) , suggesting that the cell cycle might be arrested at g2/m phase in tgev-infected cells. in addition, the levels of cdk2 significantly decreased from 4 h p.i. in tgev-infected cells compared to mock-infected cells, but cdk4 and cdk6 did not show significant difference between in mock-infected and tgevinfected pk-15 cells (fig. 5a, left panel) . the levels of cyclin b1 began to significantly decrease at 12 h p.i. in tgev-infected pk-15 cells compared to mock-infected cells, but cyclin a, cyclin d and cyclin e did not show significant difference between mock-infected and tgev-infected pk-15 cells (fig. 5b, left panel) . in parallel experiments, western blot analysis showed similar or analogous results in the expression of these cdks and cyclins in tgev-infected st cells ( fig. 5a and b, right panel) . among them, the reduction of cyclin b1 was more significant in tgev-infected st cells than in tgevinfected pk-15 cells when compared with mock infection. taken together, these results suggest that tgev infection lead to an accumulation of cells in the s and g2/m phases of the cell cycle through decreasing certain cell cycle factors that regulate the proceeding of s and g2/m phase. our previous study showed that tgev infection induced the accumulation and activation of p53 in cells. here, we examined the levels of p53 and p21 in 0.5 moi tgev-infected cells. western blot analysis showed that 0.5 moi of tgev infection could also increase the protein level of p53 and induce phosphorylation of p53 at serine 15 and 20 in pk-15 and st cells (fig. 6a) . consequently, the level of p21 increased in tgev infected pk-15 and st cells from 8 h p.i., and significantly increased at 12 h p.i., while proliferating-cell nuclear antigen (pcna), a mediator involved in p21-regulated dna replication within s phase, decreased accordingly in tgev-infected pk-15 and st cells (fig. 6a) . these results suggest that tgev infection could up-regulate p21 expression to regulate cell cycle through activation of p53 signaling. to further determine the roles of p53 in tgev-induced cell cycle arrest, we investigated the effects of pft-␣, a specific inhibitor of p53 that does not affect the mrna levels of tgev genes (huang et al., 2013) , on the cell cycle profiles and the expression of p21, cdks and cyclins in tgev-infected pk-15 and st cells. as shown in fig. 6b , pre-incubation of pk-15 and st cells with pft-␣ attenuated cell cycle arrest at s and g2/m phase induced by tgev infection. in addition, cdc2, cdk2 and cyclin b1 expression increased in tgevinfected pk-15 and st cells with pft-␣ compared to that with dmso (fig. 6c) . as expected, pre-incubation of pk-15 and st cells with pft-␣ also attenuated p21 up-regulation and pcna reduction induced by tgev infection (fig. 6c) . these results suggest that p53 and p21 might play key roles in mediation of tgev-induced cell cycle arrest. since the proportion of cells in the s and g2/m phases was greater in tgev infected cells compared to mock-infected cells, to investigate the effects of cell accumulation at s and g2/m phases on tgev replication, synchronized cells in g2/m phase, g0/g1 phase, g1/s phase or asynchronous cells were simultaneously released from their different phase of cell cycle, and then either mock infected or infected with 0.5 moi of tgev, cell cycle profiles were determined by flow cytometry and tgev grna and sgrnas levels were determined by qrt-pcr at 18 h p.i. results showed that using serum deprivation treatments, over 85% of pk-15 and 82% of st cells were synchronized at the g0 phase (fig. 7a ). using nocodazole treatments, over 90% of pk-15 cells and 80% of st cells were synchronized at the g2/m phase (fig. 7b ). using double-thymidine treatment, approximately 80% of cells were synchronized at the g1/s phase border in pk-15 and st cells (fig. 7c ). in the cells released from the g0-phase-synchronized cells, the proportion of s phase cells was greater in tgev infected cells compared to mockinfected cells at 18 h p.i. (fig. 7a) , which was in consistent with the results above. in the cells released from the g1/s and g2/m-phasesynchronized cells, the cells population at g2/m phase significantly increased in tgev-infected cells compared to that in mock-infected cells at 18 h p.i. (fig. 7b and c) . real-time rt-pcr analysis of the grna and sgrnas of tgev showed that the replication levels of grna and the synthesis of sgrna were higher in the cells released from g2/m or g1/ssynchronized cells than that in the cells released from g0/g1 phase-synchronized or asynchronous cells (fig. 8a ). in addition, the titers of the virus also were higher in the cells released from g2/m or g1/s-synchronized cells than that in the cells released from g0/g1 phase-synchronized or asynchronous cells (fig. 8b) . these results suggest that tgev induction of host cell staying at s and g2/m phase might be beneficial for virus replication. cell cycle manipulation is crucial event occurred in virusesinfected cells (davy and doorbar, 2007; kannan et al., 2011) . in this study we demonstrated that tgev-infected cells accumulated in the s and g2/m phase of the cell cycle, and the effect was not cell type specific and could be reproduced in synchronously replicating cells. the cell arrest at the s and g2/m phases was dependent upon tgev replication and was controlled by viral modulation of certain cyclins/cdks. many viruses employ a variety of mechanisms to utilize or manipulate the cell cycle pathways of infected cells (chowdhury et al., 2003; de bolle et al., 2004; li et al., 2007) . throughout the cell cycle, progression is regulated by a series of cyclins and cdks (king and cidlowski, 1998) . the g2/m transition in the cell cycle is positively controlled by complex of cdc2 (cdk1) and cyclin b. members of the cdki family of proteins (cdk inhibitors) bind to the cdk/cyclin complexes and inhibit the kinase activity, to regulate the g2/m-phase transition (king and cidlowski, 1998) . cyclin a-cdk2 complex is the main cyclin-cdk complex in the s phase, and the activity of the complex is required for s phase transition and control of dna replication (elledge, 1996) . it is possible that the regulation of s and/or g2/m phase is one of the crucial mechanisms that viruses employed to manipulate the cell cycle (li et al., 2011; martin-lluesma et al., 2008) . in tgev-infected cells, we observed that a significant decrease of cdk2, cyclin b1 and cdc2, involved in s phase and g2/m phase transition, which regulated the cell cycle to arrest at the s and g2/m phases. p53 is a key element in the induction of cell cycle arrest in viruses infected cells (casavant et al., 2006) . as a dna damage response protein, p53 transcriptionally activates numerous genes involved in dna repair and cell cycle arrest (vogelstein et al., 2000) , and p53dependent arrest of cells at g1/s or g2/m phase is an important component of the cellular response to genotoxic stress including virus infection (casavant et al., 2006; de bolle et al., 2004) . the first transcriptional target of p53 was p21, a cki of the cip/kip family, which bridges the function of p53 with the cell cycle and plays important roles in regulation of cell cycle progression or arrest (he et al., 2005) . the p21 is recognized to be an important mediator to delay transit from g1 to s phase and/or from g2 to m phase, thus preventing the effects of dna damage on gene functions (abbas and dutta, 2009). p21 has also been shown to regulate dna replication within the s phase in vitro, by binding to proliferating-cell nuclear antigen (pcna)/cdk complexes and by dissociating cyclin a/cdk2/p107/e2f transitional complexes (abbas and dutta, 2009) . in this study, we showed that tgev infection induced p53 and p21 accumulation and pcna decrease. furthermore, pft-␣, a specific inhibitor of p53, prevented the arrest of the cell cycle in tgevinfected cells. the observation that tgev infection induced cell cycle arrest at s and g2/m phases in both pk-15 and st cells virtually required the possible involvement of p53 in these processes. this observation is not consistent with the findings in other coronavirus-infectious bronchitis virus, which induced cell cycle arrest at both s and g2/m phases independent of p53 (li et al., 2007) , suggesting that p53 might play different roles in viruses-infected cells. some viruses can regulate the cell cycle progression through interaction between virus and host cells in order to induce the cell cycle arrest and provide an advantageous environment for viral replication (dove et al., 2006; li et al., 2007) . infectious bronchitis virus (ibv) could increase protein accumulation and progeny virus production in cells enriched in the g2/m phase of the cell cycle (li et al., 2007) . human immunodeficiency virus (hiv) infection also increased the transduction of hiv in the g2/m phase compared to other stages of the cell cycle (groschel and bushman, 2005) . many substrates (i.e., nucleotides) in s phase are abundantly available for viral replication (yang et al., 2006) . furthermore, there is evidence that one consequence of g2/m arrest is to establish a pseudo-s phase state for viruses to replicate of their genomes (belyavskyi et al., 1998) . to specifically investigate whether a particular stage of the cell cycle favored tgev replication, we synchronized cells in various stages of the cell cycle and compared efficiencies of virus infection. results showed that tgev replication levels, sgrna synthesis were greater in the cells released from g1/s phase or g2/m phase of the cell cycle after 18 h p.i., when compared to either cells released from the g0/g1 phase or asynchronous cells. our results also provided evidences that tgev titer in the cells released from g1/s phase or g2/m phase synchronized cells was higher than that in cells released from g0/g1 phase synchronized cells. this might be that cells synchronized at g0/g1 phase need longer time to reenter cell cycle progression than synchronized cells at the g1/s phase, which support the hypothesis that s phase state was beneficial for virus replication. therefore, we favor the hypothesis that tgev induces s and g2/m phase cell cycle arrest in order to provide a more favorable condition for virus production. in conclusion, our study revealed that tgev infection induced the host cells to arrest at s and g2/m phases of the cell cycle, which were responsible by the certain key players in the s and g2/m transition. moreover, the cell cycle arrest induced by tgev infection might provide a more favorable condition for viral replication. li ding and yong huang designed the experiments and interpreted the data and wrote the article. li ding performed the experiments with assistance and advice from meiling dai, xiaomin zhao, qian du, feng dong, lili wang, ruichao huo, wenlong zhang and xingang xu. yong huang and dewen tong revised the manuscript. all authors have read the manuscript and approved to submit it to your journal. there is no conflict of interest of any authors in relation to the submission. p21 in cancer: intricate networks and multiple activities the structural protein odv-ec27 of autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin 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early steps of infection by human immunodeficiency virus induction of p21 by p53 following dna damage inhibits both cdk4 and cdk2 activities influenza a virus replication induces cell cycle arrest in g0/g1 phase transmissible gastroenteritis virus infection induces cell apoptosis via activation of p53 signaling hepatitis c virus infection causes cell cycle arrest at the level of initiation of mitosis cell cycle regulation and apoptosis 1 cell cycle arrest and apoptosis induced by the coronavirus infectious bronchitis virus in the absence of p53 human herpesvirus 6 suppresses t cell proliferation through induction of cell cycle arrest in infected cells in the g2/m phase cell cycle, cdks and cancer: a changing paradigm hepatitis b virus x protein affects s phase progression leading to chromosome segregation defects by binding to damaged dna binding protein 1 a simple method of estimating fifty per cent endpoints the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclindependent kinase complex and blocks s phase progression in mammalian cells surfing the p53 network hcv ns2 protein inhibits cell proliferation and induces cell cycle arrest in the s-phase in mammalian cells through down-regulation of cyclin a expression human immunodeficiency virus type 1 vpr induces dna replication stress in vitro and in vivo this work was supported by grants from the national natural science foundation of china (grant no. 31072108/c1802), the doctoral program of higher education of china (grant no. 20110204110014), and the scientific research program of northwest a&f university (no. z111021103). key: cord-291754-1zxztadu authors: zhao, ye; cheng, jinlong; xu, gang; thiel, volker; zhang, guozhong title: successful establishment of a reverse genetic system for qx-type infectious bronchitis virus and technical improvement of the rescue procedure date: 2019-10-15 journal: virus res doi: 10.1016/j.virusres.2019.197726 sha: doc_id: 291754 cord_uid: 1zxztadu in this study, a pathogenic avian infectious bronchitis virus (ibv) qx-type strain yn was successfully rescued by vaccinia virus based reverse genetic technology. ten fragments contiguously spanning the complete ibv genome were amplified and cloned into the vaccinia virus genome by homologous recombination. the full-length genomic cdna was transcribed in vitro, and its transcript was transfected into bhk-21/n cells that could stably express ibv n protein. at 48 h post transfection, the culture medium was harvested and inoculated into 10-day-old specific-pathogen-free embryonated chicken eggs to replicate the rescued virus. this strategy was chosen to facilitate the rescue procedure and to ensure that the recombinant ryn virus will not require any cell culture adaptations. after only one in ovo passage, the recombinant yn virus (ryn) was successfully recovered and confirmed to possess the introduced silent marker mutation in its genome. biological characteristics of ryn such as the eid(50), tcid(50), replication in ovo, and replication kinetcs in vitro were tested and all were similar to its parental strain yn. our findings demonstrate the successful construction of highly-pathogenic qx-type ibv using a modified rescue procedure, allowing for future studies of the molecular biology and pathogenicity of ibv field strains. avian infectious bronchitis is a highly contagious disease caused by infectious bronchitis virus (ibv) that affects poultry production worldwide (cavanagh, 2007) . ibv belongs to the gammacoronavirus genus, in the coronavirinae subfamily, of the coronaviridae family, in the order nidovirales. the ibv genome is approximately 27.6 kb in size. all coronaviruses share a similar genome organization with gene 1 located at the 5ʹ end of the genome. as the replicase gene, it has been proved to be a determinant of pathogenicity of ibv (armesto et al., 2009) . the structural genes cluster at the 3ʹ end and encode the spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins (cavanagh, 2005) . ibv also possesses two accessory genes, gene 3 and gene 5, which have been shown to be dispensable for virus replication in cell culture youn et al., 2005; hodgson et al., 2006; bentley et al., 2013) . however, it seems that the deletion of accessory genes 3a, 3b, 5a or 5b from ibv could induce an attenuated phenotype both in vitro and in vivo (laconi et al., 2018; van beurden et al., 2018) . although ibv has been controlled by routine vaccination for a long time, it remains a significant threat to the global poultry industry due to the existence of many serotypes (capua et al., 1999) . the high evolution and recombination rates of ibv result in new serotype viruses emerging in the field and often causing great losses since current vaccines only provide partial protection against new strains (cavanagh et al., 1992; de wit et al., 2011) . specifically, a new ibv variant has been circulating in china since 1998. this virus has been identified as the qx strain and has been primarily associated with various renal pathologies (wang et al., 1998) . phylogenetic analyses showed that the ibv isolates that clustered with qx were mainly chinese isolates (zhao et al., 2016) . these results further indicated that ibv isolates prevalent in china were evolutionarily distant from massachusetts (mass)-type strains (terregino et al., 2008; abro et al., 2012; yan et al., 2017) . understanding the pathogenic mechanism and molecular characteristics of circulating strains could significantly facilitate the development of vaccines that provide efficient protection against the highly variable ibv. since the initial development of a reverse genetic system for rna (taniguchi et al., 1978) , this technique has proven to be a powerful method for investigating the molecular biology of rna viruses and for studying the role of individual genes in pathogenesis. however, despite these successes, not all reverse-genetic systems of positivestranded rna viruses can be easily established. the large genome of the coronavirus poses a great obstacle to the construction of reverse genetic systems based on full-length cdna. cloning techniques using traditional plasmid dna cloning vectors cannot guarantee stability of large fragments of coronavirus cdna, and many studies have found that certain coronavirus gene sequences can have toxic effects in e. coli or cannot be cloned into conventional plasmid vectors or passaged in prokaryotic systems (gonzalez et al., 2001) . therefore, infectious fulllength clones of coronaviruses have only been established at the beginning of the 21 st century (casais et al., 2001) . subsequently, reverse genetics was successfully applied to study the role of accessory genes in viral replication youn et al., 2005; laconi et al., 2018) , the potential of ibv as a vaccine vector (britton et al., 2012; bentley et al., 2013) , the virulence determinants of the beaudette and m41 strains (fang et al., 2007; maria et al., 2009; keep et al., 2018) , and the relationship between the s gene and tissue tropism of the virus (casais et al., 2003; britton et al., 2005; promkuntod et al., 2013; shan et al., 2018) . however, to the best of our knowledge, all existing reverse genetics systems for ibv were either based on vero cell-adapted beaudette strain, which is non-pathogenic in chickens (geilhausen et al., 1973) , or the mass-type strains, which are genetic quite distant from the prevalent qx-type strains. neither of these systems therefore served as an ideal virus model to study the replication and pathogenic features of qx-type strains. therefore, the application of reverse genetics techniques to qx-type strains may allow for modification of the viral genome and may provide a powerful tool for novel vaccine development. we previously isolated and characterized a virulent ibv strain from yun nan province, china, designated yn strain. this isolate is genetically similar to most of the prevalent qx-type ibv strains found in china, albeit with increased pathogenicity compared with previously characterized strains (feng et al., 2012) . in this study, we describe the establishment of a reverse genetic system of the virulent yn strain. we also developed a modified rescue strategy, which combines full length rna electroporation with in ovo virus amplification, thus avoiding cellculture adaption and providing improved rescue efficiency of ibv. these advances will enable us to modify specific gene regions and investigate their influence on gamma coronavirus replication and pathogenicity. ibv strain yn was isolated and propagated in the allantoic cavities of 10-day-old specific-pathogen-free (spf) embryonated chicken eggs (ece) (feng et al., 2012) . the complete genome of yn has been sequenced and submitted to the genbank database under accession no. jf893452. vaccinia virus vnoti/tk, cv-1, and d980r, and bhk-21 cells were described previously hertzig et al., 2004) . vaccinia virus vnoti/tk and vaccinia virus recombinants were propagated, titrated, and purified on monkey kidney fibroblasts (cv-1) by standard procedures . all cells were maintained in minimum essential medium containing 10% fetal bovine serum (fbs, gibco, grand island, ny, usa), 100 u/ml penicillin, and 100 mg/ml streptomycin. human embryonic kidney (hek) 293 t cells (american type culture collection) were cultured in dulbecco's modified eagle's medium (dmem) (hyclone, logan, ut, usa) supplemented with 10% fbs, 100 u/ml penicillin, and 100 mg/ml streptomycin. viral rna was extracted from yn-infected allantoic fluid with a genmark rna purification kit (genmark, tai-chung, taiwan) according to the manufacturer's directions. reverse transcription was performed with the primescript™ rt master mix (takara, tokyo, japan). vaccinia y. zhao, et al. virus research 272 (2019) 197726 viral dna was extracted from strain vnoti/tk according to a previously described method (bridgen, 2013) . primers used for amplifying the yn strain complete sequence (genbank, accession number jf893452) were designed according to the intrinsic restriction enzyme sites in the genome. silent mutations were introduced into the genome by primers if no restriction sites were present, and also served as molecular markers of the recombinant viruses. each dna fragment was amplified from cdna templates by pcr using the cloneamp hifi pcr premix (takara). pcr primer pairs used to amplify genomic regions are listed in table 1 . nucleotide (nt) changes and restriction sites have been presented as boxes and italicized typeface in table 1 , respectively. pcr amplification of cdna fragments was performed under the following conditions: denaturation at 98℃ for 1 min, then 29 cycles of 98℃ for 10 s, 55°c -59℃ for 10 s, and 72℃ for 30-60 s depending on the size of the products (10 s/kb), and a final extension at 72℃ for 2 min. the pcr products were purified using a nucleospin® gel and a pcr clean-up kit (macherey-nagel, düren, germany) and cloned into pgpt-1 vector, which contains a dominant selective marker gene, escherichia coli guanine phosphoribosyltransferase (ecogpt) (mulligan and berg, 1981) , under the control of the vaccinia virus p7.5 k early/late promoter. in order to construct the recombinant vaccinia virus containing complete ibv genome through vaccinia virus-mediated homologous recombination, the first step was to construct flanking region belonging to the vaccinia virus genome into the pgpt-1 plasmid. the left flank vvt7 was 557 bp in size, an xbai and an bamhi restriction site were added at the 5ʹand 3ʹ end for the ligation into pgpt-1, and an additional type iis restriction site bambi and five nucleotides 'actta' (shown in red in table 1 ) from ibv 5′utr beginning was added at the 3ʹ end of vvt7 for the further ligation with ibv fragements. between the vaccinia dna sequence and bsmbi site was a bacteriophage t7 rna polymerase promotor and an additional g nucleotide for the initiation of the in vitro transcription reaction. the pcr product vvt7 was digested with xbai and bamhi to ligated into pgpt-1 plasmid, next, we constructed the ibv segment 1 f (corresponding to nt 1 to 3285 in the yn genome) by rt-pcr. upstream of the segment 1 f 5′-end was a bsmbi restriction site, downstream of the segment 1 f 3′-end was a bamhi site, thus to allow insertion of the 1 f cdna downstream of vvt7 in pgpt-1 by restriction endonuclease digestion and t4-ligation. on the other side of pgpt-1, a 362 bp vaccinia virus flanking region vv-3′ was cloned into the plasmid following digestion with xhoi and ecori, and an additional noti restriction site was added before the ecori site to stop in vitro transcription after the poly(a) tail. the obtained plasmid, pgpt-1 f, was integrated into the vaccinia virus genome by homologous recombination under the positive selection pressure of mycophenolic acid (mpa) in the presence of xanthine and hypoxanthine, to which only recombinant vaccinia viruses with the ecogpt gene show resistance (falkner and moss, 1990) . ecogpt-selected viruses were purified and sequenced to ensure that ibv segment 1 f and the ecogpt gene had been inserted into the correct position in the vaccinia genome, and verified virus was termed vv-yn-1 f. next, to generate a complete 3ʹ untranslated region (utr) with a 25-bp poly(a) tail in strain yn, a dna was synthesized and cloned in the puc57 plasmid containing a 500 bp fragment 1 f (ibv-yn nts 2785-3285) plus 304 nts of the ibv-yn 3ʹutr sequence (27,378-27,657) followed by a 25-bp poly(a) tail and 508 bp of vaccinia virus flanking sequence. we also incorporated several restriction sites for further modification in the order: 5ʹ-saci-1 f (2785-3285)-bamhi-sali-3ʹutr (27,378-27,657)-poly(a) (25 bp)-noti-vv (508 bp)-xhoi-3ʹ. this fragment was digested with saci and xhoi and ligated into the digested pgpt-1 plasmid to replace the ecogpt gene. next fragment 1r corresponding to the ibv-yn genomic region from nt 3286 to 6559 containing bamhi and sali sites was amplified and inserted between 1 f (2785-3285) and the 3ʹutr (27, 657) in pgpt-1 (fig. 1a) . the homologous recombination between pgpt-1r-3ʹutr and vv-yn-1 f was performed by exerting negative selection pressure on d980r cells due to the presence of 6-thioguanine (hertzig et al., 2004) . after successful construction of the ibv-yn fragment 1 f1r containing the 5ʹ-t7 promoter and the 3ʹ-synthetic poly(a) tail in the vaccinia virus genome, the other eight remaining fragments (2 f, 2r, 3, 4 f, 4r, 5 f, 5r, 6) were inserted into the vaccinia virus genome by vaccinia virus-mediated homologous recombination using the method described above. the individual recombination steps are summarized in fig. 1b . the final recombinant vaccinia virus was purified and sequenced by deep sequencing. the vaccinia virus possessing the complete ibv-yn genome was amplified on a large scale in bhk-21 cells and designated vvibv-ryn. the recombinant vaccinia virus vvibv-ryn was then used to construct recombinant vvibv, vvibv yn-5a/egfp, by two rounds of vaccinia virus-mediated homologous recombination. this virus consisted of direct replacement of ibv gene 5a sequence, beginning at the orf 5a initiation codon, and ending at the orf 5a termination codon in order to maintain the genomic sequence upstream of the 5b gene. the introduced reporter gene egfp, replacing orf 5a, was expressed utilising the existing gene 5 trs. first, plasmid pgpt△5a was constructed to encode ibv-yn nt 25038-25539 and 25733-26629 upstream and downstream of the gpt gene. recombination of vvibv-ryn with plasmid pgpt△5a resulted in the isolation of the recombinant vaccinia virus vvibv-gpt△5a. vvibv-gpt△5a was used for recombination with the plasmid pibv-gfp, encoding ibv-yn nt 25038-25539 upstream and ibv-yn nt 25733-26629 downstream of the gfp sequence. the resulting recombinant vaccinia virus vvibv yn-5a/egfp was obtained after gpt negative selection. to improve the rescue of recombinant ibv a cell line stably expressing the ibv n protein (bhk-21/n) was generated using a lentivirus-based system. the construction procedure is shown in fig. 2 . briefly, the complete n gene of ibv-yn was amplified by rt-pcr. the primers used were as follows: ibv-n-bamhi-f: ttaaggatc cgccac catg gcaagcagtaaggcat; ibv-n-not i-r: aaa agc ggc cgc tca aag ttc att ttc acc aag tg. two restriction sites, bamhi and noti, were introduced into the pcr product to digest and insert it into a plasmid with the hsv-tk promoter just upstream of the insertion site, and the obtained n gene plus tk promoter plasmid was designated ptk-n. then, the n gene plus tk promoter was amplified using the ptk-n plasmid as template with primers: tk-n-f-ecori: aacatacgctctcc atcaaaacaaaa; tk-n-r-bamhi: gatgcaatttcct cattttattag gaaag g. the psmpuw-sv40-puro plasmid was cut with enzymes ecori and bamhi and the fragment containing the n gene plus the tk promoter was inserted by an in-fusion reaction. after the n gene and tk promoter were inserted into the plasmid expression system psmpuw-sv40-puro (cell biolabs, san diego, ca, usa) ( fig. 2a) , the plasmid was cotransfected along with pcmv-dr8.74 and pmd2g-vsvg (cell biolabs, san diego, ca, usa)into 293 t cell lines to package the lentiviral particles. lentiviral vector production was performed according to standard protocols. briefly, subconfluent 293 t cells in a t25 flask were cotransfected with 5 μg of psmpuw-sv40-puro-n, 2 μg of pcmv-dr8.74, and 2 μg of pmd2g-vsvg using the transfection reagent xtremegene 9 (sigma-aldrich, st. louis, mo, usa). after 6 h, the medium was changed and the lentiviruses were harvested after 72 h and filtered through a 0.45 μm filter. stable transduction of the lentiviruses was carried out as follows: 10 6 of bhk-21 cells were seeded in a 6-well plate and transduced at day 2 with 10 6 of the lentiviral vector. selection was performed from day 3 using 2 μg/ml puromycin until the parental cells from a parallel experiment died (fig. 2b ). the expression of n protein in the bhk-21/n cell line was detected by immunofluorescence analysis (ifa). bhk-21/n cells were cultured in 6-well plates for 48 h at 37℃ and 5% co 2 , the medium was then y. zhao, et al. virus research 272 (2019) 197726 removed, and cells were fixed with 80% (v/v) cold acetone for 15 min in an ice bath. next, 500 μl of chicken polyclonal anti-ibv positive serum diluted 1:200 in pbs was added and the reaction was incubated for 1 h at 37℃. the cells were washed, stained with anti-chicken fitc-labelled conjugate (sigma) for 1 h at 37℃, and directly examined by fluorescence microscopy (olympus, tokyo, japan). parental bhk-21 cells were treated in the same way as a control. to compare the rescue efficiency of our lentivirus transduced bhk-21/n cell line with commonly used bhk-21 cells, we further constructed a recombinant vvibv strain (vvibv yn-5a/egfp) expressing the reporter genes enhanced green fluorescent protein (egfp), in which gene 5a was replaced with reporter genes egfp. 10 μg of yn-5a/egfp full-length rna was used for electroporation of 10 7 bhk-21/n cells or bhk-21 cells respectively. afte 48 h incubation at 37°c, the green fluorescence in both electroporated cell lines were observed under fluorescence microscopy. 2.6. in vitro transcription, transfection, and generation of the rescued virus ryn ibv-yn full-length rna transcript was generated in vitro using purified genomic dna isolated from recombinant vaccinia virus (vvibv-ryn) containing the 27.9-kb full-length ibv cdna, that was digested with noti and in vitro transcribed by the ribomax™ large scale rna production system-t7 kit (promega, madison, wi, usa) according to the manufacturer's instructions in brief, 10 μg of ibv-ryn full-length rna was used for electroporation of 10 7 bhk-21/n cells as described previously (eriksson et al., 2008) . the transfected bhk-21/n cells were incubated at 37°c for 48 h in dmem supplemented with 10% fbs. after 48 h, the virus-containing medium was collected and inoculated into six 10-day-old spf embryonated chicken eggs (ece). after incubation in ece at 37°c for 48 h, allantoic fluid from three ece was harvested for further rt-pcr detection. the other three eggs were kept at 37°c until 144 h post-inoculation to check for embryo curling caused by ibv. the allantoic fluid that cause embryo curling and rt-pcr detection positive was further passaged in ece for 10 times to get the stable virus stock (p10 virus stock) that reproducibly induced embryo curling. this stock was then used for further analyses, such as determination of the 50% egg infective dose (eid 50 ), the 50% tissue culture infective dose (tcid 50 ) as well as replication in vitro and in ovo. to differentiate the rescued viruses from the parental viruses, viral rnas were extracted from allantoic fluid (ryn and parental yn) as described above. rt-pcr was performed with selected primer pairs designed around the silent molecular markers that had been introduced into the ryn genome (5ʹ-gtgtgctatgtaagg tggtg-3ʹ, 5ʹ-ggagc atttttaactcgtagg-3ʹ), and the pcr product was digested to verify the existence of the sali silent nucleotide change at position 6555-6560. the pcr product was also sequenced for further confirmation. to determine eid 50 of ryn, serial 10-fold dilutions (10 −1 -10 -8 ) of the amplified virus were inoculated into 10-day-old spf ece via the allantoic sac route. for each dilution, 0.1 ml of virus solution was injected into each egg and five eggs were used for each dilution. inoculated eggs were incubated for 144 h at 37℃ to check for classical embryo curling caused by ibv. eggs that died within 24 h of inoculation were discarded. eggs that died between 24-144 h were considered as infected. the viral titer was also assessed by determining tcid 50 . for this procedure, chicken embryo kidney (cek) cells were prepared and seeded into 24-well plates two days before virus infection as previously described (hennion and hill, 2015) . serial 10-fold dilutions (10 −1 -10 -6 ) of the amplified virus were added onto 80% confluent cek cells, and each dilution of virus was inoculated into four replicate wells on 24well plates. after 48 h incubation at 37℃, 5% co 2 , the 24-well plates were assessed for ibv infection by an ifa assay as described above. the eid 50 and tcid 50 calculations were based on the reed and muench method (reed and muench, 1938) . to compare the in ovo replication of the rescued virus ryn and its parental strain yn in ece, 200 μl pbs containing 10 2.0 eid 50 of ryn or yn virus were inoculated into the allantoic cavities of 10-day old eces, and 200 μl allantoic fluid of five eggs from each group were harvested at the time points of 12, 24, 36, 48, 60 and 72 h post inoculation (hpi) and pooled for the real-time pcr detection assay. in brief, the total rna of 200 μl allantoic fluid were extracted using the viral rna isolation kit (foregene co., ltd, chengdu, china) according to the manufacturer's instructions. reverse transcription was conducted at 37 ℃ for 15 min, with 3 μg of total rna using the primescript™ rt reagent kit (takara bio inc, beijing, china). the cdna samples were submitted for sybr green i real-time rt-qpcr to detect the viral load for ibv n gene as described before (zhao et al., 2015) . all quantitative pcr reactions were carried out in triplicate and repeated at least twice, and the expression of ibv was calculated according to a standard curve. to compare the in vitro replication of the rescued virus ryn and its parental strain yn on cek cells, 200 μl pbs containing 10 3.0 tcid 50 of ryn or yn virus were inoculated onto the cek cells in 24-well plates, and 200 μl supernatants from three wells from each group were harvested at the time points of 6, 12, 24, 36, 48, and 60 hpi for a real-time pcr detection assay for ibv n gene as described above. the virus copy numbers for each time point were detected in triplicate and calculated according to a standard curve. unless otherwise stated, all data are represented as the mean values ± standard derivations obtained from experiments performed at least in triplicate. data were analyzed using graphpad prism 6 (graphpad software, san diego, ca, usa). the statistical significance of differences between groups was verified using one-way anova followed by bonferroni's multiple comparison tests. for all tests, the following notations are used to indicate significant differences between ryn and yn virus: * p < 0.05; ** p < 0.01. to construct a cdna clone covering the complete ibv-yn genome we generated ten overlapping dna fragments by rt-pcr from ibv-yn rna and chemical dna synthesis. the fragment corresponding to the ibv-yn 5′-end was engineered to contain a promoter for the t7 rna polymerase that was placed immediately upstream of the ibv-yn 5′terminal a nucleotide. the fragment corresponding to the ibv-yn 3′end contained a polya sequence of 25 nts followed by a noti restriction endonuclease recognition site. this strategy has been successful for other full-length coronavirus cdna clones (casais et al., 2001; van den worm et al., 2012; tekes, 2016) . it allows to generate an in vitro transcribed rna containing one additional g nucleotide at the 5′-end (required as transcription start site for the t7 rna polymerase) and a polya tail at the 3′-end (fig. 1a) . the full-length cdna was sequentially assembled into the vaccinia virus genome by six rounds of homogenous recombination using the e. coli guanine phosphoribosyltransferase as selection marker for positive or negative selection as appropriate (fig. 1b) . the integrity of the resulting vaccinia viruses was assessed by rt-pcr and sequencing analysis of the recombined regions after each recombination step. the final recombinant vaccinia virus, vvibv-ryn, containing the complete ibv-yn genome was deep sequenced and the result showed 100% identity with the parental virus genome, with the exception of three silent nucleotide substitutions, t3281a, a3284c, and y. zhao, et al. virus research 272 (2019) 197726 t6557c, which are diagnostic for the recombinant ibv genomic cdna (fig. 1a) . as demonstrated previously, the coronavirus n protein greatly facilitates the rescue of recombinant coronaviruses (yount et al., 2000; casais et al., 2001) . we therefore constructed a lentivirus-transduced bhk-21/n cell line that stably expresses the ibv-yn n protein. the complete n gene of yn strain was amplified by rt-pcr and cloned downstream of the hsv-tk promoter. this expression cassette was then inserted into the lentivirus vector psmpuw-sv40-puro and the resulting plasmid was used to generate lentiviral vector particles ( fig. 2a, b) . following lentivirus vector transduction and puromycin selection the resulting bhk-21/n cells were assessed for n protein expression. as shown in fig. 2c , the expression of ibv n protein was readily detected by ifa, whereas no specific fluorescence was observed in parental bhk-21 cells. expression of reporter genes by ibv has been previously described and used to facilitate the studies of virus replication and transcription in target cells (bentley et al., 2013) . in this study we replaced the accessory 5a gene by genes encoding the egfp, resulting in the recombinant viruses vvibv yn-5a/egfp (fig. 3a) . after electroporation of 10 μg of yn-5a/egfp full-length rna into 10 7 bhk-21/n cells or bhk-21 cells and incubation at 37℃ for 48 h, green fluorescence were observed in both electroporated cell lines under fluorescence microscopy, confirming the expression of gfp (fig. 3b ). however, we were able to detect cells displaying green fluorescence in bhk-21 cell lines, but only a very low number; the numbers of cells displaying green fluorescence in bhk-21/n cell lines were obviously higher than in bhk-21 cell lines, indicating a higher rescue efficiency by using the bhk-21/n cell lines. the recovery of infectious ibv-yn from cloned cdna was performed using a protocol that was successfully used for the recovery of recombinant hcov-229e . briefly, vvibv-ryn dna purified from virus particles was cleaved with noti and used as a template for in vitro transcription with the t7 rna polymerase in the presence of a cap analog. the rna transcripts (10 μg) of the ibv cdna were electroporated into 10 7 bhk-21/n cells, and the bhk-21/n cell supernatant was harvested after incubation at 37 ℃ in 5% co 2 for 48 h. the supernatant was subsequently inoculated into the allantoic cavities of ece for replication of the rescued virus. this strategy was chosen to facilitate the rescue procedure and to ensure that the recombinant ibv-ryn replicate efficiently in ovo. after 48 h incubation in the allantoic cavities, the recovery of ibv-ryn was confirmed by rt-pcr amplifying and sequencing the genomic region that contained the molecular markers to distinguish ibv-ryn from ibv-yn. as shown in fig. 4a, b , the presence of a silent mutation that gave rise to a sali restriction site at position t6557c was verified by restriction enzyme digestion as well as sequence analysis. this analysis showed that the pcr product of ibv-ryn can be digested into two segments corresponding to the mutation introduced into the genome, whereas the pcr product of parental yn strain was resistant to cleavage by sali (fig. 4a) . accordingly, the t6557c mutation was confirmed by sequencing analysis (fig. 4b) . collectively, these results demonstrate the successful rescue of the pathogenic ibv strain yn from cloned cdna by using electroporation of full-length ibv in vitro transcripts into n-protein expressing cells and subsequent virus amplification in the allantoic cavities of ece. ece that have been inoculated with supernatants of ibv-ryn rnaelectroporated bhk-21/n cells were also assessed for chicken embryo development, and as shown in fig. 5a , after only two passages in ece, typical embryo lesions such as curling, stunting, and dwarfing were observed. a similar phenotype was also observed when ece were inoculated with wild-type ibv-yn. to compare replication of ibv-ryn with the parental strain ibv-yn in ece, we first produced virus stocks that reproducibly induced embryo curling by passaging the viruses 10 times in ece. 100 μl of collected p10 ibv-ryn allantoic fluid stock or wild-type ibv-yn stock that has been proven to be positive for ibv has been serially diluted (10 −1 -10 -8 ) and inoculated into 10-day-old spf ece. five eggs were used for each dilution. after 144 h incubation at 37 ℃, inoculated eggs were evaluated for classical embryo curling caused by ibv and the eid 50 titer was calculated according to the reed and muench method (reed and muench, 1938) . ibv-yn and ibv-ryn were pathogenic to chicken embryos up to a dilution of 10 6 , and the eid 50 y. zhao, et al. virus research 272 (2019) 197726 titer of ibv-ryn (10 7.17 /100 μl) was comparable to that of its parental virus ibv-yn, (10 6.83 /100 μl). we also assessed the virus titers of the same virus stocks in cek cells to determine the tcid 50 . 100 μl of collected p10 ibv-ryn allantoic fluid stock or wild-type ibv-yn stock that has been proven to be positive for ibv has been serially diluted (10 −1 -10 -6 ) and added onto 80% confluent cek cells. four replicate wells on 24-well plates were used for each dilution. after 48 h incubation at 37℃, 5% co 2 , the 24-well plates were assessed for ibv infection by an ifa assay as described above. titers were determined by observing infected cells under a fluorescence microscope and calculating the tcid 50 per 0.1 ml. the tested tcid 50 titer of ryn was almost equal to that of yn at 10 3.33 and 10 3.00 , respectively. finally, we assessed growth kinetics of ibv-ryn and ibv-yn in eces and cek cells. for the replication profile comparison in ece, 200 μl pbs containing 10 2.0 eid 50 of ibv-ryn or ibv-yn were inoculated into the allantoic cavities of 10-day old eces, and the allantoic fluid of five eggs from each group were harvested at the time points of 12, 24, 36, 48, 60 and 72 hpi and pooled for the real-time pcr detection assay (zhao et al., 2015) . as shown in fig. 5b , the recombinant ibv-ryn replicated in eces with similar growth kinetics as the parental ibv-yn virus. although the ibv-ryn had a relatively low replication speed at the first 24 h, both viruses could reach the peak titers after 36 h and stay at a high level as long as 72 h. to assess the replication kinetics in cek cells, 200 μl pbs containing 10 3.0 tcid 50 of ibv-ryn or ibv-yn were used to infect cek cells in 24well plates, and the supernatants from three wells from each group y. zhao, et al. virus research 272 (2019) 197726 were harvested at the time points of 6, 12, 24, 36, 48, and 60 hpi. viral load was determined by a real-time pcr detection assay quantifying ibv n gene as previously described (zhao et al., 2015) . as shown in fig. 5c , growth kinetics and peak titers of the recombinant ibv-ryn were indistinguishable from those of ibv-yn (fig. 5c) . collectively, these results show that the recombinant ibv-ryn has the same in ovo and in vitro phenotype as the parental ibv-yn. in 2000, the first full-length infectious clone of coronavirus was successfully rescued using the bac system (almazán et al., 2000) . the advantage of the bac system is that it can accommodate larger foreign genes or dna fragments, and methods for modification and screening are well established (almazán et al., 2015) . nevertheless, for certain coronaviruses stability of full-length bac clones remained a problem. an alternative system was described in the same year by yount and colleagues (yount et al., 2000) . they assembled a series of smaller subclones into an intact full-length tgev cdna by in vitro ligation (yount et al., 2000) . viral mrna was obtained by in vitro transcription, and then electroporated into bhk21 cells to successfully rescue recombinant tgev. notably this study also revealed that expression of the n protein facilitates the rescue of recombinant coronaviruses. one year later a third reverse genetic system for coronaviruses was described that is based on the use of vaccinia virus as eukaryotic cloning vector. this system has been first described for the generation of recombinant hcov-229e and was subsequently applied to the generation of recombinant mouse hepatitis virus (mhv; coley et al., 2005) , ibv (casais et al., 2001) , feline infectious peritonitis virus (fipv; tekes et al., 2008) , and sars coronavirus (van den worm et al., 2012). as a large dna virus, vaccinia virus has the following advantages: 1) vaccinia virus itself can accommodate large fragments of foreign genes (at least the size of coronavirus genomes), and the virus' own infectivity and replication ability are not affected. 2) the inserted foreign dna can be stably maintained and amplified in the vaccnia virus genome without the need to rely on prokaryotic cloning systems. 3) vaccinia virus can be modified by homologous recombination, making it simple and easy to introduce mutations, insertions or deletions into cloned coronavirus genomes. several studies have employed the reverse genetic system of ibv to study pathogenicity, tissue tropism, and functionality of accessory genes, as well as to explore the potential for a vector-based vaccine of ibv (casais et al., 2003 armesto et al., 2009; bentley et al., 2013) . however, to the best of our knowledge, all existing reverse genetics systems for ibv were either based on the non-pathogenic beaudette strain (casais et al., 2001) or the mass-type attenuated vaccine strains (zhou et al., 2013; beurden et al., 2017) . while according to previous researches, the prevalent serotype of ibv circulating in china and many other countries is qx type (monne et al., 2008; zhao et al., 2016) , and the qx-type strains typically share < 80% nucleotide identity with mass-type strains. therefore, the application of currently available mass-type ibv reverse genetics systems cannot provide full insight into the immunogenicity and pathogenicity of qx-type strains. in this study, we successfully established a reverse genetic system based on a highly virulent qx-type (gi-19) strain yn isolated in china. we could show that the recombinant ibv-ryn replicates with the same kinetics, reaches the same peak titers, and displays comparable pathogenicity as the parental ibv-yn in eces. this indicates that this system will be a powerful tool to elucidate crucial viral and host factors that impact the high pathogenicity of qx-type ibv strains, and it may also serve as a starting point for the generation of novel vaccine candidates directed against ibv-qx-type strains. we also developed a modified rescue strategy by combining full length rna electroporation into a bhk-21/n cell line and subsequent virus amplification in ovo. this strategy was highly efficient for the rescue of field strains. by introduction of reporter gene egfp into the ibv-ryn genome, we could easily compare the rescue efficiency of bhk-21 and bhk-21/n cell lines. after electroporation with same amount ryn-egfp/5a full-length rna, bigger numbers of green fluorescent cells were observed in the bhk-21/n cell line than bhk-21 cell line, indicating the higher rescue efficiency of it. also, in our study, a remarkable difference of virus infectivity in ece (eid 50 could reach 10 6.0 -10 7.0 per 0.1 ml) was observed compared to cek cells (tcid 50 could only reach 10 3.0 per 0.1 ml) for ibv-yn, suggesting that ece are more susceptible to ibv infection and thus greatly facilitate the rescue of recombinant viruses. by combining electroporation of ibv rna into bhk-21/n cells and subsequent virus amplification in ece we could indeed reach a rescue efficiency of 100% and only one passage in ece was required for virus recovery, which makes the procedure for generating recombinant ibv field strains feasible and less labor intensive. in conclusion, we demonstrate the successful development of a vaccinia virus-based qx-type ibv reverse genetics system. this system allows for the generation of defined, genetically modified viruses to study ibv molecular biology and pathogenesis. we expect that the improved rescue procedure, involving rna transfection into bhk21/n cells and virus amplification in ece, is readily applicable to other ibv field strains, and may lead to the development of vaccines to prevent or control infections of newly emerging ibv field strains. the authors declare that they have no conflicts of interest. characterization and analysis of the full-length genome of a strain of the european qx-like genotype of infectious bronchitis virus engineering the largest rna virus genome as an infectious bacterial artificial chromosome engineering infectious cdnas of coronavirus as bacterial artificial chromosomes the replicase gene of avian coronavirus infectious bronchitis virus is a 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pathogenicity reveals major differences among qx-like infectious bronchitis viruses and other serotypes 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 safety and efficacy of an attenuated chinese qx-like infectious bronchitis virus strain as a candidate vaccine evolution of infectious bronchitis virus in china over the past two decades establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain h120 this study was supported by the national key research and development program of china (2017yfd0500700). we thank kate fox, dphil, from liwen bianji, edanz group china (www.liwenbianji. cn/ac), for editing the english text of a draft of this manuscript. key: cord-284866-66azyje4 authors: d’ andrea, lucía; pintó, rosa m.; bosch, albert; musto, héctor; cristina, juan title: a detailed comparative analysis on the overall codon usage patterns in hepatitis a virus date: 2011-02-04 journal: virus res doi: 10.1016/j.virusres.2011.01.012 sha: doc_id: 284866 cord_uid: 66azyje4 hepatitis a virus (hav) is a hepatotropic member of the family picornaviridae. hav has several unique biological characteristics that distinguish it from other members of this family. recent and previous studies revealed that codon usage plays a key role in hav replication and evolution. in this study, the patterns of synonymous codon usage in hav have been studied through multivariate statistical methods on 30 complete open reading frames (orfs) from the available 30 full-length hav sequences. effective number of codons (enc) indicates that the overall extent of codon usage bias in hav genomes is significant. the relative dinucleotide abundances suggest that codon usage in hav can also be strongly influenced by underlying biases in dinucleotide frequencies. these factors strongly correlated with the first major axis of correspondence analysis (coa) on relative synonymous codon usage (rscu). the distribution of the hav orfs along the plane defined by the first two major axes in coa showed that different genotypes are located at different places in the plane, suggesting that hav codon usage is also reflecting an evolutionary process. it has been very recently described that fine-tuning translation kinetics selection also contributes to codon usage bias of hav. the results of these studies suggest that hav genomic biases are the result of the co-evolution of genome composition, controlled translation kinetics and probably the ability to escape the antiviral cell responses. due to the degeneracy of the genetic code, most amino acids are coded by more than one codon (synonymous codon usage). these synonymous codons are not used randomly. rather, there are some codons that are used more frequently than others. mutational pressure and translational selection are thought to be among the main factors that account for codon usage variation among genes in different organisms (sharp and li, 1986a; karlin and mrazek, 1996; lesnik et al., 2000) . understanding the extent and causes of biases in codon usage is essential to the comprehension of viral evolution, particularly the interplay between viruses and the immune response (shackelton et al., 2006) . hepatitis a virus (hav) is a hepatotropic member of the family picornaviridae (wimmer and murdin, 1991) , and its viral genome consists of a 7.5-kilobase (kb), positive-stranded rna with a single open reading frame (orf). the orf, which codes 2227 amino acids is organized into three functional regions termed p1, p2 and p3. p1 encodes the capsid polypeptides vp1 to vp4, whereas p2 and p3 encode non-structural polypeptides. the orf is preceded by a 5 untranslated region (utr) and is followed by a 3 utr with a short poly a tail (hollinger and emerson, 2001) . the structure of hav, its tissue tropism, and genetic distance from other members of the family picornaviridae indicate that hav is unique within this family (martin and lemon, 2006; cristina and costa-mattioli, 2007) . hav has been shown to possess a single conserved immunogenic neutralization site, and isolates from different parts of the world belong to a single serotype (stapleton and lemon, 1987; hollinger and emerson, 2001) . nevertheless, the study of the hav evolution in cell culture revealed the presence of some antigenic variants in the mutant spectra that were generated even in the absence of immune selection (sanchez et al., 2003) . furthermore, several escape mutants, representing antigenic variants, have been selected for their resistance to different monoclonal antibodies (mabs), suggesting the occurrence of severe structural constraints in the hav capsid that prevent the more extensive substitutions necessary for the emergence of a new serotype (nainan et al., 1992; ping and lemon, 1992) . very recent in vitro studies have shown the occurrence of highly conserved clusters of rare codons in the hav capsid-coding region and that substitutions in these clusters are negatively selected, suggesting that the need to maintain such clusters play a role in the low antigenic variability of hav (aragones et al., 2008) . moreover, recent studies suggest that fine-tuning translation kinetics selection is also underlying codon usage bias in this specific genome region (aragones et al., 2010) . these results reveal that codon usage plays a key role in hav replication and evolution. however, our knowledge of other factors also contributing to shaping synonymous codon usage bias and nucleotide composition in human hav in vivo is rather scarce. in order to gain insight into these matters, we analyzed the codon usage and base composition of all available orfs from 30 human hav isolates, and investigated the possible key evolutionary determinants of codon usage bias. full-length orfs nucleotide sequences (corresponding to 2227 amino acids) were obtained for 30 human hav isolates by mean of the use of arsa at ddbj database (available at: http://arsa.ddbj.nig.ac.jp/) and embl database (available at: http://www.ebi.ac.uk/embl/access/index.html). for strain names, accession numbers, geographic location of isolation and genotypes, see supplementary material table 1 . in order to investigate the extent of codon usage bias in hav, we first aligned the complete orf code sequences from the hav strains, using the muscle program (edgar, 2004) . once aligned, the relative synonymous codon usage (rscu) values of each codon were determined in order to measure the synonymous codon usage (sharp and li, 1986b) . this was done using the codonw program (available at: http://mobyle.pasteur.fr). the rscu is the observed frequency of a codon divided by the frequency expected, if all synonymous codons for that amino acid were used equally. if rscu value is close to 1.0, it indicates a lack of bias (tsai et al., 2007) . rscu values are largely independent of amino acid composition and are particularly useful in comparing codon usage between genes that differ in size and amino acid composition. the rscu of hav orfs were compared with corresponding values of human cells (international human genome sequencing consortium, 2001). the effective number of codons (enc) and the frequency of use of gc 3 s (g+c at synonymous variable third position codons, excluding met, trp, and termination codons) were also calculated by the use of the codon w program. enc was used to quantify the codon usage bias of an orf (wrigth, 1990) , which is one of the best overall estimator of absolute synonymous codon usage bias (comeron and aguade, 1998) . the enc values range from 20 to 61. the larger the extent of codon bias in a gene, the smaller the enc value is. in an extremely biased gene where only one codon is used for each amino acid, this value would be 20; in an unbiased gene, it would be 61. similarly, the fraction of the g+c nucleotides not involved in the gc 3 s fraction (gc 12 ) was also calculated. all these indices were also calculated using the codon w program. the relative frequencies of dinucleotides were also calculated using this program as implemented in the mobile server (http://mobyle.pasteur.fr). coa is an ordination technique that identifies the major trends in the variation of the data and distributes genes along continuous axes in accordance with these trends. coa creates a series of orthogonal axes to identify trends that explain the data variation, with each subsequent axis explaining a decreasing amount of the variation (greenacre, 1984) . each orf is represented as a 59dimensional and each dimension is related to the rscu value of each triplet (excluding aug, ugg and stop codons). this was done using the codonw program. correlation analysis was carried out using spearman's rank correlation analysis method (wessa, 2010 ; available at: www.wessa.net). in order to study the extent of codon bias in hav orfs, the average codon usage values for all triplets were calculated. the results of these studies are shown in table 1 . interestingly, the frequencies of codon usage in hav orfs are significantly different than the ones used by human cells. particularly, extremely highly biased codon frequencies were found for phe, his, asn, asp, cys and arg (see table 1 ). almost all extremely high preferred codons were u-ended (see table 1 ). table 2 . due to the fact that almost all enc values are less than 40, the results obtained for the hav orfs studied reveal that codon usage in hav is biased. in order to investigate the patterns of synonymous codon usage, the correlations between the positions of the orfs along the first principal axis generated by the coa and the respective gc 3 s and gc 12 values of each strain were analyzed. the first principal axis in coa accounts for 45.34% of the total variation, while the next three principal axes in account for 10.14%, 8.63% and 6.56% of the variability, respectively. the first axis in coa is highly correlated with the gc 3 s and gc 12 values in hav orfs. this result reveals that nucleotide composition plays an important key role in the codon usage bias observed in hav orfs (see table 2 ). in order to detect the possibility of codon usage variation of different hav genomes, the hav orfs were divided according to their hav genotype (ia, ib, iia, iib, iiia and iiib). coa was performed on the rscu values of each hav orf and the distribution of the six genotypes along the first two principal axes of coa was determined. the results of these studies are shown in fig. 1 . surprisingly, the distribution of the six genetic groups in the plane defined by the first two major axes showed that different genotypes were located at different places, suggesting that different hav genotypes exhibit differences in their codon usage patterns (see fig. 1 ). in order to gain insight into these findings, the average codon usage values for all codons were calculated for genotype ia and iiia strains enrolled in these studies, accounting for 24,416 and 31,108 codons, respectively. the results of these studies are shown in supplementary material table 3 . interestingly, the frequencies of codon usage in the different hav genotypes show significant different frequencies in cca (pro) and cgc (arg) codons. in order to observe if different frequencies of codon usage are found in different hav genome regions, the same studies were repeated for the structural (p1) and non-structural regions (p2 + p3) of the hav genome. the results of these studies are shown in supplementary material table 4 . roughly similar values are obtained for both regions for most codons, although significant differences were found for arg codons (cgc and agg). it has been suggested that dinucleotide biases can affect codon bias (tao et al., 2009) . to study the possible effect of dinucleotide composition on codon usage of the hav orfs, the relative abundances of the 16 dinucleotides in the orfs of the 30 hav strains were established. the results of these analyses are shown in table 3 . the occurrences of dinucleotides are not randomly distributed and no dinucleotides were present at the expected frequencies (table 3 ). the relative abundance of cpg showed a strong deviation from the "normal range" (mean ± sd = 0.063 ± 0.009) and was markedly underrepresented. on the other had, the frequency of upu was above the expected value (mean ± sd = 1.891 ± 0.051) ( table 3) . among the 16 dinucleotides, 14 are highly correlated with the first axis value in coa (table 4 ). these observations indicate that the composition of dinucleotides also plays a key role in the variation found in synonymous codon usage among hav orfs. to study the possible effects of cpg under-representation on codon usage bias of hav orfs, the rscu value of the eight codons that contain cpg (ccg, gcg, ucg, acg, cgc, cgg, cgu, cga) were analyzed. these eight codons [ccg (mean 0.05), gcg (mean 0.03), ucg (mean 0.11), acg (mean 0.10), cgc (mean 0.09), cgg (mean 0.03) and cgu (mean 0.23), gcc (mean 0.13)] were markedly suppressed. besides, the position of each codon in each of the four major axes of coa was determined for the 30 hav orfs. table 5 shows the codons for which the maximum and minimum values were obtained for each of the axes studied (i.e. the most divergent codons values), indicating bias in their use by hav. as it can be seen in the table, most of the divergent codons were triplets coding for arg. in order to observe if dinucleotides frequencies may vary among different genotypes, the same studies were repeated using genotype ia and iiia strains. the results of these studies are shown in supplementary material table 5 . no significant differences were observed among the two genotypes or using all 30 hav strains representing all known hav genotypes. a similar study conducted in order to study dinucleotide frequencies in structural and nonstructural regions of the hav genome also found no significant differences among the different regions of the hav genome (see supplementary material table 6 ). the results of these studies revealed that codon usage in hav orfs is quite different from that of human genes (see table 1 ). this is in agreement with previous results found for the capsid structural region of hav (sanchez et al., 2003) . in other members of the family picornaviridae, like poliovirus or foot-and-mouth disease virus (fmdv) the codon usage is very similar to that of their hosts, implying competence for trnas among virus and host (sanchez et al., 2003) . in these cases, competition is avoided by the induction of cellular shutoff of protein synthesis through carboxy cleavage of translation initiation factor 4g (eif4g) by 2a and l proteases, respectively (racaniello, 2001) . hav lacks mechanisms of inducing cellular shutoff and needs an intact eif4g factor for the initiation of translation (racaniello, 2001; ali et al., 2001) . moreover, hav has a very inefficient ires (whetter et al., 1994) . for these reasons, hav may be able to synthesize its proteins by adapting their codon usage to those less commonly used cellular trnas. this may also account for its low replicative rate (pinto et al., 2007; moratorio et al., 2007) . in this study, we analyzed synonymous codon usage and nucleotide compositional constraints in hav orfs. interestingly, contrary to previous results found for other viruses such h5n1 influenza a virus (mean enc = 50.91) (ahn et al., 2006; zhou et al., 2005) ; sars (mean enc = 48.99) (zhao et al., 2008) ; footand-mouth disease virus (mean enc = 51.42) (zhong et al., 2007) ; classical swine fever virus (mean enc = 51.7) (tao et al., 2009 ) and duck enteritis virus (mean enc = 52.17) (jia et al., 2009) , the enc values found for human hav are comparatively low (mean enc value of 39.78), indicating that the overall extent of codon usage bias in hav is significant. this is in agreement with recent in vitro studies on hav capsid variability constraints (aragones et al., 2008) . a general correlation between codon usage bias and base composition was observed in these studies. moreover, highly significant correlations between the first axis of coa and gc 3 s and gc 12 values were obtained for all hav orfs studied. these results suggest that mutational pressure significantly contributes to the codon usage bias in hav strains. nevertheless, as previously suggested for other viral systems, when significant distance among expected and actual enc values are found, other factors additional to mutational bias may be also contributing to codon usage bias (shackelton fig. 1 . positions of the 30 hav orfs in the plot of the first two major axes by correspondence analysis (coa) of relative synonymous codon usage (rscu) values. the first and second axes account for 45.34% and 10.14% of the total variation, respectively. the hav orfs are divided according to their hav genotype, genotype ia strains are indicated by a white circle ( ), genotype ib by a white square ( ), genotype iia by a black circle (᭹), genotype iib by a black square ( ), genotype iiia by a black diamond ( ) and genotype iiib by a black triangle ( ). , 2006) . this is in agreement with very recent studies on hav populations adapted to propagate in cells with impaired protein synthesis in which fine-tuning translation kinetics selection rather than translation selection was identified as the underlying mechanism of codon usage bias in the capsid coding region (aragones et al., 2010) . thus, both mutation pressure, as well as selection pressure for correct protein folding, play a critical role shaping hav codon usage, indicating that hav genomic bias is multi-factorial. in order to detect possible codon usage variation of different genomes, the hav orfs were divided according to their genotype. unexpectedly, the distribution of the six genetic groups along the first two major axes in coa showed that different genotypes are distantly located in the plane defined by the first two axes of the analysis (fig. 1) . moreover, the frequencies of codon usage of genotype ia and iiia showed significant differences in pro and arg codons (see supplementary material table 3 ). since species with a close genetic relationship always present a similar codon usage pattern (sharp et al., 1988) , these findings suggest that codon usage in hav is undergoing also an evolutionary process, probably reflecting a dynamic process of mutation and selection to re-adapt its codon usage to different environments (see fig. 1 material 3). the structural and non-structural regions of the genome roughly share the same frequencies of codon usage, except for some of the arg codons (see supplementary material table 4 ). this is in agreement with coa analysis, were most of the divergent codons were triplets coding for arg (see table 4 ). this reveals that the use of arg codons plays also a role in the evolution and the variability observed among hav strains. the frequencies of occurrence for dinucleotides were not randomly distributed and most dinucleotides did not follow the expected frequencies in hav orfs (table 3 ). the high correlation found between the first axis of coa and the relative dinucleotide abundances (table 4) suggests that codon usage in hav orfs can also be strongly influenced by underlying biases in dinucleotide frequencies. all cpg containing codons are markedly suppressed (table 3) in the 30 hav strains included in the study, confirming what has been very recently noted . marked cpg deficiency has been also observed in coronaviruses (woo et al., 2007) , vertebrate-infecting members of the family flaviviridae (lobo et al., 2009) , poliovirus (rothberg and wimmer, 1981) and other rna viruses (karlin et al., 1994) . moreover, polioviruses synthetically deoptimized either by codon deoptimization or codon pair deoptimization are generally marked by a higher content of cpg (and also upa) dinucleotide (burns et al., 2006 (burns et al., , 2009 mueller et al., 2006; coleman et al., 2008) , indicating that polioviruses have naturally evolved to eliminate these dinucleotides. cpg deficiency was proposed to be related to the immunostimulatory properties of unmethylated cpg, which were recognized by the host's innate immune system as a pathogen signature (shackelton et al., 2006; woo et al., 2007) . escaping from the host antiviral response may act as another selective pressure contributing to the multifactorial codon usage shaping (vetsigian and goldenfeld, 2009 ). thus, the results of these studies suggest that hav genomic biases are the result from the coevolution of genome composition, the need to a controlled translation kinetics and probably the need to escape the antiviral cell responses, and thus is a model in agreement with the evolution rhetoric theory proposed by vetsigian and goldenfeld (2009) in which genome biases emerge by the need to increase communication with the ever changing cell environment without changing the message. genomic analysis of influenza a viruses, including avian flu (h5n1) strains activity of the hepatitis a virus ires requires association between the cap-binding translation initiation factor (eif4e) and eif4g fine-tuning translation kinetics selection as the driving force of codon usage bias in the hepatitis a virus capsid hepatitis a virus mutant spectra under the selective pressure of monoclonal antibodies: codon usage constraints limit capsid variability coding biases and viral fitness genetic inactivation of poliovirus infectivity by increasing the frequencies of cpg and upa dinucleotides within and across synonymous capsid region codons modulation of poliovirus replicative fitness in hela cells by deoptimization of synonymous codon usage in the capsid region virus attenuation by genome-scale changes in codon pair bias an evaluation of measures of synonymous codon usage 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neutralization site on hepatitis a virus analysis of synonymous codon usage in classical swine fever virus analysis of codon usage bias and base compositional constraints in iridovirus genomes genome rhetoric and the emergence of compositional bias free statistics software, office for research development and education, version 1.1.23-r5 low efficiency of the 5 nontranslated region of hepatitis a virus rna in directing cap-independent translation in permissive monkey kidney cells hepatitis a and the molecular biology of picornaviruses: a case for a new genus of the family picornaviridae cytosine deamination and selection of cpg suppressed clones are the two major independent biological forces that shape codon usage bias in coronaviruses the "effective number of codons" used in a gene analysis of synonymous codon usage in 11 human bocavirus isolates mutation pressures shapes codon usage in the gc-rich genome of foot-and-mouth disease virus analysis of synonymous codon usage in h5n1 virus and other influenza a viruses authors acknowledge support by pedeciba and agencia nacional de investigación e innovación (anii, fce 2007 722), uruguay. we acknowledge anonymous reviewers for important suggestions regarding this work. supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2011.01.012. key: cord-275182-cmjfqkjz authors: cruz, jazmina l.g.; zúñiga, sonia; bécares, martina; sola, isabel; ceriani, juan e.; juanola, sandra; plana, juan; enjuanes, luis title: vectored vaccines to protect against prrsv date: 2010-06-25 journal: virus res doi: 10.1016/j.virusres.2010.06.017 sha: doc_id: 275182 cord_uid: cmjfqkjz prrsv is the causative agent of the most important infectious disease affecting swine herds worldwide, producing great economic losses. commercially available vaccines are only partially effective in protection against prrsv. moreover, modified live vaccines may allow virus shedding, and could revert generating virulent phenotypes. therefore, new efficient vaccines are required. vaccines based on recombinant virus genomes (virus vectored vaccines) against prrsv could represent a safe alternative for the generation of modified live vaccines. in this paper, current vectored vaccines to protect against prrsv are revised, including those based on pseudorabies virus, poxvirus, adenovirus, and virus replicons. special attention has been provided to the use of transmissible gastroenteritis virus (tgev) as vector for the expression of prrsv antigens. this vector has the capability of expressing high levels of heterologous genes, is a potent interferon-α inducer, and presents antigens in mucosal surfaces, eliciting both secretory and systemic immunity. a tgev derived vector (rtgev) was generated, expressing prrsv wild type or modified gp5 and m proteins, described as the main inducers of neutralizing antibodies and cellular immune response, respectively. protection experiments showed that vaccinated animals developed a faster and stronger humoral immune response than the non-vaccinated ones. partial protection in challenged animals was observed, as vaccinated pigs showed decreased lung damage when compared with the non-vaccinated ones. nevertheless, the level of neutralizing antibodies was low, what may explain the limited protection observed. several strategies are proposed to improve current rtgev vectors expressing prrsv antigens. prrsv is the causative agent of the most important infectious disease affecting the porcine herds worldwide. the immune response to prrsv is poorly understood but, in spite of this, some vaccines are being commercialized. commercial vaccines are mostly modified live vaccines based on attenuated european or north american prrsv strains (i.e., ingelvac ® -prrs from boehringer ingelheim, amervac ® -prrs from hipra, or pyrsvac-183 from syva labs). nevertheless, some inactivated vaccines are also available (i.e., progressis ® from merial, ingelvac ® -prrs kv from boehringer ingelheim, or suipravac ® -prrs from hipra). modified live vaccines have been preferentially used, as they can establish protective immunity, measured by viral load in blood and tissues. nevertheless, current vaccines against prrsv have several limitations. in general, modified live vaccines protect against challenge with homologous isolates. they could also protect against heterologous viruses (diaz et al., 2006; zuckermann et al., 2007) . furthermore, live vaccines provide partial protection against clinical disease but did not prevent infection (osorio et al., 1998) and, more importantly, they can revert to virulence (botner et al., 1997; nielsen et al., 2001) . as the attenuated vaccines induce an immune response resembling that induced by prrsv natural infection, they do not induce high levels of neutralizing antibodies. killed prrsv vaccines, on the other hand, in general, have been less effective in prevention of both infection and disease (ostrowski et al., 2002) . the innate immune response against prrsv is very weak, probably contributing to the delay in subsequent humoral and cellular immune responses, and also to virus persistence (kimman et al., 2009 ). prrsv does not induce interferon (ifn)-␣ production (albina et al., 1998; calzada-nova et al., 2010) , a key element in host antiviral response, leading to a minimal production of inflammatory cytokines and activation and recruitment of natural killer (nk) cells (murtaugh et al., 2002) . prrsv-induced suppression of type i ifn production is due to the interference in the activation of ifn-␤ promoter stimulator 1 (ips-1), located downstream of sensor molecule rna helicase rig-i. the inactivation of ips-1 avoids ifn regulatory factor (irf) 3 activation and, consequently, type i ifn production (luo et al., 2008) . therefore, to design an effective vaccine against prrsv, it would be advisable to increase the production of type i ifn. to date, different adjuvants promoting the production of ifn have been tested, in addition to the current vaccines formulations, with limited success (charerntantanakul, 2009) . a hallmark of the swine humoral response against prrsv is the production of non-neutralizing antibodies detected early in the infection, followed by a low neutralizing antibody (nab) titer that is detected more than 3 weeks after infection (kimman et al., 2009; murtaugh et al., 2002) . one possible explanation for the late detection of nabs is the difference on technique sensitivity, as elisa has higher sensitivity than neutralization assays. therefore, the presence of very low titers of nabs early in the infection cannot be completely discarded. early non-neutralizing antibodies are mainly induced by nucleocapsid (n), m and gp5 proteins, and have been involved in antibody-dependent enhancement of prrsv infection (mateu and diaz, 2008; murtaugh et al., 2002) . nabs are induced by gp3, gp4, gp5 and m proteins, although the ones recognizing gp5 are the most relevant for protection (kim and yoon, 2008; ostrowski et al., 2002) . two b cell epitopes were identified in gp5 protein ectodomain: an immunodominant epitope (ide), that has been proposed to act as a decoy epitope, and an epitope critical for neutralization (ecn), that is recognized by nabs (ostrowski et al., 2002) . several hypothesis have been proposed to explain the delay in nabs induction by gp5 protein, such as the presence of the ide, and glican-shielding of the ecn (lopez and osorio, 2004) . the role of nabs in protection was demonstrated by passive transfer of these antibodies . protection of swine against prrsv infection correlated with the level of nabs and it was proposed that an efficient vaccine must induce nab titers of 1:32 to prevent prrsv infection (lopez et al., 2007) . prrsv infection results in a weak and delayed t cell mediated immune response that should be necessary for the elimination of the virus (mateu and diaz, 2008; murtaugh et al., 2002) . it has been shown that the induction of ifn-␥ secreting cells, complementing neutralizing antibodies, provides partial protection against prrsv (zuckermann et al., 2007) . as interleukin (il)-10 levels inversely correlate with ifn-␥ response, it has been proposed that the expression of il-10 may be responsible for the suppression of t cell responses (charerntantanakul et al., 2006; kimman et al., 2009 ). m protein is the most potent inducer of t cell proliferation, followed by gp5, gp3 and gp2 (bautista et al., 1999) , and may play a role in protection. different vaccine adjuvants have been tested to improve t cell responses to prrsv. nevertheless, in addition to the adjuvants included in vaccine formulation, only il-2 and cpg oligodeoxynucleotides enhanced protection conferred by current vaccines (charerntantanakul, 2009 ). there are three main problems for the development of more efficient vaccines against prrsv: the correlates of protection are not well known, prrsv may induce negative regulatory signals for the immune system, and there is a extremely large antigenic variability in prrsv structural proteins. as indicated above, the prrsv heterodimer gp5-m must be the main inducer of protective humoral and cellular responses. nevertheless, minor structural proteins are also required for prrsv virion infectivity (wissink et al., 2005) and may play a role in protection. also, there is limited information about the t cell epitopes implicated in the induction of a protective t cell response (mateu and diaz, 2008) . one of the mechanisms used by viruses to suppress or evade the host immune response is the induction of regulatory t cells (treg). porcine treg phenotype is cd4 + cd25 + foxp3 + (kaser et al., 2008) , as that described for human and mice (belkaid, 2007) . tregs have been classified in natural and induced. the latter ones can be subdivided in three subtypes: treg1 (tr1) secreting il-10, t helper 3 (th3) secreting transforming growth factor (tgf)-␤, and converted tregs (belkaid, 2007) . it has been recently described that american type prrsv-infected dendritic cells induced tregs, an effect that was reverted by the addition of ifn-␣. the induced treg population is a th3 type, as it promotes tgf-␤ but not il-10 expression (silva-campa et al., 2009) . in contrast, dendritic cells infected with eu type prrsv viruses did not induce treg cells, although they exhibited an unbalanced ability to stimulate t cell immune responses (silva-campa et al., 2010) . the impact of treg induction on delayed immune responses after prrsv infection remains to be established, as well as the viral proteins involved in this process. prrsv strains are extremely diverse, even when they belong to the same genotype. among the structural proteins, m protein is the most conserved one, while gp5 is the most variable one (dea et al., 2000) . this high antigenic variability represents a problem for the development of universal vaccines against prrsv, as shown by the low efficacy of current vaccines against heterologous challenge. to solve this problem, common critical b and t cell epitopes must be identified (mateu and diaz, 2008) . nevertheless, it has been reported that the ability of a vaccine to induce a strong cellular immune response may be more important for protection than the genetic similarity with the challenge strain (diaz et al., 2006) . an additional problem is the difference in the immune responses elicited by prrsv in animals with different host genetic background (lewis et al., 2007) . therefore, the knowledge of host responses to prrsv infection is required for the development of an efficient vaccine. as mentioned above, both modified live and inactivated vaccines have been developed for prrsv. live vaccines led to better results than killed-virus based vaccines. nevertheless, the live attenuated vaccines have several problems such as incomplete protection, virus shedding and possible reversion to virulence (kimman et al., 2009 ). this problem was increased by the use of potentially hazardous methods to control the disease, such as the use of live field virus to vaccinate pigs. vector-based vaccines could represent an advantage to stimulate both humoral and cell immune responses against prrsv, and for the design of a marker vaccine. nevertheless, the results reported to date using viral vectors are not fully satisfactory and new vectors, or antigenic combinations, must be explored. prv, also known as aujeszky's disease virus (adv) is an alphaherpesvirus, classified within the family herpesviridae. prv is the causing agent of pseudorabies that was a worldwide-spread economically important disease. swine is the natural host of prv, but the virus also infects a broad range of vertebrates, including farm animals (pomeranz et al., 2005) . in order to eradicate the virus, modified live vaccines have been successfully used. all the vaccine strains were ge − phenotype, i.e., have a ge gene deletion. the elimination of ge causes virus attenuation by reducing the virus transmission, but does not reduce virus production in cell culture nor the induction of protective immunity (nauwynck et al., 2007) . these live attenuated prv have been used as vectors to protect against swine infectious diseases, such as classical swine fever (hooft van iddekinge et al., 1996) , or porcine circovirus (ju et al., 2005) . a recombinant prv, based on the attenuated bartha strain, was constructed expressing prrsv gp5 protein (qiu et al., 2005) . protection was evaluated by inoculation of 4-week-old piglets and homologous challenge with prrsv ch-1 strain. none of the animals, even those inoculated with a commercially available inactivated vaccine, developed anti-gp5 antibodies before challenge. after challenge, the production of anti-gp5 antibodies was detected in all animals. nevertheless, none of them produced neutralizing antibodies against prrsv (qiu et al., 2005) . reduced lung lesions and viremia, and faster virus elimination from tissues was observed in animals inoculated with prv vaccine vector, similar to that found in animals inoculated with the commercial vaccine (qiu et al., 2005) . alternative recombinant attenuated prv vaccine vectors expressing different combinations of prrsv antigens have also been generated. these vectors expressed gp5 alone or together with m protein, or modified gp5 (gp5m), containing a pan dr thelper cell epitope (padre) between the decoy epitope and the ecn, recognized by nabs (fang et al., 2006) , alone or co-expressed with m protein. the gp5-m heterodimer was detected in the recombinant prvs co-expressing both proteins, suggesting that prrsv antigenic structures were not changed (jiang et al., 2007c) . the prv co-expressing gp5m and m proteins was the most promising candidate in the induction of neutralizing antibodies and lymphocyte proliferation, as tested in the mouse model. as a consequence, the protection conferred by this vector was evaluated in the porcine respiratory model, in relation to the protection provided by a commercially available prrsv killed vaccine. animals inoculated with the recombinant prv expressing prrsv proteins developed nabs before the challenge. furthermore, after challenge, the nab titer was up to 4-fold higher in animals inoculated with recombinant prrsv compared with those inoculated with the killed vaccine. none of the animals inoculated with the empty prv developed neutralizing antibodies at any time during the experiment (jiang et al., 2007c) . lymphocyte proliferative responses were also higher in animals inoculated with the recombinant prv expressing gp5m and m proteins. accordingly, lung lesions and viremia were lower in these animals, indicating a certain protection against the homologous challenge (jiang et al., 2007c) . adenoviruses are currently one of the most extended systems for gene delivery. as vectors, they have high capacity for the insertion of foreign genes (from 5 kb up to 36 kb, depending on the system), and are able to transduce a broad range of cell types (bantounas and uney, 2007) . different replication-defective recombinant adenoviruses (rad) have been used as vectors for prrsv, both for vaccine development and for analysis of immunogenic properties of prrsv wt or modified structural proteins. a set of rads expressing prrsv gp5, m and a m-gly-thr-thr-gp5 fusion protein were generated. these rads were tested in the mouse model. the rad expressing m-gp5 fusion protein induced and increased neutralizing antibodies humoral immune response, compared with mice inoculated with rad expressing gp5 and m proteins independently or empty adenovirus vector . the rad expressing m-gp5 fusion protein also induced enhanced lymphocyte proliferation and cytotoxic t-lymphocyte (ctl) responses . unfortunately, protection conferred by these vectors was not evaluated in the porcine system. the same authors also generated a set of rads expressing other prrsv structural protein combinations, such as gp3, gp4 or gp5 alone, and gp3-gp5, gp4-gp5 or gp3-gp4-gp5 fusion proteins . mice inoculated with rads expressing fusion proteins developed higher nab titers and lymphocyte proliferation responses than those inoculated with rads expressing independent prrsv proteins. interestingly, specific ctl responses were higher in mice inoculated with rads expressing gp3-gp5 or gp3-gp4-gp5 fusion proteins . in fact, authors selected the recombinant rad gp3-gp5 as the best vaccine candidate for testing protection in pigs. this recombinant induces nabs in vaccinated piglets before challenge, and higher lymphocyte proliferation responses, il-4 and ifn-␥ production. nevertheless, the rad expressing gp3-gp5 fusion protein did not fully protect against homologous challenge, as only a moderate decrease in lung lesions and viremia was observed wang et al., 2009) . to improve the efficacy of the rad-based vaccine, heat shock protein (hsp) 70 and granulocyte-macrophage colony stimulating factor (gm-csf) were co-expressed as genetic adjuvants wang et al., 2009) . a set of rads was obtained, expressing hsp70-5xgly-gp3-gp5 and hsp70-2a-gp3-gp5 fusion proteins, with a five glycine or a 2a protease linker, respectively. piglets inoculated with rad expressing fusion proteins induced higher nab titers, produced higher ifn-␥ levels, and presented reduced lung lesions, than those inoculated with rad expressing gp3-gp5 protein . introduction of 2a protease between the hsp70 and prrsv fusion protein resulted in a better production of il-4 by inoculated animals, and also lower viremia. this could be due to the release of native hsp70 with higher adjuvant activity . gm-csf has been widely used as an effective mucosal adjuvant (toka et al., 2004) . intranasal inoculation of vectors expressing gm-csf stimulates ifn-␥ and il-12 production in lung tissues (bukreyev et al., 2001) . a rad expressing gm-csf-leu-glu-gp3-lys-leu-gp5 fusion protein was generated. a moderate increase in nab levels was observed before challenge in piglets inoculated with this rad vector, compared with animals inoculated with empty rad or rad expressing gp3-gp5 fusion protein alone. after challenge, animals inoculated with rad expressing the fusion protein containing the adjuvant developed significantly higher nabs than the control animals . lymphocyte proliferation responses and ifn-␥ and il-4 production were also enhanced in those animals. these enhanced immune responses correlated with a significant decrease in the viremia and lung lesions, indicating that gm-csf enhanced the immunogenicity of rad-based gp3-gp5 vaccine . adenovirus vectors have also been used to evaluate the antigenicity of prrsv structural proteins, such as gp3 and gp5, and the role of gp5 glycosylation on immune responses (jiang et al., 2007a,b) . unfortunately, these studies have been performed using the mouse model. the effect of ifn-␣ for protection against prrsv has recently been analyzed using rad. piglets were inoculated with a rad expressing porcine ifn-␣ and challenged with prrsv (brockmeier et al., 2009) . results obtained indicate that the presence of ifn-␣ has a moderate protective effect against prrsv infection. poxviruses are the largest known animal dna viruses. they have been extensively used as expression vectors for vaccination, allow expression of large foreign genes, induce strong cell mediated and humoral immune responses, and safe poxvirus vectors are available (paoletti, 1996; wang et al., 2007) . fowlpox was the first poxvirus used as vaccine vector for prrsv (guoshun et al., 2007) . fowlpox virus (fpv) belongs to the avipoxvirus genus, and its replication is restricted to avian species. nevertheless, attenuated strains of fpv have been used as vectors for poultry and mammals, resulting in strong and protective immune responses (paoletti, 1996; wang et al., 2007) . a gp5-pro-pro-ser-gp3 fusion protein, alone or combined with porcine il-18, was expressed using recombinant fpv. piglets inoculated with recombinant fpv expressing prrsv antigens induced neutralizing antibodies at 42 dpi, and higher lymphocyte proliferation response, than those inoculated with the empty vector (guoshun et al., 2007) . vaccinated animals also showed increased ifn-␥ production, compared with non-vaccinated ones. piglets vaccinated with fpv co-expressing prrsv antigens and il-18 produced higher ifn-␥ amount than those inoculated with fpv expressing gp5-gp3 fusion protein alone (guoshun et al., 2007) . partial protection was also observed after challenge with an homologous strain, as viremia was decreased in vaccinated animals (guoshun et al., 2007) . modified vaccinia virus ankara (mva), a member of the orthopoxvirus genus, has also been used as a vector for prrsv (zheng et al., 2007) . mva was used as the vaccine agent for the prevention of smallpox, and has been extensively used as viral vector for infectious diseases and cancer. mva is highly attenuated, even in immunosuppressed animals, but induces strong humoral and cellular immune responses (wang et al., 2007) . four recombinant mva viruses expressing prrsv antigens were constructed, expressing gp5 or m proteins alone, gp5-m fusion protein, or coexpressing gp5 and m proteins (zheng et al., 2007) . these vectors were tested in the mouse model. mice inoculated with recombinant mva expressing heterologous antigens developed prrsv neutralizing antibodies, with the highest antibody titers found in mice inoculated with the recombinant mva co-expressing gp5 and m proteins. similar results were obtained when ifn-␥ and il-2 production was analyzed, indicating a th1 type cellular immune response (zheng et al., 2007) . unfortunately, authors did not perform protection experiments in piglets. therefore, the usefulness of mva as vector for prrsv vaccination remains to be determined. expression vectors have been engineered using different viral replicons, by replacing the virus structural genes by heterologous ones. these rna vectors, or replicons, express high levels of the foreign proteins, and replicate but are not packaged into virus-like particles unless structural proteins are provided in trans. therefore, replicons do not spread into neighbor cells and are safe for their use as vaccines (nagai et al., 2007; rayner et al., 2002) . an alphavirus replicon derived from venezuelan equine encephalitis virus (veev) has been successfully used as a vaccine against different pathogens, including swine influenza . veev-derived vaccines induce robust humoral, mucosal and cellular immunity (rayner et al., 2002) . it has been recently described that a veev replicon expressing prrsv gp5 and m proteins reduced viremia after prrsv challenge and provides partial protection (mogler et al., 2008 . replicons from classical swine fever virus (csfv) and vesicular stomatitis virus (vsv) expressing gp5 and m proteins have also been generated, expressing high levels of prrsv antigens (n. ruggli, personal communication). coronaviruses have several advantages as vectors over other viral expression systems: (i) they are single-stranded rna viruses that replicate in the cytoplasm without a dna intermediary, making integration of the virus genome into the host cell chromosome unlikely (lai and cavanagh, 1997) ; (ii) these viruses have the largest rna virus genome and, in principle, have room for the insertion of large foreign genes (enjuanes et al., 2001 (enjuanes et al., , 2005 ; (iii) a pleiotropic secretory immune response is best induced by the stimulation of gut associated lymphoid tissues. since coronaviruses in general infect both respiratory and enteric mucosal surfaces, these viruses may be used to target the antigen to the enteric and respiratory areas to induce a strong secretory immune response; (iv) the tropism of coronaviruses may be engineered by modifying the s gene (ballesteros et al., 1997; kuo et al., 2000; sanchez et al., 1999) ; (v) non-pathogenic coronavirus strains infecting most species of interest (human, porcine, bovine, canine, feline, and avian) are available and therefore are suitable to develop safe virus vectors; and (vi) infectious coronavirus cdna clones are available to design expression systems. our group obtained the first infectious coronavirus cdna clone, for tgev. this cdna was propagated as a bacterial artificial chromosome (bac) (almazán et al., 2000; gonzalez et al., 2002) . vectors based on this infectious cdna were engineered by cloning foreign genes in the place previously occupied by non-essential genes 3a and 3b, leading to high (>50 g/10 6 cells) and stable (>30 passages) expression levels of specific heterologous genes (enjuanes et al., 2005; ortego et al., 2003; sola et al., 2003) . foreign gene expression levels were optimized by the study of the transcription-regulating sequences (trss), involved in coronavirus gene expression. our group has generated a set of trss ranging from intermediate to high foreign gene expression levels (alonso et al., 2002) , a combination of these trss could be used to drive the expression of two or three heterologous genes from just one infectious cdna (i.e., dicistronic or tricistronic vectors). tgev derived vector biosafety was improved by the generation of replicationcompetent, propagation-deficient viruses (ortego et al., 2002) . porcine respiratory coronavirus (prcv) is a mutant of tgev that replicates in the respiratory tract and causes no or mild clinical signs. prcv is spread worldwide and induces antibodies that can also neutralize tgev (saif et al., 1994) . therefore, preexisting immunity against the tgev vector could have been a problem. nevertheless, in vivo experiments showed that antibody titers against tgev increased even after two re-infections of pigs with rtgev vector (alonso s., sola i. and enjuanes l., unpublished results). one of the main advantages of recombinant tgev (rtgev) as a vector for prrsv is that tgev is a potent inducer of ifn-␣ in a process that is mediated by the virus transmembrane (m) protein (calzada-nova et al., 2010; charley and laude, 1988 ). in addition, as mentioned above, tgev vectors may present antigens at mucosal sites, eliciting mucosal and systemic immune responses. therefore, rtgev vectors will represent a novel strategy to study the induction of protection against prrsv. prrsv structural proteins gp5 and m accumulate in the endoplasmic reticulum of infected cells, where they form disulfidelinked heterodimers that are incorporated into the virion. m protein fig. 1 . predicted gp5-m heterodimer topology. the prrsv gp5-m heterodimer may be anchored in membranes, with both proteins exposing to the surface a short nterminal ectodomain. the gp5 protein ectodomain contains the protein motives relevant in antigenicity, such as the epitope critical in neutralization (ecn, purple) and the decoy immunodominant epitope (ide, green). signal peptide (red) cleavage is represented by a black arrowhead. both gp5 and m proteins contain predicted glycosylation sites (yellow), although only gp5 protein is glycosylated (represented by orange circles). m protein contains in its c-terminal an endoplasmic reticulum retention signal (dark green). homodimers are also detected in infected cells, but are not incorporated into the virus particle (dea et al., 2000; meulenberg, 2000) . gp5 and m proteins are essential for the production of viral particles, although additional minor envelope proteins are required for virion infectivity (wissink et al., 2005) . according to the accepted topology of the gp5-m heterodimer (fig. 1) , both gp5 and m proteins expose a short ectodomain on the virion surface, being involved in receptor recognition. gp5 ectodomain contains several glycosylation sites, depending on the viral strain. it has been described that gp5-m protein heterodimer formation is previous to gp5 glycosylation (mardassi et al., 1996) . gp5 glycosylation sites are close to the ecn epitope, and it has been proposed that the steric hindrance caused by the glycosylation is one of the causes for the potential delay in the production of nabs after prrsv infection (see below). it has been recently described that gp5-m heterodimer interacts with the prrsv receptor, porcine fig. 2 . design of rtgev expressing prrsv antigens. scheme of the tgev infectious cdna clone, cloned in a bac (pbac-tgev fl ). after transfection of cells, a full-length virus genome is generated (rtgev). cmv, cytomegalovirus immediate-early promoter; polya, tail of 24 a residues; hdv, hepatitis delta virus ribozyme; bgh, bovine growth hormone termination and polyadenylation sequences. the tgev derived vectors are based on a tgev genome in which non-essential 3ab genes were deleted (rtgev-3ab). genes encoding prrsv heterologous proteins were cloned in this position. expression of the foreign genes was driven by transcription regulatory sequences (trss) from genes 3a and n. ). an additional mutant, lacking n46 glycosylation site and decoy epitope, was obtained (n46s-ide). in all cases, rtgev viruses were recovered with high titers. (b) st cells were infected with the rtgevs and double immunofluorescence staining was performed. tgev n protein specific monoclonal antibodies and a secondary antibody staining red were used to identify virusinfected cells. expression of gp5 was detected with rabbit antiserum specific for a gp5 peptide coupled to a secondary antibody staining green (upper panels). expression of m protein was detected with a rabbit antiserum specific for an m protein peptide, coupled to a secondary antibody staining green (lower panels). the percentage of infected cells expressing prrsv antigens was estimated by the analysis 10 different microscopic fields. sialoadhesin, and that this interaction is dependent on gp5 glycosylation, most likely at the glycosylation site overlapping with the epitope recognized by neutralizing antibodies (van breedam et al., 2010) . as described above, gp5 and m proteins have been involved in the induction of prrsv neutralizing antibodies and a strong cellular immune response, respectively (bautista et al., 1999; ostrowski et al., 2002) . these data indicate that the gp5-m heterodimer is the most promising antigenic structure that could be used in the construction of an efficacious vaccine against prrsv. a dicistronic tgev cdna encoding prrsv gp5 and m proteins was engineered (fig. 2) . prrsv genes were cloned in the place of non-essential genes 3a and 3b. gp5 expression was driven by the transcription-regulating sequence of gene 3a (trs3a), while m protein was expressed from an optimized trs partially derived from gene n (trs22n) (alonso et al., 2002) . therefore, prrsv genes were expressed from independent subgenomic mrnas. the recovered virus expressed gp5 and m proteins in 85% and 95% of the to study if gp5 and m proteins expressed by rtgevs also colocalize, confocal microscopy analysis was performed. ma-104 or st cells were infected with prrsv and the rtgevs, respectively, and double immunofluorescence staining was performed. expression of gp5 was detected with a monoclonal antibody specific for gp5, coupled to a secondary antibody staining red (upper panels). expression of m protein was detected with a rabbit antiserum specific for an m protein peptide, coupled to a secondary antibody staining green (medium panels). as shown in the merge, colocalization of gp5 and m proteins was observed both in the prrsv and rtgev infected cells (lower panels). mutant gp5 proteins (gp5-n46s and gp5-ide-n46s) expressed by rtgevs also colocalized with m protein. rtgev infected cells, respectively (fig. 3(b) ). expression levels were maintained even in virus recovered from tissues after infection of piglets with the rtgev. this result substantially advanced the efficacy of previous rtgevs expressing individually prrsv antigens, showing high expression levels of gp5, but with limited stability. co-expression of m protein with gp5 reduced gp5 toxicity and probably will elicit a better t cell immune response. the protection conferred by this vector was tested in vivo. one-week-old piglets were inoculated with 1 × 10 8 pfu of the rtgev by three routes: oral, nasal and intragastric. nine weeks later, a challenge was perfomed with 1 × 10 7 tcid 50 of a virulent european prrsv strain. blood samples were collected at different times post-inoculation, and humoral immune responses were evaluated by elisa. all animals presented a high antibody response against tgev, therefore, the vector infected target tissues as expected. vaccinated animals also showed a clear humoral response against prrsv gp5 and m proteins. a fast recall of the immune response was observed after the challenge, as vaccinated animals induced higher antibody titers against prrsv antigens and earlier than control ones. nevertheless, the immune response elicited by this rtgev provided very limited protection, and antibody titers decreased before challenge. the lack of protection against challenge was likely due to the relatively low levels of neutralizing antibodies produced before challenge. nevertheless, results using rtgev as a platform were promising, as a humoral immune response against prrsv antigens was elicited. gp5 antigenicity may be a problem for the obtention of efficient vaccines. therefore, several strategies to change gp5 antigenic structure were performed (fig. 3(a) ). in all cases, the gp5 mutants were co-expressed with m protein using a dicistronic vector, to minimize toxicity problems due to gp5 production. the ectodomain of gp5 protein is n-glycosylated. there are three or four predicted glycosylation sites in the gp5 from the north american strains of prrsv, whereas there are only two sites in the gp5 protein from european strains (wt, fig. 3(a) ). the relevance of the n-glycans in gp5 antigenicity is not clear. it has been proposed that the removal of the glycosylation sites could lead to the improvement of the immune response against prrsv, due to the elimination of the steric hindrance raised by the carbohydrate on the epitope inducing nabs (ansari et al., 2006) . elimination of the glycosylation sites present only in the north american strains, both in engineered and natural prrsv mutants, led to an increase in the levels of nabs induced by the mutant viruses (ansari et al., 2006; faaberg et al., 2006) . nevertheless, it is worth noting that these sites are not present in european prrsv strains. although the elimination of the glycosylation site overlapping the epitope critical for neutralization (g1) (fig. 3(a) ) often leads to non-infectious viruses (ansari et al., 2006; wissink et al., 2005) , natural north american strain mutants lacking this glycosylation site were found (2.1% of the sequenced gp5 proteins). surprisingly, one of this natural mutants elicited lower neutralizing antibody response than the wild-type prrsv strain (faaberg et al., 2006) . this is in contrast with the data obtained with lactate dehydrogenase-elevating virus (ldv), where deletion of the n-glycan enhanced the nabs response (plagemann and moenngin, 1997) . elimination of the most conserved n-glycosylation site (g2, fig. 3(a) ) (only 0.2% of the sequenced north american gp5 proteins lack this motif) led to higher levels of neutralizing antibodies compared with the response elicited by the wild-type virus (ansari , 2006) . as glycosylation of gp5 is probably also involved in virus infectivity, it is difficult to analyze the influence of n-glycans on the immunogenicity of the protein. in the rtgev system, prrsv gp5 and m are not involved in infectivity and, therefore, the relevance of these proteins antigenicity in protection could be analyzed in our laboratory using the rtgev vector. gp5 mutants lacking glycosylation site g1 (n46s), g2 (n53s) or both (n46,53s) were generated (fig. 3(a) ). the mutation asn by ser was selected in all cases, as this substitution most likely introduced little secondary structure modifications. also, some prrsv field strains bear similar asn by ser aminoacid mutations in putative glycosylation sites. all rtgev viruses were recovered with high titers (fig. 3(a) ). nevertheless, only the n46s mutant, lacking the glycosylation site partially overlapping the ecn, was stable ( fig. 3(b) ). this rtgev vector expressed high levels of gp5-n46s and m prrsv proteins in 75% and 90% of the infected cells, respectively (fig. 3(b) ). several b cell epitopes have been found in gp5 protein. an immunodominant epitope is located in the endodomain and, therefore, has probably limited effect on the antigenicity of the ecn epitope, as it is not exposed in the viral surface (dea et al., 2000; oleksiewicz et al., 2002; rodriguez et al., 2001) . a second immunodominant epitope (ide) was described in the ectodomain of gp5, close to the ecn (fig. 3(a) ) (ostrowski et al., 2002) . it has been suggested that this immunodominant site could be responsible for the delay in the production of nabs against prrsv acting as a decoy epitope. antibodies against ide and ecn epitopes were found in the sera of prrsv infected pigs, appearing at different times post-infection. furthermore, an increase in the titers against ecn correlates with a decrease in the level of antibodies specific for ide (lopez and osorio, 2004; ostrowski et al., 2002) . an enhanced immunogenicity of a recombinant gp5 protein in which a synthetic sequence spacer has been introduced between ide and ecn epitopes, to better display the neutralizing epitope has been reported. the data suggests that ide is in fact acting as a decoy epitope (fang et al., 2006) . rtgev vectors were engineered expressing gp5 mutants lacking ide, in order to clarify whether this epitope is acting as a decoy epitope, enhancing the production of prrsv specific nabs. this approach represents an advance over similar constructions made in a prrsv infectious cdna clone, as in this case the deletion of the decoy epitope prevents the recovery of the recombinant virus (ansari et al., 2006) . two gp5 modifications were combined within the same construct, expressing gp5 protein lacking the decoy epitope and the glycosylation site overlapping the epitope recognized by neutralizing antibodies (n46s-ide, fig. 3(a) ). the rtgev virus was recovered with high titer (fig. 3(a) ), and expressed modified gp5 and m proteins in 65% and 93% of the infected cells, respectively ( fig. 3(b) ). the data obtained in cultured cells suggest that rtgev vectors expressing prrsv antigens were not fully stable, mainly due to gp5 protein toxicity resulting in a significant lost of gp5 expression after 8-10 virus vector passages in cell culture. in contrast, m protein expression was fully stable, with at least 95% of infected cells expressing m protein for more than 10 passages in tissue culture. a decrease in gp5 expression was also observed after the introduction of modifications in this protein (upper panels, fig. 3(b) ). again, m protein expression remained constant, independently of gp5 mutant co-expressed (lower panels, fig. 3(b) ). the reduction in gp5 expression could be responsible of the modest results in protection observed with the live rtgev vectors, in comparison to the protection elicited with non-infectious antigens expressed using rtgev vectors. as described above, the rtgev vector expressing prrsv gp5 and m proteins represents a substantial advance on the efficacy of previous rtgevs expressing prrsv antigens (i.e., gp5 alone). we postulated that co-expression of m protein with gp5 reduces gp5 toxicity by the formation of gp5-m heterodimer. to clarify this issue, confocal microscopy analysis was performed (fig. 4) . ma-104 or st cells were infected with prrsv and the rtgevs, respectively, and double immunofluorescence staining was performed. as shown in the merge (fig. 4, lower panels) , colocalization of gp5 and m proteins was observed both in the prrsv and rtgev infected cells. this result suggests that the gp5-m heterodimer is formed in both cases. the decrease in gp5 expression levels by the introduction of gp5 mutations suggested that the modifications could affect heterodimer formation. colocalization of gp5 and m proteins was also observed when a mutant gp5 protein (i.e., gp5-n46s, or gp5-ide-n46s) was expressed by the rtgev vector (fig. 4) , suggesting that a heterodimer was also formed by mutant gp5 proteins. coimmunoprecipitation of gp5 and m proteins to fully demonstrated gp5-m heterodimer formation is in progress. 4.5. protection conferred by rtgev derived vaccines 4.5.1. formulation of a killed vaccine expressing gp5 mutants with alterations in the glycosylation pattern as a complementary approach, a killed vaccine was developed based on the rtgev-gp5-n46s-m virus, co-expressing gp5 lacking the first glycosylation site and m proteins. st cells were infected with this rtgev, and the culture medium was harvested at 48 hpi. soluble antigens were inactivated by incubation with binary ethylenimine (bei), and a vaccine was formulated. groups of six 1-week-old piglets were intramuscularly inoculated with the formulation to evaluate the protection conferred by this vaccine. a boost was performed 3 weeks after inoculation. six weeks after the first inoculation, animals were challenged by intranasal inoculation with 10 7 tcid 50 of prrsv/olot91 strain. blood samples were collected at different times post-inoculation to determine the levels of specific antibodies by elisa. vaccinated animals induced higher and faster antibody titers against prrsv antigens than control animals (fig. 5(a) , left panel). neutralizing antibody titers were also higher in the vaccinated animals when compared with non-vaccinated animals (fig. 5(a) , right panel). viremia, gross lesions, and histopathology in the lungs of vaccinated and nonvaccinated animals were analyzed. a clear degree of protection samples were analyzed by enzyme-linked immunosorbent assays (elisas) specific to detect antibodies against tgev, gp5 and m. to evaluate response against gp5, gp5 protein from prrsv olot91 strain was expressed and purified from insect cells and used as antigen for the elisa. (b) lung damage caused by prrsv infection. the lungs from animals inoculated with empty rtgev vector, or rtgev expressing gp5-n46s and m proteins, were analyzed. lung lesions observed in all the pigs, with different degree of severity, included a craneo-ventral consolidation of apical and medial lung lobes. was observed, as the lungs from vaccinated animals showed a significantly lower degree of lung damage than those from nonvaccinated ones (fig. 5(b) , left panel). furthermore, a reduction in viremia was also observed in vaccinated animals (fig. 5(b) , right panel). altogether, these data suggested that the elimination of the glycosylation site close to the neutralizing epitope improves protective immune response against prrsv. the protection conferred by rtgev-gp5-n46s-m was tested in vivo. one-week-old piglets were inoculated with 1 × 10 8 pfu of the rtgev by three routes: oral, nasal and intragastric. a boost was performed 3 weeks after inoculation. six weeks later, a challenge was performed with 1 × 10 7 tcid 50 of prrsv/olot91 strain. blood samples were collected at different times post-inoculation, and humoral immune responses were evaluated by elisa. all the animals produced a high antibody response against tgev (data not shown), therefore, the vector infected target tissues as expected. after challenge, vaccinated animals showed a clear humoral response against prrsv antigens (fig. 6(a) ). a moderately faster recall response was observed, as vaccinated animals induced higher antibody titers against prrsv antigens and earlier than control animals (fig. 6(a) ). the protection conferred by this tgev based vaccine was also evaluated. a certain degree of protection was observed, as the lungs from vaccinated animals showed a lower degree of lung damage than those from non-vaccinated ones (fig. 6(b) ). nevertheless, the immune response was not strong enough to provide full protection, probably because the levels of neutralizing antibodies were similar in vaccinated and non-vaccinated animals (data not shown). to date, rtgev expressing prrsv antigens only provided partial protection. this could be due to the fact that the expression of prrsv antigens by rtgev vectors was not fully stable, mainly due to gp5 protein toxicity resulting in a significant lost of gp5 expression in 8-10 passages. in contrast, m protein expression was fully stable, with at least 95% of infected cells expressing m protein for more than 10 passages in tissue culture. the lack of full protection using rtgev expressing prrsv antigens could also be due to the presence of domains in the expressed proteins inducing negative regulatory t cells (treg). as the vector used in the immunization (rtgev) efficiently induced the production of ifn, it is likely that either prrsv gp5 or m proteins could contain negative signals inducing treg. this negative regulation of the immune response elicited could also be a major cause for the delay in the development of a protective immune response against prrsv. to improve rtgev vector stability different strategies can be developed, such as the expression of small domains of gp5 containing the epitopes relevant for protection but lacking domains responsible for instability in their expression. alternatively, the generation of a library of point mutants in gp5 fragments in which the epitopes eliciting negative treg have been eliminated may overcome what we consider the second most relevant limitation in the protection against prrsv. these approaches are currently in progress in our laboratory. an improvement of vaccination strategies against prrsv is required, as current vaccines have limited efficacy. best results have been obtained using modified live vaccines and virus vectored vaccines could represent an advantage to stimulate immune responses against prrsv. the results reported to date using viral vectors are not fully satisfactory and new vectors must be explored. tgev based vector vaccines expressing 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for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus co-expressing gp5 and m proteins under different promoters in recombinant modified vaccinia virus ankara (rmva)-based vaccine vector enhanced the humoral and cellular immune response of porcine reproductive and respiratory syndrome virus (prrsv) assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge we thank c.m. sánchez, s. ros, and m. gonzález for technical assistance. this work was supported by grants from the comisión interministerial de ciencia y tecnología (cicyt, bio2007-60978), the national pork board , and the european union (fp7, plaprova-227056, and porrscon-245141). s.z., i.s. and m.b. received contracts from the eu. j.l.g.c. received contract from community of madrid. key: cord-346264-hnwgzt6v authors: nagai, makoto; wang, qiuhong; oka, tomoichiro; saif, linda j. title: porcine sapoviruses: pathogenesis, epidemiology, genetic diversity, and diagnosis date: 2020-05-26 journal: virus res doi: 10.1016/j.virusres.2020.198025 sha: doc_id: 346264 cord_uid: hnwgzt6v the first porcine sapovirus (sav) cowden strain was discovered in 1980. to date, eight genogroups (giii, v-ix) and three genogroups (giii, gv, and gvi) of porcine savs have been detected from domestic pigs worldwide and wild boars in japan, respectively based on the capsid sequences. although giii cowden strain replicated in the villous epithelial cells and caused intestinal lesions in the proximal small intestines (mainly in duodenal and less in jejunum), leading to mild to severe diarrhea, in the orally inoculated neonatal gnotobiotic pigs, the significance of porcine savs in different ages of pigs with diarrhea in the field is still undetermined. this is due to two reasons: 1) similar prevalence of porcine savs was detected in diarrheic and non-diarrheic pigs; and 2) co-infection of porcine savs with other enteric pathogens is common in pigs. diagnosis of porcine sav infection is mainly based on the detection of viral nucleic acids using reverse transcription (rt)-pcr and sequencing. much is unknown about these genetically diverse viruses to understand their role in pig health and to evaluate whether vaccines are needed to prevent sav infection. the first porcine sapovirus (sav), the cowden strain was discovered by electron microscopy in the intestinal contents of a 27-day-old diarrheic nursing pig in the united state in 1980 (saif et al., 1980 . later it was classified as a genogroup iii (giii) sav based on the complete genomic sequence analysis (guo et al., 1999) . sapoviruses belong to the sapovirus genus within the family caliciviridae. they are non-enveloped viruses that possess a single-stranded, positive-sense rna genome. sapovirus particles are small and round with a diameter of 30-40 nm, exhibiting a typical star-of-david structure and cup-shaped surface depressions by electron microscopy (em) or immune em (iem) (alhatlani et al., 2015; oka et al., 2015; saif et al., 1980) . the genome length is 7-8,000 nucleotides (nt) excluding a 3'-end polyadenylated [poly(a)] tail. the 5'-end of the genome covalently links to a small virus-encoded protein (vpg). sapovirus genomes have two overlapping open reading frames (orfs): orf1 and orf2 (oka et al., 2015) . orf1 encodes the nonstructural proteins ns1-ns2-ns3 (putative ntpase)-ns4-ns5 (vpg)-ns6 (protease)-ns7 (rna-dependent rna polymerase: rdrp) and the capsid protein, vp1. orf2 encodes the minor structural protein, vp2. sapoviruses are genetically highly diverse and have been classified into 19 genogroups based on the vp1 sequences (farkas et al., 2004; oka et al., 2016; scheuer et al., 2013; yinda et al., 2017) . among them, eight genogroups (giii, gv, gvi, gvii, gviii, gix, gx, and gxi) and 3 genogroups (giii, gv, and gvi) of savs have been detected from pigs and wild boars, respectively. in this review, we will summarize current knowledge on the pathogenesis of giii cowden strain, the epidemiology and genetic diversity of porcine savs, and the diagnosis of sav infection in pigs. the pathogenesis of most genogroups of porcine savs is unknown, except for giii cowden strain. the original field sample for the discovery of cowden strain contained not only sav particles (33 nm in diameter), but also rotavirus (55 nm and 70 nm in diameter for single-and double-capsid particles, respectively) and 23nm virus-like particles (saif et al., 1980) . saif et al. successfully removed rotavirus from the sample using selective membrane ultrafiltration before serial passage in gnotobiotic pigs. the 23-nm virus-like particles failed to replicate in the experimentally inoculated gnotobiotic pigs. at the 12th and above passages, the intestinal contents of the inoculated pigs contained only sav particles by immune electron microscopy (iem). flynn et al. (flynn et al., 1988) studied the pathogenesis of porcine sav cowden strain in 4-day-old gnotobiotic pigs. they inoculated orally (po) 18 pigs with the 12th passage of the virus, monitored clinical signs for 14 days, and euthanized pigs at different days post-inoculation (dpi) to examine histopathological changes compared to mock-inoculated pigs at similar ages. they found that sav cowden strain caused diarrhea in all the pigs by 3 dpi and persisted for 3-7 days. most pigs had mild diarrhea during the infection and two pigs (2/18) had severe diarrhea at 4-5 dpi. porcine sav replicated in the villous epithelial cells, but not crypt cells, mainly in duodenum, less in jejunum and the least in ileum, but not in the large intestines as determined by immunofluorescent assays (ifa) using pig hyperimmune antisera against porcine sav cowden strain. histologically, porcine sav-inoculated pigs showed mild to severe villous atrophy in the duodenum with short and flat villi with areas of denudation. typical sav particles were detected from the feces and large intestinal contents (lic) of sav-inoculated pigs at 1-7 dpi using iem. later guo et al. (guo et al., 2001) found that infectious porcine sav entered the blood stream during the acute phase of infection of orally inoculated gnotobiotic pigs. using more sensitive taqman real-time rt-pcr assay for the detection of porcine sav rna, fecal viral rna shedding in virus-inoculated pigs started at 1-3 dpi, reached the highest titers [10.8±0.4 log10 genomic copy equivalent (ge)/ml] at 6-10 dpi and lasted for 30±4 days (lu et al., 2016) . these observations are similar to the pathogenesis of bovine nebovirus, an enteric calicivirus belonging to the nebovirus genus, that replicated in the proximal portion of the small intestine of calves (hall et al., 1984; smiley et al., 2002) . the 13th passage of porcine sav cowden strain from the lic of a gnotobiotic pig was successfully isolated in primary porcine kidney cells (flynn and saif, 1988) . for decades, posav had been the only culturable enteric calicivirus until the successful cultivation of human noroviruses in b cells in 2014 and in intestinal stem cell-derived human enteroids in 2016 (ettayebi et al., 2016 jones et al., 2014) . interestingly, j o u r n a l p r e -p r o o f initial adaptation of posav in primary porcine kidney cells and the subsequent adaptation in llc-pk, a continuous swine kidney epithelial cell line, required the supplementation of intestinal contents collected from mock-infected gnotobiotic pigs (flynn and saif, 1988; parwani et al., 1991) . later, the essential components in the intestinal contents for posav replication were identified as bile acids (chang et al., 2004) . several human novs were grown in enteroids, which occurred exclusively when the culture medium was supplemented with bile or bile acids (ettayebi et al., 2016) . bile acids are synthesized in the liver, released with bile into the duodenal lumen, and most of them are recycled back into the liver in the ileum. so, the concentration of bile acids is much higher in the proximal intestine than in other organs and this may be one of the restriction factors for posav replication mainly in duodenum. using the llc-pk cell culture system, α2,3-and α2,6-linked terminal sialic acids on o-linked glycoproteins have been identified as the binding receptor for porcine sav cowden strain (kim et al., 2014) . in the same study, it was also confirmed that these sialic acids are the binding receptor on piglet small intestinal tissues. recently, the same group found that the tight junction (tj) protein occludin is a functional receptor for porcine sav in llc-pk cells (alfajaro et al., 2019) . the binding of porcine sav or virus-like particles or bile acids alone to llc-pk cells caused the dissociation of tjs and exposed occludin for posav binding. then sav and occludin form a complex and move to late endosomes via rab5-and rab7-dependent trafficking to start replication. the fact that more than one receptor is involved in sav binding and entry is similar to findings for some other caliciviruses. feline calicivirus (fcv) f9 strain uses a2,6-linked sialic acids on an n-linked glycoprotein as binding factors (stuart and brown, 2007) and junctional adhesion molecule 1 (jam-1) for virus entry into cells (makino et al., 2006) . some murine noroviruses use sialic acid linked to ganglioside (cw3 like strains) or protein (cr3 strain) (taube et al., 2009) for binding and protein receptors cd300lf and/or cd300ld for entry (haga et al., 2016; orchard et al., 2016) . taken together, cellular receptors (α2,3-and α2,6-linked sialic acids on o-linked glycoproteins and occludin) and bile acids are some of the restriction factors of porcine sav replication in the proximal small intestine. it may also explain why porcine sav cowden strain did not replicate in other organs when piglets were inoculated intravenously (iv) with the virus (guo et al., 2001) . to date, porcine savs have been detected in the fecal samples of domestic pigs with and without diarrhea worldwide and of wild boars without diarrhea in japan (table 1) . pigs in all growing stages can be infected with porcine savs; however, pigs are infected with savs early in life and post weaning pigs have higher sav infection rates than other age groups (barry et al., 2008; jeong et al., 2007; reuter et al., 2010; valente et al., 2016; wang, q.h. et al., 2006a) . this can be explained by lactogenic immunity in nursing pigs and environmental factors (valente et al., 2016) . suckling piglets are protected passively by maternal antibodies against savs until weaning and post weaning pigs become susceptible to sav infections when maternal antibodies decline (alcalá et al., 2010; barry et al., 2008; martínez et al., 2006) . on the other hand, nutritional, environmental and social changes during the post-weaning period add significant stress on these animals (valente et al., 2016) . although porcine savs induced diarrhea and intestinal lesions in experimentally inoculated gnotobiotic piglets (guo et al., 2001; flynn et al., 1988; lu et al., 2016) , there were no significant j o u r n a l p r e -p r o o f differences in the prevalence of savs between the same age groups of pigs with diarrhea and without diarrhea in the field (table 1) . currently, giii is the predominant genogroup of porcine savs ( savs (giii, gv, and gviii) in both trees, suggesting that these porcine savs possess a common ancestor and are distantly related to other savs in the porcine population ( fig. 1) . although the end of vp2 of porcine savs as well as other savs is highly variable (table 2) , neither deletion nor insertion in the region, like that of the s indel strains of porcine epidemic diarrhea virus, is reported. the currently, conventional or real-time rt-pcr are the most widely used routine laboratory diagnostic assays for the detection of porcine savs from fecal samples, with the advantages of specificity, high sensitivity, broad reactivity, and convenience. many primers used for the screening of porcine savs have been designed (table 3) . almost all primers are designed targeting the partial rdrp region, which presents conserved motifs that are useful for molecular diagnosis of genetically highly diverse savs ( seroepidemiology of porcine enteric sapovirus in pig farms in venezuela early porcine sapovirus infection disrupts tight junctions and uses occludin as a coreceptor functions of the 5′ and 3′ ends of calicivirus genomes high genetic diversity in rdrp gene of brazilian porcine sapovirus strains bile acids are essential for porcine enteric calicivirus replication in association with down-regulation of signal transducer and activator of transcription 1 detection of multiple genotypes of calicivirus infection in asymptomatic swine in taiwan metagenomic analysis of the rna fraction of the fecal virome indicates high diversity in pigs infected by porcine endemic diarrhea virus in the united states detection and characterization of porcine sapoviruses from asymptomatic animals in irish farms salaries and research support for qw and ljs were provided by state and federal funds appropriated to the ** these primers are universal primers for calicivirus, but not posav-specific. so, their rt-pcr products should be sequenced for confirmation. *** these primers also detected porcine kobuvirus.j o u r n a l p r e -p r o o f key: cord-335706-lopcb77c authors: tan, feifei; wei, zuzhang; li, yanhua; zhang, rong; zhuang, jinshan; sun, zhi; yuan, shishan title: identification of non-essential regions in nucleocapsid protein of porcine reproductive and respiratory syndrome virus for replication in cell culture date: 2011-03-24 journal: virus res doi: 10.1016/j.virusres.2011.03.011 sha: doc_id: 335706 cord_uid: lopcb77c nucleocapsid (n) protein of porcine reproductive and respiratory syndrome virus (prrsv) plays a central role in virus replication. in this study, serial nand c-terminal truncations of n protein were performed in the context of type 2 prrsv infectious cdna clone, and our results revealed that a stretch of inter-genotypic variable n terminal residues aa 5–13 ((5)ngkqqkkk(13)k) and the last four inter-genotypic variable aa residues ((120)sps(123)a) at the c terminus of n protein were dispensable for type 2 prrsv infectivity. all the recovered deletion mutant viruses had spontaneous mutations in the n coding region, including substitution, deletion and insertion. we re-engineered the additional internal deletion with or without the original c-terminal deletion back into wild-type aprrs and found that the internal domain spanning the inter-genotypic variable residues 39–42 ((39)kgp(42)g) and conserved residues 48–52 ((48)knpe(52)k), respectively, were dispensable for type 2 prrsv viability. these results demonstrated that n protein contains non-essential regions for virus viability in cell culture. such dispensable regions could be utilized as insertion site for foreign tag expression and the rescued viruses could be the candidates for marker vaccine. porcine reproductive and respiratory syndrome virus (prrsv) is a single-strand, positive-sense rna virus, and is a member of the family arteriviridae, together with equine arteritis virus (eav), lactate dehydrogenase-elevating virus (ldv), and simian hemorrhagic fever virus (shfv). arteriviridae and coronaviridae are classified as members of the order nidovirales, mainly based on their similar replication and transcription strategy (den boon et al., 1991; gorbalenya et al., 2006; snijder and meulenberg, 1998) . two genotypes of prrsv have been classified, (european) type 1 and (north american) type 2, lelystad virus (lv) (wensvoort et al., 1992) and vr2332 (benfield et al., 1992) are the representative strains of two types, respectively. prrsv virion consists of at least seven structural proteins, including glycoprotein 2 (gp2) , gp3 (de lima et al., 2009) , gp4 (van nieuwstadt et al., 1996) , gp5, and unglycosylated envelope (e) protein (wu et al., 2005) , membrane (m) protein, and nucleocapsid (n) protein (bautista et al., 1996) . structural proteins of arteriviruses are expressed via a nested set of subgenomic mrnas (sgmrnas) formed by a unique discontinuous transcription process (de vries et al., 1990; meng et al., 1996; nelsen et al., 1999) . the common 5 leader sequence of sgmrnas is derived from the 5 proximal untranslated region (utr). the leader-body junction sequence can be amplified by sgmrna-specific rt-pcr (chen et al., 1993; den boon et al., 1996; meulenberg et al., 1993; nelsen et al., 1999; zheng et al., 2010) . n protein is the most abundant component of the virion and plays a crucial role in virus assembly. generally, type 1 and type 2 prrsv n proteins consist of 128 and 123 amino acids (aa), respectively. however, stadejek et al. (2008) reported that n protein of type 1 exhibits size polymorphism, the number of amino acid residues varies from 124 to 130. although n protein is one of the inter-genotypically conserved proteins, lv and vr2332 share only 64% identity at the amino acid level (dea et al., 2000) . n protein is assembled into an icosahedral nucleocapsid in the form of disulfide-linked homodimer , and the cterminal half of prrsv n protein is believed to be the dimerization domain (spilman et al., 2009) . the n-terminal half of n protein is enriched in basic amino acids and intrinsically disordered, which are common properties of rna-binding proteins (daginakatte and kapil, 2001; doan and dokland, 2003; yoo et al., 2003) . the crystal structure of the c-terminal dimerization domain comprises two antiparallel ␤-sheet floors superposed by two long ␣ helices and flanked by two n-and c-terminal ␣ helices (doan and dokland, 2003) , as shown in fig. 1a . the structure is homologous to that of eav (deshpande et al., 2007) , and surprisingly, has a fold similar to that of severe acute respiratory syndromeassociated coronavirus (yu et al., 2006) . by replacing each amino (doan and dokland, 2003) (pdb id: 1p65). alignment of the n sequence from type 2 strain vr2332 (genbank id: ay150564), the wild-type aprrs (genbank id: gq330474) in this study and type 1 strain lv (genbank id: m96262). (b) sequence alignment of the first 100 nt sequence of orf7 of lv, aprrs and n-terminal deletion mutants, the identical sequence with lv is denoted as dot while natural sequence deletion compared with lv designated as dash line. the engineered deletion is represented by asterisk. the 34 nt that was documented to be essential for rna replication of lv (verheije et al., 2002) is indicated in the open box, the 7 nt sequence which had kissing interaction with 3 utr is indicated by shaded box. acid with proline, wootton et al. (2001) demonstrated that the 111-117 aa substitution of type 2 prrsv n protein reduces the conformational-dependent monoclonal antibody (mcab) binding significantly. therefore, the c-terminus of n protein is important for maintaining the local and/or the overall configuration of the prrsv n protein. verheije et al. (2001) reported that c-terminal six amino acids of n protein are non-essential for virus infectivity of type 1 prrsv. these authors also indicated that the n-terminal and internal regions of n protein cannot tolerate deletion. as shown in fig. 1a , n protein is intra-genotypically conserved and only one (d61) out of the 123 aa of type 2 prototypic strain, vr2332, is different from the aprrs strain (y61) used in this study. several inter-genotypically conserved domains were observed too. in addition, crystal structure of c-terminal dimerization domain except for the last 5 aa of vr2332 n protein was determined (doan and dokland, 2003) . in this study, to further illustrate the roles of the terminal and internal aa residues of n protein in type 2 prrsv replication, particularly in viral viability. we performed serial deletions at the n-and c-termininal of n protein in the context of full-length cdna clone paprrs. we found that type 2 prrsv contained extensive regions that were dispensable for virus viability in cell culture. these results are of great significance for foreign gene expression and genetic engineered vaccine development. marc-145 cells were grown in minimal essential medium (mem) (sigma) complemented with 10% fetal bovine serum (fbs) (invitrogen), and maintained in mem with 2% fbs at 37 • c with 5% co 2 . type 2 prrsv infectious cdna clone paprrs (yuan and wei, 2008) was used as the wild-type control in all experiments. its complete genomic sequence was available as genbank accession number gq330474. pbsx was a shuttle plasmid that cloned the spe i-xho i region (nt 13117-15520) of aprrs genome into the pbluescript sk(+) vector (fermentas). p7usc was constructed by inserting asc i immediately after the stop codon of orf7. n-terminal deletion was performed from the fifth amino acid to retain the integrity of orf6. fragments containing the corresponding deletion region were generated using the mutagenic pcr method as described before (yu et al., 2009) , pbsx was the template. then the spe i-xho i fragment of paprrs was replaced by the analogous fragments derived from mutagenic pcr products. internal deletion mutants were constructed in the same manner. c-terminal deletion mutants were constructed based on the fulllength clone p7usc. the fragments containing the desired deletion were amplified by pcr, asc i was introduced in anti-sense primers (table 1) , and the sense primer was spef (table 1 ). the spe i-asc i fragment of p7usc was replaced by the pcr fragments carrying the desired deletion. for the construction of plasmids that contained primary engineered deletions and spontaneous mutations, appropriate fragments were prepared by rt-pcr products from the viral cell culture supernatants. the amplified fragments between xba i and xho i were swapped into the corresponding region of paprrs. all full-length clones of mutants were verified by sma i mapping and nucleotide sequencing. full-length plasmids were prepared with qiaprep spin miniprep kit (qiagen). marc-145 cells were seeded in six-well plates and grown to 80% confluence. the monolayer cells were transfected with 1 g plasmid using 3 l fugene hd regent (roche) according to the manufacturer's instructions. after visible cpe was observed (about 72 h posttransfection for wild-type aprrs) and 80% of the monolayer cells were detached, the culture supernatants were collected and labeled as passage (p) 0 viruses. the wildtype and recovered mutant viruses were passaged in marc-145 cells five times to gain p1-p5 virus stocks. at least three further passages were performed if no visible cpe was observed after transfection. marc-145 cells were transfected with plasmids of wild-type or mutants. intracellular expressions of nsp2 and n were visualized by immunofluorescence staining at 48 hpt and 72 hpt, respectively, the protocol was same with that described before (yu et al., 2009 ). the mcabs against nsp2 and n protein (mr40) (wootton et al., 1998) were kindly donated by dr. ying fang at south dakota state university. alexa fluor 568 goat anti-mouse igg (h + l) (invitrogen) was used as a secondary antibody. viral rna was extracted from cell supernatants using qiaamp viral rna kit (qiagen), and treated with turbo dnase (ambion) to eliminate input genomic dna according to the manufacturers' instructions. first-strand cdna was synthesized using reverse transcriptase xl (amv) (takara) and anti-sense primer qst (table 1 ). the fragment containing the n gene was amplified by taq dna polymerase (takara) using primer pairs sf14413 and sr15497. the pcr products were purified using tiangel mini purification kit (tiangen) and sequenced directly. the full-length genome of the recovered viruses was amplified by five primer pairs as described previously (yuan and wei, 2008) , and the primer sequences were available upon request. the leader-body junction sites of prrsv were detected by sgmrna-specific rt-pcr (nelsen et al., 1999; zheng et al., 2010) . as shown in fig. 3b , sf12 (nt 12-32) and sr15284 (nt 15284-15306) are the primer pairs of sgmrna7 amplification of aprrs as described before (yu et al., 2009 ). for the recovered p0 viruses that had a mixed population, plaque purification was performed. the transfected cell supernatants of c 3/121-123 and n 48-52 were used to infect marc-145 cells at a moi of approximately 0.01. after 1 h incubation, cell monolayer was overlaid with 2× mem (invitrogen) supplemented with 4% fbs and 2% low melting agarose (promega). well-isolated plaques were passaged onto fresh marc-145 cells. rna was extracted and rt-pcr was performed as previously described, then the n gene was sequenced. marc-145 cells were infected with wild-type and p5 rescued viruses at ∼0.01 moi. cell culture supernatants were collected at various times after infection. titers were determined by standard tcid 50 assay. three independent titrations were performed and the mean value was used for determination of the viral multi-step growth-curve. overlapping genes are a dominant feature of arterivirus genome, and there are 11 nucleotides that overlap between orf6 and orf7. therefore, n-terminal deletion mutants were truncated from the fifth residue of orf7 to ensure the integrity of orf6. intergenotypic aa sequence alignment indicated that the lv strain (type 1) contains four extra aa residues (tapm) than aprrs (type 2) immediately upstream of the inter-genotypically conserved region from 14 g of aprrs (fig. 1a) . it was therefore possible that this variable region plays little role in type 2 prrsv n functionality. accordingly, we constructed five deletion mutants that comprised residues 5-11, 12, 13, 14 and 20 at the n terminus, which were designated as pn 7/5-11, pn 8/5-12, pn 9/5-13, pn 10/5-14 and pn 16/5-20, respectively ( fig. 2a) . we also wanted to investigate the c-terminal requirements of n protein for type 2 prrsv viability. to this end, a series of c-terminal truncation mutants were constructed. firstly, a recombinant full-length clone p7usc containing an asc i immediately after the stop codon of orf7 was constructed (fig. 2b, d) . transfection assay demonstrated that the asc i insertion did not affect viral infectivity, and the rescued virus 7usc had similar growth properties with the wild-type virus aprrs (fig. 4b ). therefore, p7usc was used as the backbone for constructing c-terminal truncations, which contained residues 121-123, 120-123, 119-123, and 118-123 deletions, named as pc 3/121-123, pc 4/120-123, pc 5/119-123 and pc 6/118-123, respectively (fig. 2b) . to define whether the n-terminal deletions affect virus replication, full-length clones pn 7/5-11, pn 8/5-12, pn 9/5-13, pn 10/5-14, pn 16/5-20, and the wild-type paprrs, were transfected into marc-145 cells. intracellular expression of nonstructural protein 2 (nsp2) and n protein was determined by immunofluorescence assay (ifa) at 2 and 3 days post-transfection (dpt), respectively, which indicated the genomic rna replication and sgmrna transcription properties of the mutants. as shown in fig. 3a , all but pn 16/5-20 displayed nsp2 and n protein expression, but the fluorescence intensity was different. it was noteworthy that there were only several single cells stained for mutant pn 10/5-14, suggesting virus spreading between neighboring cells was affected. moreover, no fluorescent signal was detected in the case of pn 16/5-20 (even after prolonged incubation at 7 dpt; data not shown), indicating that deletion of residues 5-20 ( 5 ngkqqkkkkgdgqpv 20 n) might have blocked both virus replication and transcription. in the parallel transfection experiments, visible cytopathic effect (cpe) appeared for the wild-type paprrs as well as for mutants pn 7/5-11, pn 8/5-12 and pn 9/5-13. the longer deletion mutants pn 10/5-14 and pn 16/5-20, however, produced no infectious particles even after three further passages. these results demonstrated that the nterminal residues 5-13 of n protein were non-essential for virus infectivity in cultured cells. we next investigated the possible blockage for the n-terminal deletion mutants that failed to produce progeny viruses. two sgm-rnas (sgmrna7.1 and sgmrna7.2) of orf7 have been defined in type 2 prrsv infected cells (nelsen et al., 1999; zheng et al., 2010) . as outlined in fig. 3b , total rnas of cells transfected with pn 9/5-13, pn 10/5-14, pn 16/5-20 and paprrs, respectively, were extracted and sgmrna7-specific rt-pcr was performed as previously described (yu et al., 2009; zheng et al., 2010) . as shown in fig. 3c , the two expected bands representing sgm-rna7.1 and sgmrna7.2 were amplified from cells transfected with wild-type paprrs, pn 9/5-13 and non-viable pn 10/5-14, but the band of pn 10/5-14 was weaker than others, indicating the lower transcription level than others. this was consistent with the ifa result and further demonstrated that sgmrna transcription of pn 10/5-14 was down-regulated. however, pn 16/5-20 showed no sign of sgmrna7 transcription (fig. 3c ) which was coincident with the ifa results against n protein expression (fig. 3a) . to assess their genetic stability, the rescued viruses n 7/5-11, n 8/5-12 and n 9/5-13 were serially passaged five times to establish virus stocks of p 1-5 virus. the supernatants of each passage were collected, and the cdna fragment [nucleotides (nt) 14413-15497] containing orf7 was amplified by rt-pcr. nucleotide sequencing analysis confirmed that the engineered deletions were retained in all of the passaged viruses. however, several spontaneous mutations appeared when compared with wild-type virus aprrs. another independent transfection experiment was carried out to confirm the occurrence of spontaneous mutations. the spontaneous mutations of the n-terminal deletion mutant viruses at p5 in two independent experiments were summarized in fig. 5a . almost all of the spontaneous mutations were different in two experiments. the spontaneous mutation nt t15355c (aa s120p) existed in n 9/5-13 was identical with that in mutant virus n 8/5-12 in experiment 1. most of the spontaneous mutations emerged at p1 could be stably passaged to p5. given that n plays multiple functions in virus replication process, it was necessary to investigate whether additional genetic alterations were present in other genomic regions. in doing so, a total of five overlapping cdna fragments were amplified by rt-pcr, followed by direct nucleotide sequencing. the consensus sequence of the full-length genome of mutant virus n 9/5-13 (p5) was assembled and showed no other detectable mutations besides the detected s120p mutation in n coding region. we also determined the sequence of 5 utr, 3 utr and m coding region of other mutant viruses, and found no additional genetic alterations. to further quantitatively assess the growth behavior of the recovered n-terminal deletion mutant viruses in cultured cells, multiple-step growth curves were determined for p5 of rescued viruses n 7/5-11, n 8/5-12 and n 9/5-13. as depicted in fig. 3d , virus n 7/5-11 and n 8/5-12 had similar growth kinetics with the parental aprrs. the mutant virus n 9/5-13 reached the peak titer at 72 h post-infection (hpi), which was 12 h delayed compared with aprrs, and the titer was nearly 100-fold lower than that of the wild type aprrs (fig. 3d) . this suggested that a larger deletion at the n-terminal of the n protein adversely affected virus replication process, and the spontaneous mutations in the rescued viruses may account for the growth difference. we next attempted to define the role of the c-terminal unstructured residues for virus replication. upon transfection of c-terminal deletion mutants, the intracellular expression of nsp2 and n protein were ifa positive for all mutants (fig. 4a ), though only a few positive cells were detected for pc 5/119-123 and pc 6/118-123, indicating the rna synthesis level was reduced. moreover, the marc-145 cells transfected with mutants pc 3/121-123 and pc 4/120-123 developed typical prrsv cpe, albeit it was delayed by 48 h compared with the parental p7usc. no cpe was observed for the larger deletion mutants pc 5/119-123 and pc 6/118-123, in spite of three additional passages. this was confirmed by rt-pcr with sf14413/sr15497 which showed no sign of infectious particles. these results indicated that the last 4 aa ( 120 sps 123 a) at the c terminus of type 2 prrsv n protein were dispensable for virus viability, which was 2 aa upstream of the previous result based on lv strain (verheije et al., 2001) . the recovered c-terminal deletion viruses were also passaged for assessment of their genetic stability. the presence of the engineered deletions was confirmed by rt-pcr and nucleotide sequencing at all five passages for the recovered viruses c 3/121-123 and c 4/120-123. however, direct nucleotide sequencing of the rt-pcr products of c 3/121-123 displayed ambiguous sequence at some positions of orf7, suggesting that the rt-pcr products contained a mixed population. we then performed plaque purification assay for transfectant virus c 3/121-123 (p0). a total of 20 plaques were picked and nucleotide sequencing of rt-pcr product (nt 14413-15497) displayed no ambiguity for an individual plaque. collectively, we found three different types of spontaneous substitutions including nt t15178c, a15179g and a15319g, which resulted in aa y61h, y61c and t108a substitution, respectively (fig. 5b) . surprisingly, two types of additional deletions (aa 39-42 and 48-52) were found (fig. 5b) . in an effort to assess the variety of the spontaneous mutations, we repeated the transfection experiment, plaque purification and rt-pcr, which showed only one type of additional mutation (nt a15179g (aa y61c)) (fig. 5b) . mutant virus c 4/120-123 was analyzed in the same manner. the analysis revealed different spontaneous mutations in two independent transfections, including e51g substitution and a 3-aa insertion (fig. 5b) . the full-length genome sequence of c 4/120-123 (p5) containing the e51g mutation showed no further mutation in the other genomic regions. surprisingly, the 9-nt sequence (acagtgctt) was inserted between 15341t and 15342t. the fusion sequence rendered the encoded aa 115 i unchanged, and followed by a stretch of 3 aa (qcf) insertion before the last 4 truncated aa ( 116 rvt 119 a) (fig. 5b) . nucleotide sequence comparison showed that the inserted 9-nt was a direct repeat of nt 1171-1179 (gq330474) of nsp1 coding region, implying possible non-homologous rna recombination between these two regions. growth kinetics were assessed for two plaque-purified viruses (ppvs) of c 3/121-123 generated in the experiment 1 (fig. 5b) , including ppv7 (c 3/121-123 containing the y61c mutation) that also was present in the repeated experiment, and ppv10 (c 3/121-123 containing residues 48-52 deletion) that had the maximal internal deletion (fig. 5b ). for c 4/120-123, the p5 virus containing the e51g mutation was analyzed. fig. 4b depicts the growth curves of these mutant viruses, compared with the parental 7usc and aprrs. the c 3/121-123 ppv7 had a slightly lower titer than 7usc at each time point. however, the titers of c 3/121-123 ppv10 and c 4/120-123 were approximately 100-fold lower than that of 7usc, and their peaks appeared at 84 and 72 hpi, respectively, which was noticeably delayed (fig. 4b ). this suggested that the absence of the last four residues ( 120 sps 123 a) and the internal region ( 48 knpe 52 k) could adversely affected virus replication. to further investigate whether the emergence of internal aa deletions was the result of the primary engineered deletion, or the internal aa residues per se were not required for n protein functionality, we introduced two internal deletions found in c 3/121-123 ppvs back into the wild-type paprrs, named as pn 39-42 and pn 48-52, respectively (fig. 2c) . meanwhile, two double mutants containing the originally engineered c-terminal 3-aa deletion, together with second-site mutation y61c and the residue 48-52 deletion were reconstructed. the resultant plasmids were desigfig. 7 . multiple alignment of the first 60 aa of n protein of five different ppvs of recovered n 48-52, the right panel indicates the plaque numbers of each ppv. aprrs is the wild-type, whereas pn 48-52 indicates the engineered deletion. n 48-52 ppv2, ppv3, ppv5, ppv8 and ppv20 represent the different ppvs of the mutant virus n 48-52. the residues that match aprrs are obscured, and differed from the sequence of pn 48-52 are labeled. the short line indicates the deleted residues in pn 48-52 and recovered viruses. the 5-aa insertion in ppv20 is indicated by the brackets. nated as pc 3/y61c and pc 3/n 48-52, respectively (fig. 2d) . all of these mutant plasmids displayed nsp2 and n expression (fig. 6a) and developed visible cpe after transfection, suggesting that the internal aa residues ( 39 kgp 42 g) and ( 48 knpe 52 k) were not essential for virus viability, regardless of the presence or absence of the primary deletion. in view of the frequent occurrence of the second-site mutations, the genetic stability of these reconstructed mutants was also investigated, but no further additional mutation was identified for recovered viruses n 39-42 and c 3/y61c at all passage levels in the two independent transfection assays. however, mutant virus c 3/n 48-52 had yet one additional point mutation, nt a15085g (aa i30v), which was genetically stable for at least five passages. intriguingly, no additional mutation appeared within n in the independent transfection experiment 2 in all five passages. in the case of n 48-52, sequence analysis indicated that the recovered viruses consisted of a mixed population in both independent experiments; viral plaque purification was therefore performed for n 48-52 viruses in experiment 1. eighteen plaques were selected and sequence analysis showed five different second-site mutations, including g40r&k44e (ppv5 in fig. 7) , and four different substitutions together with 48/52k/k repaired, as summarized in fig. 7 . we further determined the master sequence of the full-length genome sequence of ppv3, no detectable genetic alteration was found in the other region of the viral genome besides n gene. it is also worth noting that the five aa (pgpvm) insertion between residues 21 and 22 of the n protein in virus n 48-52 ppv20 resulted in an unusual length of n protein (125 aa). sequence comparison showed that the inserted 15-nt (cccgggccctgtcat) was identical to a region in orf1 (nt 12141-12155, gq330474), which coded for nsp12 (ziebuhr et al., 2000) . whether genomic recombination between these two regions or some other factors plays a role in this process is under investigation. two of the p5 ppvs of n 48-52 (ppv3 and ppv20 in fig. 7) , together with recovered viruses n 39-42, c 3/y61c and c 3/n 48-52 containing the i30v substitution, were used to assess their growth properties. these viruses exhibited indistinguishable growth kinetics from the wild type aprrs (fig. 6b) , which suggested that the introduced or induced internal deletions did not affect virus replication significantly. the n protein could tolerate the internal deletion per se, which might not be related to the c-terminal engineered deletion. n protein is the most abundant and important structural protein in prrsv virion, and plays a crucial role in virion assembly. in this study, we found that the n-terminal residues 5-13 and last four c-terminal residues were non-essential for type 2 prrsv viability. in addition, we also demonstrated that deletion in the middle region of n protein did not block the production of infec-tious virus. our study is believed to be the first report that the inter-genotypically variable n terminal and internal residues of n protein could tolerate deletion without affecting type 2 prrsv viability in cultured cells, while discrete inter-genotypically conserved terminal residues play crucial roles in viral rna synthesis and/or virus growth. the nonessential regions identified here could be further utilized as insertion site for foreign gene tag and the rescued viruses could be the candidates for genetic marker vaccine. the n protein of coronavirus has been shown to participating virus rna synthesis through interacting with the transcription regulatory sequence (grossoehme et al., 2009) or as the rna chaperone (zuniga et al., 2007 (zuniga et al., , 2010 . for arterivirus, it was documented that all of the structural proteins are not required for genomic rna replication and sgmrna transcription of eav (molenkamp et al., 2000) . in this study, we demonstrated that both terminal and internal domains contained aa residues that are nonessential for virus viability. however, the replication and transcription level of some mutants were reduced, indicating prrsv n protein and/or its coding sequence may affect viral rna synthesis. in comparison with the prrsv n amino acid sequence (fig. 1a) , we found that the inter-genotypically conserved residue g14 was the only difference between viable pn 9/5-13 and non-viable pn 10/5-14. it was possibly that the g14 sequence specificity was required for virus viability, which needs further site-directed mutagenesis. we also proved that the internal residues ( 39 kgp 42 g) and ( 48 knpe 52 k), respectively, were not essential for virus viability. interestingly, these regions sandwiched the reported nls ( 41 pgkk (n/s) k 47 k) of n protein (rowland et al., 1999 . lee et al. (2006) demonstrated the nls-null mutant clone produced infectious virus, and the rescued virus displayed a titer 100-fold lower than that of wild-type virus. the mutagenized nls underwent strong selection pressure in pig that resulted in partial or complete reversion and reacquisition of nls function (pei et al., 2008) . in our study, the rescued virus n 48-52 also displayed additional mutation when transfected into the marc-145 cells. whether the rescued virus n 48-52 undergoes selective pressure in cells still needs further study. the maximal deletion mutant virus n 9/5-13 and c 4/120-123 had severe defect on virus growth kinetics, as shown in figs. 3d and 4b, suggesting these deletions might adversely affect virus growth. it was also worth noting that the clone pc 3/n 48-52 had identical sequence of n protein with ppv10 of c 3/121-123. however, the growth kinetics of ppv10 was much lower than that of c 3/n 48-52 (figs. 4b and 6b); the only difference was that the latter had one more additional mutation i30v in n protein. further investigation on whether the i30v substitution determines the different growth property is under way. there are at least two experimentally identified rna signals involving in rna synthesis at the 3 -terminus of arterivirus. one is the pseudoknot interaction between two 3 -proximal stem loops (sl4 and sl5) demonstrated in eav system snijder, 2006, 2007) . however, the nucleotides involved in the pseudoknot interaction in prrsv are located in 3 utr. another is the kissing loop structure identified in lv strain, verheije et al. (2002) reported that a stretch of 34 nt within lv orf7 is essential for rna replication because of a kissing interaction between the 7-nt sequence in orf7 and its complementary counterpart in 3 utr. in this study, the mutant pn 16/5-20 deleted part of this corresponding structure, including the putative 7-nt kissing-loop sequence in aprrs orf7 (fig. 1b) . therefore, we deduced that such a kissing-loop interaction might also play a role in type 2 prrsv replication, which was supported by the failure of fluorescent signal and sgmrna7 detection in transfected cells (fig. 3a, c) . as a multi-functional protein, viral n protein is known for interacting with other structural protein for virion assembly (kuo and masters, 2002) , and with rna for genome encapsidation (doan and dokland, 2003) . therefore, it is necessary to investigate whether genetic alterations exist in other genomic regions. we determined the nucleotide sequences of 5 utr, 3 utr and m region of all mutant viruses, however, no obvious genetic alterations were observed in these regions. therefore, the n protein domains that may be responsible for protein-rna and protein-protein interactions are most likely located outside of the mutated region. intriguingly, we found that a stretch of 9 nt sequence (acagtgctt, nt 1171-1179) insertion in n coding region of mutant virus c 4/120-123 was a direct repeat from nsp1 coding region. meanwhile, another 15-nt (cccgggccctgtcat, nt 12141-12155) found in virus n 48-52 ppv20 was part of the nsp12 coding sequences. as the 9 nt insertion was out-of-frame, we speculate that such recombination most likely happened at the rna level via non-homologous rna recombination. we demonstrated that the n protein truncated viruses were accompanied by multiple patterns of spontaneous mutations, whereas few missense mutations were found in other genomic regions of the p5 mutant viruses n 9/5-13, c 4/120-123, n 48-52 ppv3, and wild-type aprrs. we cannot completely rule out the possibility that quasispecies nature may account for some of the additional mutations, as we essentially determined the consensus sequences of the mutant viruses by direct sequencing of the rt-pcr products. another reason for the lack of mutation is that the parental virus, aprrs, is highly adapted on marc-145 cells, which could limit the number of the quasispecies. more importantly, these spontaneous mutations are often in the forms of deletions and insertions, which could not be simply attributed to the quasispecies nature. our results suggested that the spontaneous mutations in n protein were caused by the introduced deletion rather than replication error. n protein is one of the genetically conserved structural proteins. on the contrary, we found various patterns of spontaneous mutations generated from the initial terminal deletion of n protein. in present study, we did repeat transfection to investigate the properties of the spontaneous mutations. however, almost all of the recovered viruses had different additional mutations in the independent transfection assay. for example, point mutation and out-of-frame insertion were found in the recovered virus c 4/120-123 in two experiments (fig. 5b) . also the same spontaneous mutation might respond to different primary deletions, such as s120p that arose simultaneously in the recovered viruses n 8/5-12 and n 9/5-13. on the other hand, most of the spontaneous mutations were located at the n-terminal domain of n protein, the structure of which is still unresolved. therefore, it was difficult to illustrate any spatial relationship between the primary deletion and spontaneous mutations described in our study. in addition, we did not find a clear pattern of local charge compensation for the spontaneous mutations. another explanation of the spontaneous mutations would be the role of the underlying rna secondary structure. we also analyzed the local secondary structure of all mutants, but no significantly change was found. except for pn 16/5-20, none of the other mutants and the recovered viruses affects the potential kissing loop structure of n protein. therefore, the inherent mechanism still needs further manipulation of n protein and structural analysis of the n-terminal domain of n protein. taken together, we dissected the prrsv structure-function relationship and found that (1) a stretch of inter-genotypic variable n terminal residues aa 5-13 ( 5 ngkqqkkk 13 k) were nonessential for virus viability; (2) the last four inter-genotypic variable aa residues ( 120 sps 123 a) at the c terminus of n protein were dispensable for type 2 prrsv viability; (3) the internal aa residues ranging from inter-genotypic variable aa 39-42 ( 39 kgp 42 g) and inter-genotypic conserved 48-52 ( 48 knpe 52 k), respectively, were dispensable for type 2 prrsv viability. our results indicated the non-essential regions in n protein for prrsv replication in cell culture, which lays a foundation for foreign gene expression and development of genetic tagged vaccine. structural polypeptides of the american (vr-2332) strain of porcine reproductive and respiratory syndrome virus rna signals in the 3 terminus of the genome of equine arteritis virus are required for viral rna synthesis an rna pseudoknot in the 3 end of the arterivirus genome has a critical role in regulating viral rna synthesis characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr-2332) sequences of 3 end of genome and of 5 end of open reading frame 1a of lactate dehydrogenase-elevating virus and common junction motifs between 5 leader and bodies of seven subgenomic mrnas mapping of the rna-binding domain of the porcine reproductive and respiratory syndrome virus nucleocapsid protein gp3 is a structural component of the prrsv type ii (us) virion all subgenomic mrnas of equine arteritis virus contain a common leader sequence current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily equine arteritis virus subgenomic mrna synthesis: analysis of leader-body junctions and replicativeform rnas structure of the equine arteritis virus nucleocapsid protein reveals a dimer-dimer arrangement structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus nidovirales: evolving the largest rna virus genome coronavirus n protein n-terminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication a nested set of six or seven subgenomic mrnas is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus identification and characterization of a sixth structural protein of lelystad virus: the glycoprotein gp2 encoded by orf2 is incorporated in virus particles subgenomic rnas of lelystad virus contain a conserved leader-body junction sequence the arterivirus replicase is the only viral protein required for genome replication and subgenomic mrna transcription porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association the localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus the molecular biology of arteriviruses cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid definition of subtypes in the european genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in europe proteins encoded by open reading frames 3 and 4 of the genome of lelystad virus (arteriviridae) are structural proteins of the virion viable porcine arteriviruses with deletions proximal to the 3 end of the genome kissing interaction between 3 noncoding and coding sequences is essential for porcine arterivirus rna replication lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: a review of mystery swine disease research at lelystad homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein the 2b protein as a minor structural component of prrsv colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae reverse genetic manipulation of the overlapping coding regions for structural proteins of the type ii porcine reproductive and respiratory syndrome virus construction of infectious cdna clones of prrsv: separation of coding regions for nonstructural and structural proteins recombinant prrsv expressing porcine circovirus sequence reveals novel aspect of transcriptional control of porcine arterivirus virus-encoded proteinases and proteolytic processing in the nidovirales coronavirus nucleocapsid protein is an rna chaperone coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription this study was co-sponsored by the natural science foundation of china (#30972204) and the european union (seventh framework program; project no. 245141) to s.y. key: cord-285180-32bxx94u authors: lee, sunhee; lee, changhee title: functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus date: 2015-10-02 journal: virus res doi: 10.1016/j.virusres.2015.06.013 sha: doc_id: 285180 cord_uid: 32bxx94u porcine deltacoronavirus (pdcov) is a newly discovered enterotropic swine coronavirus that causes enteritis and diarrhea in piglets. like other coronaviruses, pdcov commonly contains 4 major structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. among these, the n protein is known to be the most abundant and multifunctional viral component. therefore, as the first step toward understanding the biology of pdcov, the present study investigated functional characteristics and expression dynamics of host proteins in a stable porcine cell line constitutively expressing the pdcov n protein. similar to n proteins of other coronaviruses, the pdcov n protein was found to interact with itself to form non-covalently linked oligomers and was mainly localized to the nucleolus. we then assessed alterations in production levels of proteins in the n-expressing pk (pk-pdcov-n) cells at different time points by means of proteomic analysis. according to the results of high-resolution two-dimensional gel electrophoresis, a total of 43 protein spots were initially found to be differentially expressed in pk-pdcov-n cells in comparison with control pk cells. of these spots, 10 protein spots showed a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots and were picked for subsequent protein identification by peptide mass fingerprinting following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. the affected cellular proteins that we identified in this study were classified into the functional groups involved in various cellular processes such as cell division, metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication. notably, two members of the heat shock protein 70 family were found to be up-regulated in pk-pdcov-n cells. these proteomic data will provide insights into the specific cellular response to the n protein during pdcov infection. porcine deltacoronavirus (pdcov) is an emerging viral pathogen that was first reported in 2012 in hong kong, china (woo et al., 2012) . in february 2014, the detection of pdcov was first announced in ohio, united states, in conjunction with outbreaks of diarrhea without other etiologic agents. since its emergence, this novel coronavirus has been detected in 17 us states, and almost 80% of the tested samples corresponded to cases of coinfection of pdcov with other enteric viral pathogens such as a rotavirus or porcine epidemic diarrhea virus (li et al., 2014; utr. orf1a/b encompasses two-thirds of the genome encoding 2 overlapping viral replicase polyproteins, 1a and 1ab, which are then proteolytically processed into mature nonstructural proteins. as in other coronaviruses, production of polyproteins 1a and 1ab requires a −1 ribosomal frameshift during translation of the genomic rna. the last third of the genome encodes the 4 structural proteins, spike (s), envelope (e), membrane (m), and nucleocapsid (n), as well as two accessory genes, nonstructural gene 6 (ns6) and ns7 gene, between m and n, and within n, respectively (lai et al., 2007; lee and lee, 2014; li et al., 2014; marthaler et al., 2014a; woo et al., 2012) . among the structural proteins of coronaviruses, the n protein is abundantly produced in infected cells and has multiple functions in viral replication and pathogenesis (mcbride et al., 2014) . as the sole structural component of the viral capsid, the n protein of coronaviruses interacts with the nucleic acid and itself for selfassociation to protect the viral genome from extracellular agents, serving as the critical basis for ribonucleoprotein (rnp) complexes during virus assembly (mcbride et al., 2014) . the entire life cycle of coronaviruses takes place in the cytoplasm of infected cells, and accordingly, the n protein is distributed mainly in the cytoplasmic compartments. in addition to their cytoplasmic localization, coronaviral n proteins are commonly localized to the nucleolus, suggesting their non-structural functions in ensuring successful virus infection mcbride et al., 2014) . although a variety of studies have confirmed that n possesses multifunctional significance in coronavirology (mcbride et al., 2014) , detailed characteristics of this protein and its role in the replication of pdcov remain unknown. in the present study, alterations in cellular gene expression that are caused by the n protein were evaluated as a first step toward understanding the biological role of the n protein in pdcov replication. to accomplish this task, stable porcine-origin cell lines constitutively expressing the pdcov n protein were generated and characterized in this study. changes in expression patterns of various cellular proteins in the n protein-expressing porcine cells in comparison with control cells were examined by proteomic analysis at different time points. our proteomic data are expected to provide novel information for better knowledge of the properties and functions of the n protein during pdcov infection. hek-293t cells (crl-1573) were purchased from the american type culture collection (atcc, manassas, va) and cultured in dulbecco's modified eagle medium (dmem) with high glucose (invitrogen, carlsbad, ca) with 10% fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions (100×; invitrogen). pk-15 cells were grown in rpmi 1640 medium (invitrogen) supplemented with 10% fbs and antibiotic-antimycotic solutions (100×). the cells were maintained at 37 • c in an atmosphere of humidified air containing 5% co 2 . pam-pcd163-n cells that stably express the n protein of porcine reproductive and respiratory syndrome virus (prrsv; sagong and lee, 2010) were cultured in rpmi 1640 medium (invitrogen) supplemented with 10% fbs, antibiotic-antimycotic solutions (100×), 10 mm hepes (invitrogen), 1 mm sodium pyruvate (invitrogen), and nonessential amino acids (100×; invitrogen) in the presence of 50 g/ml zeocin (invitrogen) and 200 g/ml g418 (invitrogen). the anti-prrsv nucleocapsid (n) monoclonal antibody (mab) and anti-myc mab were purchased from median diagnostics (chuncheon, south korea) and invitrogen, respectively. antibodies to the 6× histidine tag, glucose-regulated protein 78 (grp78), heat shock cognate 70-kda protein (hsc70), ␤-actin, ␣-tubulin, and sp1 were purchased from santa cruz biotechnology (santa cruz, ca). dna manipulation and cloning were performed according to standard procedures (sambrook and russell, 2001) . the escherichia coli strain dh5␣ (rbc bioscience, taiwan) was used as the host for general cloning. the full-length n gene was amplified from the pdcov knu14-04 strain (lee and lee, 2014) with the following primer pair: knu14-04-n-fwd (5 -gccggtcgacatggctgcaccagtag-3 ) and knu14-04-n-rev (5 -cggctctagacgctgctgattcctgc-3 ), where underlines indicate the sali and xbai restriction enzyme sites, respectively. the pcr amplicon was initially inserted into the pbudce4.1 vector (invitrogen) that contains a myc epitope and 6 repetitive histidine codons, and the resulting plasmid pbud-pdcov-n was verified by nucleotide sequencing. because the pdcov genome contains one accessory ns7 gene within the n gene in a different reading frame, the presence of ns7 could affect our functional studies of the pdcov n protein. to prevent any such side effects, the translation initiation codon and the fifth codon of the ns7 orf were modified to disrupt its expression by pbud-pdcov-n. to accomplish this, overlapping pcr was conducted to simultaneously change the atg start codon and the fifth codon of the ns7 gene to tta and taa at genomic nucleotide positions 24,096-24,098 and 24,108-24,110, respectively, using pbud-pdcov-n as a template with the following primers for the t24097c/t24109a mutation: ns7-ko-fwd (5 -ggcaacggagttccgctaaactccgccatc-3 ) and ns7-ko-rev (5 -gatggcggagtttagcggaactccgttgcc-3 ), where lowercase letters indicate the mutated nucleotides. both mutations were translationally silent with respect to the orf encoding the n protein, and the resulting plasmid pbud-pdcov-n w/ons7 was verified by nucleotide sequencing. a fragment of pdcov n w/ons7 cdna that was prepared from pbud-pdcov-n was then subcloned into the pfb-neo retroviral vector (stratagene, la jolla, ca) using the sali and ecori restriction sites to construct the pdcov n gene expression plasmid pfb-neo-pdcov-n-myc/his that produces recombinant n protein. the retrovirus gene transfer system (stratagene) was used to generate cell lines constitutively expressing the recombinant pdcov n gene or an empty vector only as described elsewhere nam and lee, 2010; oh and lee, 2012) . antibioticresistant continuous cell clones were analyzed by rt-pcr to determine the presence of the full-length n gene, and the positive clones (pk-pdcov-n and pk-neo) were amplified for subsequent experiments. pk-pdcov-n cells were grown on microscope coverslips placed in 6-well tissue culture plates. at 48 h post-seeding, the cells were 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 using 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with an anti-his tag or anti-myc antibody for 2 h. after washing 5 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). the cells were finally counterstained with 4 ,6-diamidino-2phenylindole (dapi; sigma, st. louis, mo), and the cell staining was visualized by the fluorescence leica dm il led microscope (leica, wetzlar, germany) or a confocal laser scanning microscope (carl zeiss, gattingen, germany). the n expression in pk-pdcov-n cells was analyzed by flow cytometry. the cells were trypsinized at 48 h post-seeding and centrifuged at 250 × g (hanil centrifuge fleta 5) for 5 min. the cell pellet was washed with cold washing buffer (1% bsa and 0.1% sodium azide in pbs), and 10 6 cells were resuspended in 1% formaldehyde solution in cold wash buffer for fixation at 4 • c in the dark for 30 min followed by centrifugation and incubation of the pellet in 0.2% triton x-100 in pbs at 37 • c for 15 min for permeabilization. after centrifugation, the cell pellet was resuspended in a solution of the primary anti-his tag antibody or normal mouse igg1 (santa cruz biotechnology) and the mixture was incubated at 4 • c for 30 min. the cells were washed and allowed to react with an alexa fluor 488-conjugated anti-mouse igg secondary antibody at 4 • c for 30 min in the dark. the stained cells were washed again and analyzed on the bd facsaria iii flow cytometer (bd biosciences, belford, ma). the growth properties of cells expressing pdcov n protein and control cells were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay (sigma) detecting cell viability as described previously (kim and lee, 2013) . all mtt assays were performed in triplicate. whole cell lysates were prepared from pk-pdcov-n cells grown at 5 × 10 5 cells/well in 6-well tissue culture plates at indicated time points using lysis buffer as described previously . for cell fractionation, pk-pdcov-n cells were fractionated using the nuclear/cytosol fractionation kit (biovision, mountain view, ca) according to the manufacturer's manuals. the protein concentrations of the cell lysates were determined using the 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. equal amounts of total protein were separated in a nupage 4-12% gradient bis-tris gel (invitrogen) under non-reducing or reducing conditions, and electrotransferred onto an immobilon-p membrane (millipore, billerica, ma). the membranes were blocked with 3% powdered skim milk (bd biosciences) 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 with the primary antibody against the 6× his-tag, grp78, hsc70, or ␤-actin at 4 • c overnight. the blots were then incubated with a horseradish peroxidase (hrp)-labeled goat anti-mouse igg or goat anti-rabbit igg antibody (santa cruz biotechnology) at the dilution of 1:2000 for 2 h at 4 • c. finally, the proteins were visualized by enhanced chemiluminescence (ecl) reagents (amersham biosciences, piscataway, nj) according to the instructions of the manufacturer. to analyze the expression kinetics of cellular proteins in pk-pdcov-n cells, the band density of each protein was quantified relative to ␤-actin using densitometry with the wright cell imaging facility (wcif) version of the imagej software package (http://www. uhnresearch.ca/facilities/wcif/imagej/). a chemical cross-linking assay was performed as described previously . briefly, pk-pdcov-n cells and pam-pcd163-n cells were grown in a 6-well tissue culture plate for 48 h. for cross-linking studies, the cells were washed twice with cold pbs and then incubated at rt for 30 min with a 2 mm solution of a membrane permeable and thiol-cleavable cross-linker, 3,3dithiobis(succinimidyl propionate) (dsp; pierce), dissolved in 10% dimethyl sulfoxide (v/v in pbs). the reaction was quenched with 50 mm tris-hcl [ph 7.5] and incubated for an additional 15 min. the cells were lysed with lysis buffer, and the resultant cell lysates were then subjected to a western blot assay as described above. pellets of cultured cells were washed twice with ice-cold pbs, placed in sample lysis solution consisting of 7 m urea, 2 m thiourea, 4% 3-[(3-cholamidopropyl) dimethyammonio]-1propanesulfonate (chaps), 1% dithiothreitol (dtt), 2% pharmalyte (ph 3.5-10, amersham biosciences), and 1 mm benzamidine, and were then sonicated for 10 s using a sonoplus device (bandelin electronic, germany). the samples were subsequently incubated for 1 h at 4 • c. after centrifugation at 15,000 × g for 1 h at 4 • c, insoluble cellular debris were discarded, and the supernatant was collected and stored at −80 • c until use. the protein concentrations were measured by the bradford assay as described previously (bradford, 1976) . protein samples were analyzed by 2de using commercial ipg dry strips (ph 4-10, 24 cm; genomine inc., pohang, south korea) for the first-dimensional separation (isoelectric focusing; ief) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) for the second dimension. the ipg dry strips were loaded independently with 200 g of each protein sample and rehydrated overnight in rehydration buffer (7 m urea, 2 m thiourea, 2% chaps, 1% dtt, and 1% pharmalyte). ief was performed at 20 • c using a multiphor ii electrophoresis unit and an eps 3500 xl power supply (amersham biosciences) with the following parameters: gradient increase from 150 to 3500 v for 3 h and 3500 v for a total of 96,000 v h. following ief separation, the ipg strips were incubated for 10 min at rt with gentle shaking in equilibration buffer (50 mm tris-cl ph 6.8, containing 6 m urea, 2% sds, and 30% glycerol) containing 1% dtt and were subsequently incubated in the equilibration buffer containing 2.5% iodoacetamide under the same conditions. each equilibrated strip was then inserted into 10-16% polyacrylamide gels (20 cm × 24 cm) and sds-page was run using a hoefer dalt 2d system (amersham biosciences) at 20 • c for 1700 v h according to the manufacturer's instructions. the 2d gels were stained by the silver staining method as described previously with some modifications (oakley et al., 1980) . to compensate for the variability of 2de, three independent experiments were conducted for further statistical analysis. the stained gels were imaged using a high-resolution 2d gel ccd image analyzer, dyversity (syngene, frederick, md), and quantitative analysis of the digitized gel images was carried out using the pdquest software (version 7.0, bio-rad, hercules, ca) according to the protocols provided by the manufacturer. data representing 3 independent 2de experiments for each sample were statistically analyzed using student's t-test, and p-values of less than 0.05 were considered to be statistically significant. the quantitative data from each protein spot were normalized based on the total valid spot intensity for each gel. only protein spots that showed significant differential expression (p < 0.05) with a ±2-fold consistent change in the expression level in comparison with the control sample were selected for mass spectrometry (ms) analysis. each selected protein spot was excised manually from the silverstained gels and was enzymatically digested in-gel as previously described (shevchenko et al., 1996) and using modified porcine trypsin (sequencing grade; promega, madison, wi). the gel pieces were washed with 50% acetonitrile (acn), air-dried thoroughly at rt, and then rehydrated for 8-10 h at 37 • c with modified sequencing grade porcine trypsin (8-10 ng/l). the proteolytic reaction was terminated by addition of 5 l of 0.5% trifluoroacetic acid (tfa). the tryptic peptides were recovered by combining the aqueous phase from several extractions of gel pieces with 50% aqueous acn. after concentration, the peptide mixture was desalted using c 18 ziptips (millipore), and the peptides were eluted in 1-5 l of 50% acn. an aliquot of this solution was mixed with an equal volume of a saturated solution of ␣-cyano-4-hydroxycinnamic acid (chca; sigma) in 50% acn/0.1% tfa, and 1 l of this mixture was immediately spotted onto the target plate. maldi-tof analysis was performed on a microflex lrf 20 instrument (bruker daltonics, billerica, ma) as described by fernandez et al. (1998) . the spectra were collected from 300 shots per spectrum over the m/z range 600-3000 and calibrated by two point internal calibration using trypsin auto-digestion peaks (m/z 842.5099, 2211.1046). the peak list was generated using flex analysis 3.0. we used the following threshold for peak-picking: 500 for minimum resolution of monoisotopic mass, 5 for s/n. the search program mascot from matrix science (http://www.matrixscience.com/) was used for protein identification by pmf. the following parameters were used for the database search: trypsin as the cleaving enzyme, a maximum of one missed cleavage, iodoacetamide (cys) as a complete modification, oxidation (met) as a partial modification, monoisotopic masses, and a mass tolerance of ±0.1 da. the pmf acceptance criterion was probability scoring. total rna was extracted from the lysates of pk-pdcov-n cells at 24 and 48 h post-seeding by the trizol reagent (invitrogen) and was treated with dnase i (takara, otsu, japan) 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 performed using a thermal cycler dice real time system (takara) with genespecific primer sets as described previously (sagong and lee, 2011) . the primer sequences are available upon request. the rna levels of cellular genes were normalized to porcine ␤-actin mrna, and relative quantities (rq) of mrna accumulation were calculated using the 2 − ct method (livak and schmittgen, 2001) . to detect alterations of cellular mrna levels in the presence of the pdcov n protein, the relative fold change of each cellular gene was calculated in accordance with the comparison of pk-pdcov-n and control pk-neo cells. we used the student's t-test for all statistical analyses, and differences with p-values <0.05 were considered statistically significant. a set of seven novel mammalian and avian coronaviruses was recently discovered, including one porcine coronavirus, pdcov (woo et al., 2012) . pdcov is the fifth coronavirus infecting pigs in addition to transmissible gastroenteritis virus, porcine respiratory virus, porcine hemagglutinating encephalomyelitis virus, and porcine epidemic diarrhea virus (pedv). to date, interactions between this novel virus and the host have not been studied yet. furthermore, alterations of cellular protein expression in response to pdcov or each viral protein upon infection currently remain undetermined. in the current study, the pdcov n protein was chosen in the current study for the proteomic analysis to evaluate specific host cellular responses, which is not only the most abundant viral component but also performs several biological functions in completing viral replication. for this purpose, sublines of pk-15 cells were established to stably express recombinant pdcov n under the control of a retroviral longterminal-repeat (ltr) promoter. ten generated cell clones were initially collected and subjected to rt-pcr and western blotting to confirm the n gene expression at mrna and protein levels, respectively (data not shown). according to the results of the western blot analysis, one pk-pdcov-n cell clone that constitutively expressed the highest levels of n was selected for subsequent studies. to characterize pk-pdcov-n cells, we examined intracellular expression levels of n by ifa, facs analysis and western blotting. as shown in fig. 1a , the specific cell staining was clearly evident when pk-pdcov-n cells were reacted with the anti-his tag antibody, confirming the constant high expression level of the n protein. furthermore, the majority of the cells consistently exhibited specific fluorescent signals, indicating a homogenous population of cells in terms of n expression (fig. 1b) . time-course western blot analysis revealed that the pk-pdcov-n cells stably express and accumulate robust levels of a ∼45 kda recombinant n protein, larger than its predicted molecular weight of approximately 38 kda possibly due to post-translational modifications and the presence of c-terminal myc and histidine tags (fig. 1c ). in addition, the overall growth kinetics of pdcov n gene-expressing pk cells was found to be similar to that of the parental pk-neo cells, indicating that the pdcov n expression has no effect on cell proliferation (fig. 1d) . self-association of the n protein has been observed in many viruses, including coronaviruses, and is essential for assembly of the viral core constructing the basic architecture of viruses. sequence analysis of the pdcov n protein indicated that it is composed of 342 amino acids residues and contains no cysteine residues. as expected, no band corresponding to a disulfide-linked n dimer was detected by sds-page under non-reducing conditions ( fig. 2a, lane 4) , indicating that the pdcov n protein does not undergo cysteine-linked homodimerization. as a positive control, the prrsv n protein in the n-expressing stable pam cells was clearly demonstrated to form 35-kda n-n dimers under the same conditions (sagong and lee, 2010; fig. 2a, lane 2) . the ability of n to form non-covalent dimers was further investigated in a chemical crosslinking experiment. as shown in fig. 2b , the prrsv n protein in pam-pcd163-n cells formed a number of higher-order oligomers (lane 1) as reported previously . when the n protein in pk-pdcov cells was subjected to cross-linking, numerous multimeric forms of the n protein were identified (lane 2), indicating that the n protein of pdcov exists in the form of noncovalently linked oligomers that are used for assembly the viral capsid. we next determined whether the recombinant n protein expressed in pk-pdcov-n cells is subject to nucleolar localization that is known to be a common feature of coronaviral n proteins. the staining pattern in pk-pdcov cells was found to be predominantly cytoplasmic and nucleolic; this pattern persisted for up to 60 h after seeding (fig. 3a) . nearly all cells expressing pdcov n showed distinct fluorescent signals in the nucleolus at 48 h post-seeding, and thereafter, the pdcov n protein was localized mainly to the cytoplasm at 72 h post-seeding. the nucleolar localization of pdcov n was confirmed by transient transfection of bhk-21 or st cells with the plasmid pbud-pdcov-n (fig. 3b) . the cell fractionation assay also revealed the presence of the n protein in both the cytoplasmic and nucleic fractions (fig. 3c) . these observations demonstrated that, like n proteins of other coronaviruses, pdcov n protein is mostly distributed in the nucleolus along with the cytoplasm and has a conserved subcellular localization property. cellular proteins in the parental pk-neo cells and pk-pdcov-n cells were extracted and subjected to 2de analysis to compare the host protein expression profiles. to reduce the variability of gel electrophoresis, three independent 2de analyses of cellular extracts from control or n gene-expressing pk cells were performed, and spot intensity data from triplicate gels were selected for statistical analysis. on average, 1090 ± 63 protein spots were resolved by 2de within a ph gradient 4-10 and were visualized using silver staining; the molecular weights of the spots ranged from 10 to 200 kda. these spots were used for the comparative analysis. fig. 4a shows representative images of 2de gels from control pk cells (upper panel) and n gene-expressing pk cells (middle and lower panels). a total of 43 protein spots were initially found to be differentially expressed in pk-pdcov-n cells when compared with control pk-neo cells. on the basis of the statistical comparison, only those spots that consistently showed alteration in expression levels between pk-pdcov-n cells and the control cells were chosen for further protein identification. to identify the differentially expressed cellular protein spots in pk-pdcov-n cells at different time points, 10 protein spots with a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots (fig. 4b) , were selected and manually excised from the stained gels. subsequently, the trypsin-digested spots were subjected to maldi-tof analysis. with combined pmf and database searching, identity of all 10 proteins was successfully determined. information on all these proteins in pk-pdcov-n cells, with their protein scores and sequence coverage, is summarized in table 1 . to better understand the implications of the cellular responses to the pdcov n protein, we further (c) nuclear and cytoplasmic fractionation of pk cells expressing pdcov n. each nuclear and cytosolic fraction was prepared from pk-pdcov-n cells at indicated time points post-seeding and subjected to western blot analysis with the antibody specific for his-tag (top panel), sp1 as a nuclear protein marker (second panel), or ␣-tubulin as a cytosolic protein marker (third panel). all blots were also reacted with ␤-actin antibody to verify equal protein loading (bottom panel). categorized the identified proteins by biological processes according to the gene ontology database as described previously (zhang et al., 2009) . these proteins showing altered expression were associated with various cellular functions including intracellular transport, metabolic processes, gene regulation, the stress response, protein synthesis, cytoskeleton networks, and cell division. since changes in cellular protein expression may be attributed to alterations in the corresponding mrna levels, transcriptional changes for all the identified proteins were tested by real-time rt-pcr to confirm the results of the proteomic analysis (fig. 5) . we were able to detect mrnas corresponding to nearly all proteins identified by proteomics, except for histone h4. although the altered expression levels of the remaining 9 proteins were consistent with the real-time rt-pcr results, we observed only modest increase or reduction in mrna levels. these results could be explained the case the correlation between mrna and protein abundance could be insufficient to predict protein expression levels from quantitative mrna data (gygi et al., 1999) , suggesting that the differences in protein levels might be due to post-translational modifications or protein stability rather than mrna levels. the quantity of each spot was normalized based on the total valid spot intensity for each gel and the relative fold change of each spot was then calculated between control pk-neo and pk-pdcov-n cells. data representing three independent 2de experiments for each sample were statistically analyzed and error bars represent standard deviations. *p = 0.001-0.05. two members of the hsp70 family, glucose-regulated protein 78 (grp78) and heat shock cognate 70-kda protein (hsc70), were found to be up-regulated in the pdcov n-expressing cells by the proteomic analysis. to further verify the dynamic alterations in fig. 5 . transcriptional alteration of identified cellular genes in pk-pdcov-n cells. the mrna level of each gene was assessed by quantitative real-time rt-pcr and normalized to that of porcine ␤-actin. relative quantities (rq) of mrna accumulation were evaluated by 2 − ct method and the relative fold change of each gene was then calculated between control pk-neo and pk-pdcov-n cells. results are expressed as the mean values from three independent experiments in duplicate and error bars represent standard deviations. *p = 0.001-0.05; † p < 0.001. expression of these stress-response proteins under the influence of pdcov n, we performed time-course western blot analysis. total cellular lysates were prepared from pk-pdcov-n cells at different time points, and the expression kinetics of the grp78 and hsc70 was compared between control and n protein-containing extracts. higher expression of both proteins was first evident in pk-pdcov-n cells as early as at 12 h post-seeding in comparison with control pk-neo cells, and the production of both proteins was persistently high during the later time points, suggesting that their enhanced expression is dependent on the n protein (fig. 6) . these results were consistent with the proteomic analysis of 2de gels and realtime rt-pcr, demonstrating that the synthesis of grp78 and hsc70 is indeed up-regulated in response to expression of the n protein of pdcov. since viruses are obligate intracellular parasites, they must utilize the cellular machinery and biosynthetic components of the host cell for their own replication. after viral infection, the infected cells launch a number of host antiviral defensive responses, which are turned on to eliminate the invading viruses. on the other hand, viruses employ their own evasion strategies to complete their replication and to successfully spread to neighboring cells. this series of interactions between the virus and host results in compensatory modifications in cellular gene production. accordingly, numerous studies have been extensively conducted by means of various molecular and genomic methods to understand how the altered host gene expression affects viral infection and the associated pathogenic processes. the proteomic analysis coupling high-resolution 2de and maldi-tof/ms is a tool that can generate ample data on cellular protein profiles modified by viral replication. with the proteomic techniques, it is now possible to identify relative changes in protein abundance for evaluation of host cellular fig. 6 . differential expression of the hsp70 family in pk-pdcov-n cells. cell lysates were prepared from pdcov n gene-expressing pk cells at indicated time points and immunoblotted to determine the expression profile of each protein with antibodies specific for grp78 and hsc70 (top to second panels). the blot was also reacted with anti-his tag (third panel) and anti-␤-actin (bottom panel) antibodies to confirm the status of n expression and equal protein loading, respectively. each cellular protein expression was quantitatively analyzed by densitometry in terms of the relative density value to the ␤-actin gene and pk-pdcov-n sample results were compared to pk-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. responses to viral infection or viral protein expression and to gain specific insights into the cellular mechanisms involved in viral pathogenesis (alfonso et al., 2004; brasier et al., 2004; neuman et al., 2008; oh and lee, 2012; ringrose et al., 2008; sagong and lee, 2010; zhang et al., 2008) . in the present work, a proteomic approach was applied for profiling the global protein expression changes in host cells in response to the n protein of pdcov, which is a major constituent of the virion and infected cells. therefore, we initially established a porcine cell line stably expressing the pdcov n protein and then characterized the n protein produced in those cells. due to the absence of cysteine residues, a disulfide-linked homodimeric n protein of pdcov was not detected in pk-pdcov-n cells. rather, the pdcov n protein was shown to be self-assembled via non-covalent oligomerization as a structural component for formation of the viral capsid. interestingly, the n protein of pdcov was found to be predominantly present in both the cytoplasm and nucleolus of pk-pdcov-n cells. the localization of nidovirus n proteins to the nucleolus may be necessary for control of rna synthesis or ribosome biogenesis via association with ribosomal subunits and interaction with nucleolar proteins (chen et al., 2002; wurm et al., 2001; yoo et al., 2003; ) . similarly, the pdcov n protein may participate in such a viral strategy to favor viral replication and pathogenesis. in addition, the nucleolar localization sequence detector program (http://www.compbio.dundee.ac. uk/www-nod/) predicted that pdcov n harbors a putative nucleolar localization signal (nols) consisting of a stretch of basic amino acids. therefore, further research is needed to identify a functional nols to confirm the intracellular localization of the pdcov n protein. these biological characteristics of n gene-expressing cells have yet to be confirmed using an authentic n protein in pdcovinfected cells, but according to our results, pdcov n appears to act as a multifunctional protein playing structural and non-structural roles contributing to completion of viral replication. our proteomic data revealed that 10 differentially expressed cellular proteins are identifiable in pdcov n gene-expressing porcine cells. the proteins that we identified in this study are involved in diverse cellular processes: metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication, and cell division. the significance of the functional roles of the selected host proteins affected by the interaction of the pdcov n protein with the host cell is discussed below. the most interesting finding in the present study is that two cellular chaperone proteins belonging to the hsp70 family are up-regulated concomitantly by the n protein of pdcov. the first up-regulated protein is grp78 in cells expressing the pdcov n protein, which is associated with endoplasmic reticulum (er) stress. in eukaryotic cells, the er is the major site for synthesis and folding of transmembrane and secreted proteins, and accordingly, animal viruses also use the er as a site of synthesis and processing of their own proteins. the amount of protein entering the er can differ under physiological and environmental conditions. if protein synthesis exceeds the folding capacity of the er, unfolded proteins accumulate there, resulting in er stress. to maintain the er homeostasis, cells have developed a signaling pathway known as the unfolded protein response (upr) that transmits signals across the er membrane to the cytosol and the nucleus and ultimately reduces protein translation and enhances the er folding capacity by up-regulating chaperone proteins (ron and walter, 2007) . coronavirus infection of cultured cells is known to cause er stress and to induce the upr, which then crosstalks with various cellular signaling pathways, including mitogen-activated protein kinase cascades, autophagy, apoptosis, and innate immune responses, indicating the involvement of upr activation in virus-host interactions and viral pathogenesis . more interestingly, global proteomic and microarray analyses have shown that the expression of chaperon proteins, such as grp78 and grp94, is up-regulated in cells infected with a human coronavirus or in cells expressing the s2 subunit (jiang et al., 2005; yeung et al., 2008) . furthermore, the n protein of pedv, another porcine coronavirus, overexpression was shown to trigger er stress (xu et al., 2013) . thus, it appears that pdcov infection may induce er stress and the upr, leading to the up-regulation of grp78 to counteract the er stress; hence, the n protein may be responsible for this stress response pathway. the second identified hsp70 family protein is hsc70, also known as heat shock 70-kda protein 8 (hspa8). hsc70 is a molecular chaperone with multiple functions in protein folding and trafficking in all eukaryotic cells and in protecting cells from apoptosis or a wide array of stressors such as heat and infection (morano, 2007; powers et al., 2008; takayama et al., 1999) . accumulating evidence has shown that hsc70 plays important roles in certain processes involved in viral infection by modulating cell entry, virion disassembly and assembly, and the cellular antiviral response and apoptosis (chuang et al., 2015; gutiérrez et al., 2010; ivanovic et al., 2007; liu et al., 2013; radhakrishnan et al., 2010; yan et al., 2010) . apoptosis is considered an innate defense mechanism that limits propagation of a virus by eliminating infected cells (everett and mcfadden, 1999) . therefore, many viruses have evolved to employ various strategies that inhibit apoptosis in order to prevent premature cell death, thereby securing sufficient time for progeny production. thus, a conceivable explanation is that pdcov may take the advantage of suppressing apoptosis by up-regulating hsc70 in the early phase of the infection, and the n protein seems to be involved in this mechanism. in addition, hsc70 mediates the nuclear export of the influenza virus ribonucleoprotein complex via interaction with viral proteins m1 and ns2 (watanabe et al., 2006 (watanabe et al., , 2014 . in the present study, we found that pdcov n can be localized to the nucleolus in nexpressing cells. on the other hand, n proteins must be trafficked from the nucleolus to the cytoplasm to accomplish nucleocapsid assembly as well as viral rna synthesis and virus-host interactions. according to our results and existing data, hsc70 may facilitate the export of pdcov n from the nucleolus to complete the viral life cycle. like many other viruses, coronaviruses turn off host protein translation, while continuing to the synthesis of their own gene products to finish viral replication. one of the mechanisms behind this translational suppression is through interaction of the n protein with elongation factor 1␣ (ef1␣), a major translation factor in mammalian cells . in the present study, the amount of another translational factor, ef2, was affected by the host response to the pdcov n protein. although we do not know whether the increased expression of ef2 is related to its binding to pdcov n, such differential expression of a translation factor may be associated with regulation of both host and viral protein synthesis. we also found that expression of the cytoskeletal protein ezrin is significantly enhanced by the n protein. ezrin is a member of the ezrin-moesin-radixin (emr) family of host cytoskeletal proteins that organize the cortical cytoskeleton by mediating interactions between actin and the plasma membrane proteins and function as signal transducers in numerous signaling pathways (neisch and fehon, 2011) . the emr also modulate rna virus infection by regulating stable and dynamic microtubule formation (bukong et al., 2013; haedicke et al., 2008; naghavi et al., 2007) . given the biological features of the emr proteins, the up-regulation of ezrin in our study suggests that the pdcov n protein may manipulate the host cytoskeletal network and cell signaling, possibly to facilitate the processes of viral infection and replication. in contrast, the expression of translocon-associated protein subunit delta (trapd) is suppressed in cells expressing the pdcov n protein, according to our results. trapd is a part of the trap complex that is involved in translocating proteins across the er membrane and in regulating the retention of er resident proteins (fons et al., 2003) . coronaviruses acquire their lipid envelop via budding of the nucleocapsid through the er-golgi intermediate compartment (mcbride et al., 2014) . the down-regulation of trapd may lead to the release of cellular proteins from the er during pdcov replication, which in turn, promotes translocation of envelope-associated viral structural proteins to the er membrane -the site of budding -to facilitate virus assembly. eukaryotic cells reprogram their metabolism to adapt to stress-induced damage in response to environmental stressors. among our differentially expressed proteins, some are either directly or indirectly involved in metabolism. one hypothesis that can explain this result is that production of the n protein affects cellular biosynthesis by modulating the expression of metabolism-related proteins, which in turn remodels the intracellular environment for optimal pdcov replication. in conclusion, to our knowledge, this is the first report of a proteomic analysis of cellular responses to the pdcov n protein. although definite functions of the proteins that we identified here were not determined, it is likely that alterations in their expression are involved in virus-host interactions. to resolve these questions as well as performing various pdcov research, obtaining a korean pdcov isolate that can grow in cell culture is necessary; we are currently working on this task. in future studies, we are planning to assess cellular responses to pdcov by proteomic analysis to validate our present data; the proteins that are differentially expressed in the cells overexpressing pdcov n or in the cells infected with pdcov can be analyzed in detail in order to identify their precise function in the replication of pdcov. on the other hand, one limitation of the proteomic methodology used in this study is the inability to reliably detect low-molecular-weight or low-abundance proteins such as cytokines, which are involved in the host immune response. therefore, more comprehensive works are also needed to expand our present findings and to explore other proteins that may play a role 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for prevention of apoptosis induced by wssv infection glucose-regulated protein 78 as a novel effector of brca1 for inhibiting stress-induced apoptosis colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar rna-associated protein fibrillarin proteomic profiling of hepatitis b virus-related hepatocellular carcinoma in china: a seldi-tof-ms study changes in the cellular proteins of pulmonary alveolar macrophage infected with porcine reproductive and respiratory syndrome virus by proteomics analysis the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha key: cord-325973-e3rxr6oq authors: navarro, ryan; nair, rajeev; peda, andrea; aung, meiji soe; ashwinie, gs; gallagher, christa a; malik, yashpal s; kobayashi, nobumichi; ghosh, souvik title: molecular characterization of canine parvovirus and canine enteric coronavirus in diarrheic dogs on the island of st. kitts: first report from the caribbean region date: 2017-08-15 journal: virus res doi: 10.1016/j.virusres.2017.08.008 sha: doc_id: 325973 cord_uid: e3rxr6oq although canine parvovirus (cpv) and canine enteric coronavirus (ccov) are important enteric pathogens of dogs and have been studied extensively in different parts of the world, there are no reports on these viruses from the caribbean region. during 2015–2016, a total of 104 diarrheic fecal samples were collected from puppies and adult dogs, with or without hemorrhagic gastroenteritis, on the caribbean island of st. kitts (kna). by pcr, 25 (24%, n = 104) samples tested positive for cpv. based on analysis of the complete deduced vp2 amino acid sequences, 20 of the kna cpv strains were assigned to new cpv-2a (also designated as cpv-2a-297a). on the other hand, the vp2 genes of the remaining 5 strains were partially characterized, or could not be sequenced. new cpv-2a was the predominant cpv variant in st. kitts, contrasting the molecular epidemiology of cpv variants reported in most studies from nearby north and south american countries. by rt-pcr, ccovs were detected in 5 samples (4.8%, n = 104). based on analysis of partial m-protein gene, the kna ccov strains were assigned to ccov-i genotype, and were closely related to ccov-i strains from brazil. to our knowledge, this is the first report on detection and genetic diversity of cpv and ccov in dogs from the caribbean region, and underscores the importance of similar studies in the other caribbean islands. viruses are important etiological agents of diarrhea in domestic and wild canids. among them, canine parvovirus (cpv), a member of the family parvoviridae, is a major cause of hemorrhagic gastroenteritis in dogs (decaro and buonavoglia, 2012; miranda and thompson, 2016) . cpv are small, nonenveloped viruses consisting of a single-stranded, negative sense dna (∼5.2 kb) molecule (berns and parrish, 2013; parrish, 1999; reed et al., 1988) . the cpv genome contains two large open reading frames (orf). the right orf encodes 2 structural proteins (vp1 and vp2) by alternative splicing of the same mrnas, whilst the left orf codes for 2 nonstructural proteins (ns1 and ns2). the cpv vp2 capsid protein is antigenically significant, and has been implicated in governing host range restriction, tropism, and viral-host interactions (decaro and buonavoglia, 2012; miranda and thompson, 2016; parrish, 1999) . most studies on molecular epidemiology of cpv are based on the vp2-encoding gene (decaro and buonavoglia, 2012; miranda and thompson, 2016; truyen, 2006) . cpv emerged as a new enteric pathogen of domestic dogs in the late 1970s, possibly through host switching events involving a feline panleukopenia parvovirus, or a closely related virus (berns and parrish, 2013; parrish, 1999) . as a result of accumulation of mutations in the vp2-encoding gene, the original cpv strain (strain cpv-2) eventually got replaced with antigenic variants cpv-2a, cpv-2b, cpv-2c, new cpv-2a, and new cpv-2b that are variously distributed in dog populations worldwide (decaro and buonavoglia, 2012; miranda and thompson, 2016) . although the current cpv vaccines, derived from the original cpv-2 strains, or cpv-2b strains, have been shown to confer protective immunity against cpv disease, and post-vaccination reactions have rarely been encountered in immunized dogs, the emergence of new genetic and antigenic variants underscores the importance of constant http://dx.doi.org/10.1016/j.virusres.2017.08.008 received 16 may 2017; received in revised form 10 july 2017; accepted 21 august 2017 monitoring of evolution patterns of cpv strains circulating in dogs throughout the world (decaro and buonavoglia, 2012; miranda and thompson, 2016) . canine coronavirus (ccov) (family coronaviridae, genus alphacoronavirus, species alphacoronavirus-i) usually cause mild, selflimiting enteritis in dogs, although fatal disease has been observed with a pantropic variant of ccov buonavoglia, 2008, 2011; decaro et al., 2013; pinto et al., 2014) . ccov are enveloped viruses with a single-stranded, positive sense rna (27-31 kb) genome . the ccov membrane (m) protein is the most abundant structural protein and has been shown to elicit antibodies, whilst the spike (s) glycoprotein is the main inducer of virus-neutralizing antibodies. based on analysis of the m-and/or s-protein encoding genes, ccov strains have been classified into at least two genotypes, ccov-i and ccov-ii buonavoglia, 2008, 2011) . recently, ccov-ii strains were further classified into two subtypes, ccov-iia (classical strains) and ccov-iib (strains arising from putative recombination events between ccov-ii and transmissible gastroenteritis virus of swine) buonavoglia, 2008, 2011; le poder, 2011) . ccovs have been detected in canine populations worldwide buonavoglia, 2008, 2011) . the caribbean region has a sizeable dog population, and dogs with diarrhea, including those with hemorrhagic gastroenteritis are routinely presented at veterinary clinics on these islands. although the prevalence and genetic diversity of cpv and ccov in dogs have been extensively studied in different parts of the world including nearby latin american countries, there are no reports on these important canine viruses from the caribbean region so far. we report here the detection and molecular characterization of cpv and ccov strains in dogs with diarrhea on the caribbean island of st. kitts (kna). during 2015-2016, a total of 104 diarrheic fecal samples were collected from puppies and adult dogs, with or without hemorrhagic gastroenteritis, at two veterinary clinics (the ross university school of veterinary medicine clinic, and the ponds veterinary clinic) on the island of st. kitts, caribbean region. the samples were stored at −20°c until further analysis. the present study was conducted in compliance with good laboratory practice (glp). for pcr, viral dna was extracted from the fecal samples using the qiaamp fast dna stool mini kit (qiagen sciences, md, usa). samples were screened for the presence of cpv using a pcr-based detection assay targeting a 583-bp stretch of the 3′-portion of the vp2-encoding gene, as described previously (buonavoglia et al., 2001) . in order to determine the cpv variant, a 1799 bp fragment of cpv genome containing the complete orf of vp2 gene was amplified using a newly designed primer vp2f (5′-atg agt gat gga gca gtt caa cc-3′, corresponding to nucleotide [nt] 2787-nt 2809 of reference strain cpvb), and primer 555rev (buonavoglia et al., 2001) . pcrs were performed using platinum™ taq dna polymerase (invitrogen, ca, usa) following manufacturer's instructions. pcr-grade water was used as the negative control. viral rna was extracted from fecal samples using the qiaamp viral rna mini kit (qiagen sciences, md, usa). for detection of ccovs in fecal samples, rt-pcr based on a partial stretch (409 bp) of m proteinencoding gene was performed as reported previously (pratelli et al., 1999) . rt-pcrs were carried out using superscript ® iii rt (invitrogen, ca, usa) and platinum™ taq dna polymerase (invitrogen, ca, usa) following manufacturers' instructions. we used pcr-grade water as the negative control. for nt sequencing, pcr products were purified using the qiaquick pcr purification kit (qiagen sciences, md, usa) according to manufacturer's protocol. nucleotide sequences were obtained using the abi prism big dye terminator cycle sequencing ready reaction kit (applied biosystems, ca, usa) on an abi prism 3130 genetic analyzer (applied biosystems, ca, usa.). the pcr products were sequenced in both directions. homology search for related cognate sequences was performed using standard nt blast program (basic local alignment search tool, www.ncbi.nlm.nih.gov/blastn). multiple alignments of deduced amino acid (aa) sequences were performed using the clustalw program (version ddbj, http://clustalw.ddbj.nig.ac.jp/) with default parameters. phylogenetic trees were constructed using the mega (v5.2.2) software. the genbank accession numbers for nt sequences of complete, or partial orf of vp2 genes of the kna cpv strains, and partial m-protein encoding genes of the kna ccov strains are shown in table 1 . the federation of st. kitts and nevis is a twin island nation in the lesser antilles of the caribbean region with a total human population of ∼55,000 (map is shown in supplementary fig. s1 ). although there are no official estimates on the canine population of st. kitts, different breeds of domestic dogs, including a local island breed are kept as pets in many households on the island. diarrhea, including hemorrhagic gastroenteritis is prevalent in domestic dogs on st. kitts, as evident from clinical cases that are presented now and then at the two major veterinary clinics (the ross university school of veterinary medicine clinic [rvc] , and the ponds veterinary clinic [pvc]) on the island. in the present study, cpv, or ccov were detected in 30 (28.8%) of the 104 fecal samples obtained from diarrheic dogs at the two veterinary clinics on st. kitts (table 1) . by pcr of the partial vp2 gene, a total of 25 (24%, n = 104) dogs tested positive for cpv. among them, 15 dogs were presented with hemorrhagic gastroenteritis, whilst the remaining 10 dogs had severe diarrhea. all the cpv positive dogs were sporadic cases from different households across the island of st. kitts. the age of the dogs that tested positive for cpv ranged from 3 days up to 3 years of age (table 1) . eighteen of the 25 cpv positive dogs were aged ≤ 6 months, corroborating previous observations that dogs up to 6 months may exhibit a greater risk of infection (decaro and buonavoglia, 2012; miranda et al., 2015) . most of the cpv positive samples were from mixed breeds (table 1) , contradicting a previous observation that purebreds were more susceptible to cpv disease than mixed breeds (kalli et al., 2010) . however, a few other studies have shown that breed may not be a risk factor (miranda et al., 2015) . vaccination is crucial to control and prevent cpv disease (decaro and buonavoglia, 2012; miranda and thompson, 2016) . lack of vaccination, incomplete vaccination schedules, or vaccine failures, primarily due to interference with maternal antibodies may predispose dogs to natural infection. moreover, some studies have raised questions on the complete efficacy of the current cpv vaccines against the cpv antigenic variants, as these vaccines were derived from old cpv-2 strains (decaro et al., , 2009 decaro and buonavoglia, 2012; miranda and thompson, 2016; truyen, 2006) . in the present study, nearly half (11/25) of the cpv positive dogs did not receive any vaccine, whilst 2 puppies did not attain the recommended minimum age of vaccination (6 weeks) at the time of sampling (table 1) . among the vaccinated dogs that tested positive for cpv (4/25), only one animal had received two doses of the vaccine (table 1) . therefore, lack of, or inadequate protective immunity might have predisposed these dogs to cpv disease, although the role/s of other risk factors, such as endoparasitism and unsanitary environments cannot be ruled out (miranda et al., 2015) . on the other hand, no information was available on the immunization status of the remaining cpv positive dogs (table 1) . information on vaccination status was available for only 26 of the 79 dogs that tested negative for cpv. eleven of these dogs received one, or more doses of cpv vaccine before sampling, whilst 15 were not immunized against cpv. to study the genotype nature and evolution of cpv strains circulating in st. kitts, we determined the complete orf nt sequences of vp2 genes of 20 kna strains (table 1 ). the deduced vp2 aa sequences of all the 20 kna cpv strains exhibited 297-a, 426-n and 555-v, and therefore, were classified as new cpv-2a (cpv-2a-297a) strains (table 2 ; supplementary fig. s2 ). on the other hand, the vp2 genes of the remaining 5 kna strains were partially characterized, or could not be sequenced due to insufficient volumes of fecal samples (table 1) . other relevant mutations that may influence viral antigenicity and/or host range have also been reported in many of the recent cpv-2a/2b strains, such as g300d, y324i and t440a (decaro and buonavoglia, 2012; miranda and thompson, 2016) . however, these mutations were not observed in the putative vp2 proteins of the kna new cpv-2a strains (table 2 , supplementary fig. s2 ). the vp2 of kna cpv strains shared 99-100% nt and deduced aa identities among themselves, and with those of other new cpv-2a strains. nucleotide and deduced aa identities of 99% were also observed with the vp2 of the cpv-2, cpv-2a, cpv-2b, new cpv-2b, and cpv-2c strains. by phylogenetic analysis of complete orf nt sequences of vp2 genes, the kna strains clustered together, and were found to be more closely related to new cpv-2a strains than other cpv-2 variants (fig. 1) , corroborating the observations at deduced aa level. although the present study is the first report on cpv variants from the caribbean region, the molecular epidemiology of cpv has been documented in nearby north and south american countries (miranda and thompson, 2016) . in ecuador and usa, cpv-2b and cpv-2c were more prevalent than cpv-2a. only cpv-2c strains have been reported from mexico and paraguay. cpv-2c was the major variant in argentina and brazil (calderón et al., 2012; pinto et al., 2012) . cpv-2b was the major genotype in canada (gagnon et al., 2016) . in uruguay, cpv-2c was the predominant variant during 2006-2010 (miranda and thompson, 2016) . however, new cpv-2a variants emerged in 2010 (pérez et al., 2012) , and went on to become the major cpv-2 variant in uruguay in 2011 (maya et al., 2013) . a recent study reported cpv-2a as the major variant in columbia (duque-garcía et al., 2017) . in the present study, new cpv-2a was found to be the predominant cpv variant in domestic dogs on the caribbean island of st. kitts, contrasting the molecular epidemiology of cpv reported in most studies from north and south american countries. by rt-pcr targeting a partial fragment of the m protein encoding gene, 5 of the 104 samples tested positive for ccovs (table 1) . among these sporadic cases, one sample was from an adult dog with mild enteritis, whilst the remaining fecals were from puppies exhibiting mild to severe diarrhea. in the present study, high quality nt sequences of the partial m-protein gene (369 bp, excluding the 5′-and 3′-end primers) could be obtained for 4 of the kna ccov strains (table 1) . the partial m-protein gene of the kna ccov strains shared 100% nt sequence identities between themselves. with other ccovs, the partial m protein gene sequences of the kna ccov strains shared maximum nt and deduced aa sequence identities of 99% and 100%, respectively, with those of several brazilian ccov-i strains. absolute deduced aa sequence identities were also observed with cognate stretch of reference ccov-i strain 23/03/ita. on the other hand, nt and deduced aa identities of < 91% and < 94%, respectively, were observed with those of ccov-ii (ccov-iia and −iib) strains. alignment of partial deduced m protein aa sequences of the kna ccov strains with those of ccov-i, and ccov-iia and −iib strains is shown in supplementary fig. s3 . by phylogenetic analysis of partial m protein genes, the kna ccov strains were found to be closely related to ccov-i strains from brazil within the ccov-i cluster, and were distantly related to ccov-ii and feline coronavirus strains (fig. 2) . based on analysis of partial m-protein gene, the kna ccov strains were found to belong to ccov-i genotype. whilst the molecular epidemiology of ccov has been studied in different asian and european countries (cavalli et al., 2014; buonavoglia, 2008, 2011; decaro et al., 2010 decaro et al., , 2013 erles and brownlie, 2009; jeoung et al., 2014; mcelligott et al., 2011; ntafis et al., 2013; soma et al., 2011; wang et al., 2016) , there is a dearth of data on distribution of ccov genotypes from the north and south americas. in brazil, ccov-i and ccov-ii strains, including pantropic ccov-iia variants were found to be circulating in the domestic dog population (costa et al., 2014; pinto et al., 2014) . ccov-iia and −iib genotypes have been associated with fatal enteritis in puppies in usa (licitra et al., 2014) . although ccovs were detected at lower frequencies in st. kitts, this is the first report on detection and genotyping table 2 comparison of evolutionary relevant amino acid (aa) residues of putative vp2 proteins of canine parvovirus (cpv) strains detected on the island of st. kitts (kna), caribbean region, with those of cpv-2, cpv-2a, cpv-2b, cpv-2c, new cpv-2a, new cpv-2b, and vaccine strains. the kna cpv strains are underlined. a dot '.' indicates an identical amino acid residue at cognate position of deduced vp2 aa sequence of the concerned cpv strain with that of reference strain cpv-b/usa/1978. alignment of complete deduced aa sequences of putative vp2 proteins of the cpv strains is shown in supplementary fig. s2 . amino acid position a 80 87 93 101 103 232 267 297 b 300 305 321 323 324 375 426 c 440 555 560 564 568 570 variant strain 1978. b the s297a mutation has been found to be fixed in vp2 of recent cpv-2a and cpv-2b strains, and these variants are sometimes designated as new cpv-2a and new cpv-2b strains (decaro and buonavoglia, 2012; miranda and thompson, 2016) . in a recent study, new cpv-2a and new cpv-2b strains have also been designated as cpv-2a-297a and cpv-2b-297a, respectively (zhou et al., 2017) . c amino acid residue 426 of vp2 constitutes the sole basis of differentiating the new cpv-2a (426-n), new cpv-2b (426-d), and cpv-2c (426-e) strains (decaro and buonavoglia, 2012; miranda and thompson, 2016) . d new cpv-2a strains exhibiting g300d are also referred to as cpv-2a-297a300d (zhou et al., 2017) . we reported here the detection and molecular characterization of cpv and ccov on the caribbean island of st. kitts. although pcr/rt-pcr have been used extensively for detection of cpv/ccov in many studies including recent reports, these screening assays have been shown to be relatively less sensitive than qpcr/rt-qpcr (decaro et al., 2005a,b; duque-garcía et al., 2017; kumar and nandi, 2010; miranda and thompson, 2016; ntafis et al., 2013; wang et al., 2016) . one of the limitation of this study was the use of pcr/rt-pcr as screening assays. however, considering the lack of data from the caribbean region, the present study primarily focused on detection and molecular characterization of circulating strains rather than strictly monitoring prevalence. new cpv-2a (cpv-2a-297a) was found to be the predominant cpv variant on st. kitts. cpv-2a has been shown to be the major variant in asia, whilst cpv-2c, or cpv-2b were predominant in most studies from the north and south americas (miranda and thompson, 2016; zhou et al., 2017) . however, the predominance of cpv-2a variants in recent studies from columbia, uruguay, and st. kitts pointed towards the changing epidemiology of cpv in this part of the world. the kna ccov strains were genetically closely related to brazilian ccov-i strains, which may be attributed to the geographical proximity of the caribbean region to latin american countries. although this is the first report on detection and genetic diversity of cpv and ccovs from the caribbean region, the present study was based on a single island. in order to gain vital insights into the molecular epidemiology of these important canine viruses across the entire caribbean region, similar studies are required in the other islands. fig. 2 . phylogenetic tree constructed by the maximum likelihood (ml) method from nucleotide sequences of partial m-protein encoding genes (369 bp) of the kna ccov strains with those of ccov-i, ccov-ii, and feline coronavirus (fcov) strains. the tree was statistically supported by bootstrapping with 1000 replicates, and phylogenetic distances were measured using the tamura-3parameter model of substitution. the clustering patterns of kna ccov strains were validated by creating ml trees with other mathematical models, such as the jukes-cantor model, kimura 2-parameter model, and hasegawa-kishino-yano model (data not shown). in the tree, the positions of the kna ccov strains are shown by dark circles. genbank accession numbers are shown in parentheses. bootstrap values < 70% are not shown. scale bar, 0.05 substitutions per nucleotide. parvoviridae evidence for evolution of canine parvovirus type 2 in italy study of canine parvovirus evolution: comparative analysis of fulllength vp2 gene sequences from argentina and international field strains detection and genetic characterization of canine parvovirus and canine coronavirus strains circulating in district of tirana in albania molecular 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data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2017.08.008. key: cord-332317-wrztpeb8 authors: zhang, xin; shi, hongyan; chen, jianfei; shi, da; dong, hui; feng, li title: identification of the interaction between vimentin and nucleocapsid protein of transmissible gastroenteritis virus date: 2015-03-16 journal: virus res doi: 10.1016/j.virusres.2014.12.013 sha: doc_id: 332317 cord_uid: wrztpeb8 nucleocapsid (n) protein of transmissible gastroenteritis virus (tgev) packages viral rna genome to form a ribonucleoprotein complex. in addition to its function as a structural protein, n protein is involved in cell apoptosis or cell-cycle regulation. n protein possibly interacts with host factors to modulate cellular functions. to identify cellular proteins that interacted with n protein of tgev, methods of gst pull-down and co-ip were utilized to precipitate cellular proteins of swine testicular (st). bound cellular proteins were resolved by sds-page. analysis of interacting proteins by mass spectrometry allowed identification of 15 cellular protein bands representative of 12 cellular proteins including vimentin that bound to n protein. furthermore, the function of vimentin cytoskeleton in st cells during tgev infection was examined. vimentin cytoskeleton was required for virus replication. the present study thus provides protein-related information about interaction of tgev n protein with host cell that should be useful for understanding host cell response to coronavirus pathogenesis infection and the underlying mechanism of coronavirus replication. coronaviruses (covs), a genus in the coronaviridae family, are pleomorphic, enveloped viruses (perlman and netland, 2009) . covs have been clustered in the cornavirinae subfamily, which includes three approved genera, alpha-, beta-and gammacoronavirus, as well as a tentative new genus, the deltacoronavirus (de groot et al., 2011; reguera et al., 2012) . transmissible gastroenteritis virus (tgev) is a representative cov in the alphacoronavirus genus; severe acute respiratory syndrome-related coronavirus (sars-related cov) is a representative of the betacoronavirus genus; infectious bronchitis virus (ibv) is a representative of the gammacoronavirus genus; and bulbul-cov is a representative of the deltacoronavirus genus (de groot et al., 2011) . the coronaviridae are involved in respiratory, enteric, hepatic and neuronal infectious disease in animals and humans (perlman and netland, 2009) . tgev infection causes severe diarrhea in suckling piglets (about 2 weeks old), which results in enormous economic loss in swine-producing areas in the world (kim and chae, 2001; sestak et al., 1996) . about two-thirds of the tgev genome (28.5 kb) encodes the replicase gene (rep) at the 5 end, and one-third of the genome encodes other viral genes at the 3 end in the order 5 -s-3a-3b-e-m-n-7-3 (penzes et al., 2001) . the genome of the tgev encodes four structural proteins: spike (s), membrane (m), envelope (e), and nucleocapsid (n) . n proteins of covs are highly basic with a molecular mass ranging from 40 to 63 kda, depending on the species and strains. n protein binds to the rna genome, forming a helical nucleocapsid (escors et al., 2001; sturman et al., 1980) . recently, some reports showed that n protein of tgev play an important role in host cell for virus replication. n protein of tgev facilitates template switching and is required for efficient transcription (zuniga et al., 2010) . n protein underwent proteolysis in parallel with the activation of caspases within host cell and n protein of tgev is a substrate for caspases (eleouet et al., 2000) . tgev n protein nucleolus localization was found in transfection experiments and might induced a cell cycle delay or arrest to facilitate virus replication (wurm et al., 2001) . in contrast, tgev n protein was not accumulated in the nucleus in the infection context (calvo et al., 2005) . in addition, the role of tgev n protein in cell cycle arrest has been recently reported (ding et al., 2014) . n protein of tgev may function through direct or indirect interaction with cellular proteins. for better understanding of the mechanisms associated with pleiotropic functions of n protein, cellular proteins of swine testicular (st) cells were pulled down associated with n protein using glutathione (gst)-tagged full-length n proteins immobilized on gst agarose. and cellular proteins of st cells were precipitated using n protein mab. by sds-page coupled with mass spectrometry (ms), a total of 12 cellular proteins interacting with n protein were successfully identified. information on the expanded repertoire of cellular proteins interacting with n protein will provide a framework for future biochemical analyses of these protein functions in tgev infection. st cells were grown in rpmi-1640 medium supplemented with 10% fetal calf serum under standard culture conditions (5% co 2 , 37 • c). tgev infectious strain h (accession no. fj755618) (wang et al., 2010) was propagated on an st cell monolayer. mouse mab to glyceraldehyde-3-phosphate dehydrogenase (gapdh) (ab9484) and rabbit poloclonal antibody vimentin (ab92547) were purchased from abcam. fitc-labeled goat antimouse igg was purchased from kirkegaard and perry laboratories (kpl). tritc-labeled goat anti-rabbit igg was purchased from sigma. the mab to n protein of tgev were donated from mr. wang shao (institute of animal husbandry and veterinary science, fujian academy of agricultural science, china). st cells were plated in six-well plates 1 day prior to infection with tgev infectious strain h at a multiplicity of infection (moi) of 1. tgev not-infected samples were exposed to culture medium alone. after adsorption for 1 h, cells were washed twice and incubated in fresh rpmi-1640. the prokaryotic expression plasmid pgex-tgev-n was constructed previously . escherichia coli bl21 (de3) strain containing pgex-tgev-n plasmid was expressed under induction of 1 mm isopropyl-␤-d-thiogalactopyranoside. gst pulldown assay was performed as previously described . expressed gst protein was used as a control. the lysate of tgev-infected st cells was prepared with ripa lysis buffer (50 mm tris-hcl, ph 7.4, 150 mm nacl, 1% np-40, 0.25% deoxycholate) containing a protease inhibitor phenylmethanesulfonyl fluoride (pmsf) (1 mm). after centrifugation at 12,000 × g for 15 min, lysate supernatant was pretreated with 2 l mouse igg control (beyotime) and protein a/g plus-agarose (santa cruz biotechnology) for 30 min at 4 • c to eliminate non-specific binding to agarose gel. the lysate supernatant (500 g) was incubated with 1 g of mab to n protein of tgev for overnight at 4 • c. then, 20 l resuspended protein a/g plus-agarose was added to this mixture and incubated at 4 • c on a rocker platform for 2 h. after washing four times with lysis buffer, isolated immunoprecipitated proteins (boiling 10 min with page sample loading buffer) were then analyzed by 12% page analysis. the lysate of tgev not-infected st cells was used as a control. 2.6. protein identification by matrix-assisted laser desorption/ionization time of flight (maldi-tof/tof) ms the gels described above stained with phastgel blue r (ge healthcare). protein bands of interest were manually excised from gels. maldi-tof/tof was performed as previously described (zhang et al., 2009 ). data were searched by gps explorer (ver. 3.6) with the search engine mascot (ver. 2.1). the search parameters were as follows: national center for biotechnology information non-redundant (ncbinr) database (release date, july 2011), and the database sus (41,373 sequences; 16,019,616 residues); a trypsin digest was performed with one missing cleavage, ms tolerance was set at 100 ppm and ms/ms tolerance at 0.6 da. known contaminant ions (tryptic autodigest peptides) were excluded. mascot protein scores (based on combined ms and ms/ms spectra) >59 were considered statistically significant (p ≤ 0.05). individual ms/ms spectrum, with a statistically significant (confidence interval ≥ 95%) ion score (based on ms/ms spectra), was accepted. to eliminate redundancy of proteins that appeared in database under different names and accession numbers, single protein member belonging to species sus or with the highest protein score (top rank) was separated from multi-protein family. equivalent amounts of cell lysates were subjected to 12% page and then transferred to 0.22 m nitrocellulose membranes (hybond-c extra, amersham biosciences). after blotting, membranes were incubated with mouse pab to vimentin or with mouse mab to n protein (1:2000) at 37 • c for 1 h. after washing three times with pbst, membranes were inoculated with dylight tm 800-labeled antibody to mouse igg (h+l) (1:10,000, kpl, usa) or dylight tm 800-labeled antibody to rabbit igg (h+l) (1:5000, kpl, usa) at 37 • c for 45 min. images were visualized by odyssey infrared imaging system (li-cor). st cells inoculated with tgev were cultured for 0, 1, 2, 4, 8, and 16 h. cells were washed twice with pbs and fixed with paraformaldehyde (4%) for 30 min at 4 • c, and then allowed to air dry. after blotting with 5% skimmed milk powder, the fixed cells were incubated with mab to tgev n protein (1:200) and rabbit pab to vimentin (1:100, abcam) for 1 h at 37 • c in a humidified chamber. after washing three times with pbst, the fixed cells were incubated with fitc-labeled goat anti-mouse igg (1:100, kpl) and tritc-labeled goat anti-rabbit igg (1:200, sigma). additional nuclear staining with 4 ,6-diamidino-2-phenylindole (dapi, sigma) was performed as described previously (jungmann et al., 2001) . the triple-stained cells were washed three times with pbst and subsequently examined under a leica tcs sp5 laser confocal microscopy. 2.9. transfection of sirna against vimentin sirna against vimentin (genepharma) was used for transfection. sequence of the sirna strands (two rounds of silencing) were as follows: 5 -gcuaacuaccaagacacuatt-3 (sense) and 5 -uagugucuugguaguuagctt-3 (antisense); 5 -ccucugguu-gacacccauutt-3 (sense) and 5 -aaugggugucaaccagaggtt-3 (antisense). negative control sirna strands were as follows: 5 -uucuccgaacgugucacgutt-3 (sense), 5 -acguga-cacguucggagaatt-3 (antisense). transfection with sirna was performed with lipofectamine 2000 reagent (invitrogen) by following the manufacturer's instructions. st cells were cultured overnight in six-well tissue culture plates. the sirna (20 nm) was complexed with lipofectamine 2000 reagent by incubating together at room temperature for 30 min. after removing the cell culture supernatant, the complex was added. after incubation for 36 h, the cells were infected with tgev. total rna was extracted from the st cells transfected with sirna against vimentin and negative control sirna, using the rneasy mini kit (qiagen) according to the manufacturer's protocol. cdna synthesis was performed with total cellular rna using a reverse transcriptase m-mlv kit (takara), according to the manufacturer's protocol. real time rt-pcr was performed using a lightcycler 480 ii (roche) in a total volume of 25 l containing 10 ng of cdna template, 1× sybr ® premix ex taq tm ii (perfect real time, takara), and a 0.4 m concentration of each primer. after initial denaturation at 95 • c for 2 min, the amplification was performed for 40 cycles, each consisting of denaturation at 95 • c for 5 s and primer annealing at 60 • c for 30 s. melting curves were obtained, and quantitative analysis of the data was performed in a relative quantification (2 − ct ) study model. parallel tgev notinfected st cells were used as control (relative expression = 1) and gapdh as an internal reference gene. st cells were re-plated 1 day before infection in 96 well plates for the 50% infectious dose (tcid 50 ) assays. treated samples and their paired controls were thawed as described and immediately serially diluted. cell cultures were then infected for 1 h. after 48 h of incubation, cpe was observed. tcid 50 is calculated using the method of reed and munch. virus titer assay were performed three times for each condition and were performed using the student's t-test. the expressed gst-n protein immobilized on gst-agarose beads was used as a bait to pull down cellular proteins of tgevinfected st cells that form a complex with n protein. gst protein was used as control to eliminate non-specifically binding proteins. after extensive washing the resins with ripa buffer, cellular proteins bound to n protein were analyzed by sds-page and stained with phastgel blue r (ge healthcare). as shown in fig. 1 , 10 cellular protein bands that were not seen in gst control gel. the 10 protein bands of interest were manually excised from gels and plated into 96-well microplates for ms identification. the mab to n protein was used as a bait to precipitate cellular proteins of tgev-infected st cells that form a complex with n protein. after extensive washing resins with ripa buffer, cellular proteins bound to n protein were analyzed by sds-page and stained with phastgel blue r (ge healthcare). as shown in fig. 1 , 5 cellular protein bands that were not seen in tgev not-infected st cells. the 5 protein bands of interest were manually excised from the gels and plated into 96-well microplates for ms identification. tgev n protein-associated cellular proteins were identified using in-gel tryptic digestion, and maldi-tof/tof identification. 15 protein bands binding to n protein were subjected to in-gel trypsin digestion, and peptides were analyzed by ms. database searches with the peptide masses resulted in positive identification for 8 different cellular proteins (table 1, table s1 , and fig. s1 ). two atp-dependent rna helicase proteins were identified as interacting with n protein. protein band 1 was identified as atpdependent rna helicase a (rha) and protein band 4 was identified as atp-dependent rna helicase ddx1 (dbp-rb). four cytoskeleton proteins were identified as interacting with n protein. protein band 3 and band 14 were identified as ␣-actinin-4 (actn4). protein band 5 and band 15 were identified as vimentin (vim). protein band 11 and band 12 were identified as myosin. four ribosome-associated proteins were identified as interacting with n protein: heterogeneous nuclear ribonucleoprotein u (hnrnp u) (band 2 and band 13); 40s ribosomal protein s9 (band 8); 60s ribosomal protein l26 (band 9); and 40s ribosomal protein s13 (band 10). one proteinbiosynthesis-associated protein (elongation factor 1-␣, band 6) and one cell-cycle-arrest protein (vasohibin-1, band 7) were identified as interacting with n protein. maldi-tof ms spectra and tof/tof spectra of vimentin are shown in fig. 2 . three cellular proteins, hnrnp u, actn4, and vimentin, were identified both by gst-n pull down and co-ip in tgev-infected cells, which should have more biological importance in the context of infection. to verify the proteins identified by ms, western blotting was performed. many cytoskeleton associated proteins play important roles in virus infection (dohner and sodeik, 2005) . vimentin protein was identified not only in gst pull-down assay but also in co-ip assay. therefore, the identified protein vimentin was selected for western blot analysis. cellular proteins immobilized on gst-agarose beads in gst pull-down assay were examined with specific antibodies to vimentin (fig. 3) . results validated the maldi-tof/tof identification of cellular proteins. to confirm the interaction between n protein of tgev and cellular vimentin, immunoprecipitation assay was utilized to identify whether tgev n protein interacted with cellular vimentin in tgevinfected st cells. from the immunoprecipitation results (fig. 4) , we can see that the n protein of tgev was precipitated by the pab to cellular vimentin in tgev-infected st cells but not in tgev not-infected st cells. results demonstrate that cellular vimentin interacted with n protein of tgev. subcellular localization of vimentin was investigated in tgevinfected st cells using indirect immunofluorescence confocal microscopy. results indicated that subcellular localization of vimentin was distributed in cytoplasm with enrichment in the perinuclear region after tgev infection (fig. 5) . cellular vimentin labeled with tritc was overlaid with n protein of tgev labeled with fitc, indicating that cellular vimentin was co-localized with n protein of tgev within the st cells during infection. to further investigate the role of vimentin in virus infection, vimentin protein of st cells was inhibited using sirna. the decrease level of vimentin mrna was validated by rt-pcr (fig. 6a) . st cells transfected with vimentin-specific sirna expressed lower level of vimentin by western blot analysis, compared to that transfected with the control sirna (fig. 6b ). after transfection with sirna, st cells were infected with tgev for another 16 h after transfection with sirna at an moi of 1. virus titer assay were performed three times for each condition and were performed using the student's t-test. tcid 50 of virus in control sirna group was 10 5.2 /ml and the vimentin sirna group was 10 3.1 /ml. fig. 5c shows that knock-down of vimentin resulted in significant reduction of cellassociated virus, which reflected viral replication. identifying host cell protein interacting with viral protein is a critical step for understanding protein function and viral replication. recently, affinity purification followed by ms has been widely used to find protein-protein interactions (dunham et al., 2012) . in the present study, using gst pull-down and co-ip coupled with ms identification, 15 cellular protein bands representative of 12 cellular proteins interacting with tgev n protein were identified. several cellular proteins found in this study were identical to those previously described interacting with cov n protein. ef1a, ddx1 and hnrnp u were also observed in infectious bronchitis virus (ibv) n protein using quantitative proteomic approaches (emmott et al., 2013) . interaction between ef1a and n protein of tgev and sars-cov were founded (zhou et al., 2008) . the hnrnp u protein was identified as binding preferentially tgev 3 -end (galan et al., 2009) . a very recent work describes the key role of ibv n protein interaction with ddx1 in viral transcription (wu et al., 2014) . four cytoskeleton-associated proteins were identified that associated with n protein in this study. the cytoskeleton consists of a filament scaffold within cell. filaments are dynamic and divided into three types: microfilaments (actin filaments), microtubules, and intermediate filament (naghavi and goff, 2007) . during infection, viruses interact with components of the cytoskeleton to reach their appropriate intracellular sites of replication (radtke et al., 2006) . in previous study, differentially expressed cytoskeletal proteins were found in tgev infected st cells using two-dimensional difference gel electrophoresis (zhang et al., 2013) . cytoskeletal proteins found in this study provides new insights for understanding the mechanisms of tgev replication. cov replication uses complex mechanisms that involve viral and cellular proteins. similar to other positive-strand rna viruses, cov genome replication takes place in the cytoplasm in a membraneprotected microenvironment that contains all the protein required for viral rna synthesis (enjuanes et al., 2006) . electron microscopy studies of mouse-hepatitis-virus-infected cells have shown that replication complexes of virus consist of double-membrane vesicles (gosert et al., 2002) . cov replication complex formation possibly utilizes components of cellular autophagy (prentice et al., 2004) . although cov replication essentially takes place within the cytoplasm, the virus may control cell machinery by locating some of its proteins in host cell nucleus (enjuanes et al., 2006) . in the present study, vimentin was identified in pull-down assay and co-ip assay, suggesting that it plays an important role in tgev infection. costaining of cellular vimentin and n protein on immunofluorescence microscopy also supported an association between proteins. based on these findings, we speculate that vimentin plays a role in tgev replication. some studies demonstrate that vimentin plays important role in virus infection cycle. vimentin binding is critical for virus entry, such as human cytomegalovirus (cmv) (miller and hertel, 2009 ), japanese encephalitis virus (jev) (liang et al., 2011) , and cowpea mosaic virus (cpmv) (koudelka et al., 2009) . vimentin is required for virus replication, such as bluetongue virus (btv) (bhattacharya et al., 2007) , and dengue virus (denv) (kanlaya et al., 2010) . in this study, vimentin is required for tgev replication. therefore, these may potentially serve as novel therapeutic targets for controlling tgev infection. the efficiency of tgev infection depends on the presence and integrity of vimentin cytoskeleton, which may facilitate virus infection at different steps. vimentin may promote viral replication by interaction with n protein, which may help virions to transport through a functional golgi complex for viral maturation (risco et al., 1998) . additional work will be needed to pinpoint the exact steps during viral infection. overall, this study was the first to identified cellular proteins interaction with tgev n protein using proteomics method. a total of 12 cellular proteins were identified, which provided new information to understand the mechanism of coronavirus replication. the interaction between the cellular vimentin and n protein of tgev was confirmed in tgev-infected st cells. knockdown of vimentin impairs tgev replication in st cells. the present study thus provides insights into interaction of tgev n protein with host cellular proteins that would be useful for further studying viral 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gastroenteritis coronavirus pur46-mad clone and evolution of the purdue virus cluster coronaviruses post-sars: update on replication and pathogenesis coronavirus replication complex formation utilizes components of cellular autophagy viral interactions with the cytoskeleton: a hitchhiker's guide to the cell structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies two types of virus-related particles are found during transmissible gastroenteritis virus morphogenesis contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid molecular characterization of a chinese vaccine strain of transmissible gastroenteritis virus: mutations that may contribute to attenuation nucleocapsid phosphorylation and rna helicase ddx1 recruitment enables coronavirus transition from discontinuous to continuous transcription localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division ef1a interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and plays a role in virus replication identification of cellular proteome using two-dimensional difference gel electrophoresis in st cells infected with transmissible gastroenteritis coronavirus differential proteome analysis of host cells infected with porcine circovirus type 2 the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription this work was supported by the national natural science foundation of china (grant nos. 31172350, 31101823), heilongjiang provincial natural science foundation (grant no. jc201118) and heilongjiang provincial department of education (2011td001). we thank mr. zhou xin-wen (fudan university, china) for help with maldi-tof/tof mass spectrometry and mr. wang shao (institute of animal husbandry and veterinary science, fujian academy of agricultural science, china) for the donation of mab to n protein of tgev. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2014.12.013. key: cord-276198-psjua913 authors: v’kovski, philip; al-mulla, hawaa; thiel, volker; neuman, benjamin w. title: new insights on the role of paired membrane structures in coronavirus replication date: 2015-04-16 journal: virus res doi: 10.1016/j.virusres.2014.12.021 sha: doc_id: 276198 cord_uid: psjua913 the replication of coronaviruses, as in other positive-strand rna viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. the proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the coronaviridae family members, but across the order nidovirales. taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral rna. however, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication. organelles, this has been difficult to test experimentally. however, there are several lines of experimental and genetic evidence that suggest that rna synthesis is tied to the formation of replicative organelles. viral rna accumulates in the coronavirus organelles, suggesting that the organelles may be a site of rna synthesis (knoops et al., 2008 (knoops et al., , 2012 gosert et al., 2002; hagemeijer et al., 2012) . furthermore, viral organelles are not formed when rna synthesis is stopped (stokes et al., 2010; verheije et al., 2008) . while it is clear that rna synthesis is linked with the organelles, it has proved difficult to directly test whether or to what extent the process of organelle formation is necessary for the process of rna synthesis, because of the practical difficulty in separating the two processes in an experimental setting. electron tomography studies have revealed that the replicative organelles of different nidoviruses are drawn from a repertoire of paired-membrane structures, including (paired) convoluted membranes, pouch-like double-membrane spherules, long paired membranes and double-membrane vesicles (knoops et al., 2008 (knoops et al., , 2012 maier et al., 2013a) , though studies of the more recently discovered mesoniviruses and roniviruses remain poorly characterized (zirkel et al., 2011; spann et al., 1995) . the nidoviruses that have been studied to date all induced a combination http://dx.doi.org/10.1016/j.virusres.2014.12.021 0168-1702/© 2015 elsevier b.v. all rights reserved. posthuma et al. (2008) , wood et al. (1970) , pedersen et al. (1999) , pol et al. (1997) 3a hsu et al. (2010) , richards et al. (2014) , teterina et al. (1997) , suhy et al. (2000) secoviridae p er v? nr roberts and harrison (1970) betaflexiviridae p er v nr edwardson and christie (1978) , rudzinska-langwald (1990) guix et al. (2004) , mendez et al. (2007) bromoviridae p er z,s 1a+2a pol moreira et al. (2010) , schwartz et al. (2002) , schwartz et al. (2004) fig. 1 . the common element in nidovirus-like membrane rearrangement is that the membranes are paired, usually maintaining a consistent-sized gap between the two membranes (reviewed here, angelini et al., 2014) . since protein-induced membrane fig. 1 . conservation and functional organization of the carboxyl-terminal region of nidovirus polyprotein 1a. domains that are homologous at the amino acid level are shown at left in solid colors. more distantly related potential homologs identified by genome position and comparison of predicted secondary structures are marked with stripes. positions of transmembrane regions (black bars) and hydrophobic non-transmembrane regions (striped bars) were predicted by tmhmm 2.0 (krogh et al., 2001) and amended to reflect known topologies (kanjanahaluethai et al., 2007; oostra et al., 2007 oostra et al., , 2008 wherever possible. clusters of conserved cysteine and histidine residues that may bind metal ions are marked with white ovals. a jagged line denotes the uncertain position of the amino terminus. regions that induce membrane pairing, proliferation or vesiculation in betacoronavirus sars-cov and arterivirus eav are shown above and below the domain annotation, respectively, and all annotations come from the references listed for table 1 . double-membrane organelles observed (x) or uncertainly observed (?) in infected cells are marked at right. virus names are abbreviated as follows: white bream virus (wbv), fathead minnow nidovirus (fhmnv), equine arteritis virus (eav), lactate dehydrogenase elevating virus (ldv), porcine reproductive and respiratory syndrome virus (prrsv), simian hemorrhagic fever virus (shfv) and wobbly possum nidovirus (wpnv). pairing appears to be a consistent feature associated with nidovirus replication, and in the absence of data carefully dissecting the relationship between the shape and function of these different paired membrane structures, it makes sense to refer to the resulting structures collectively as double-membrane organelles (dmo). despite a relative wealth of structural data, it has proved difficult to test hypotheses about the role of dmos in viral replication and fitness directly because dmo formation is linked so closely to replication and expression of replicase proteins. here, we will discuss the implications of two recent studies that address questions about the role of dmos in nidovirus replication (al-mulla et al., 2014) , and characterize the effects of a new dmo-blocking drug against a variety of coronaviruses (lundin et al., 2014) . further evidence of the probable importance of nidovirus replicative organelles for viral rna replication comes in the form of genetic conservation. nidoviruses, and most particularly coronaviruses, are highly genetically variable and contain several genus-specific or even species-specific genes (lauber et al., 2013) . however, there are two clusters of genes that are conserved in all known nidoviruses lauber et al., 2013) . the first is a highly conserved cluster of genes homologous to the severe acute respiratory syndrome coronavirus (sars-cov) nsp3-6 ( fig. 1) . expression of the membrane-anchored proteins nsp3, nsp4 and nsp6 is sufficient to induce the formation of sars-like paired-membrane replicative organelles (angelini et al., 2013) . the second conserved gene cluster encodes the viral rna polymerase and superfamily 1 helicase (deng et al., 2014) . the conservation of membrane-pairing genes in the context of an otherwise hypervariable group of viruses is a strong argument in favor of the importance of at least the membrane-pairing genes for rna synthesis the proteins that form sars-cov replicative organelles have several features in common with distant homologs found throughout the nidovirales. we will refer to the transmembrane proteins homologous to sars-cov nsp3, nsp4 and nsp6 a as tm1, tm2, and tm3, respectively. the relative genomic positions and functions attributed to tm1-3 in nidoviruses are shown in fig. 1 . of the three proteins involved in sars-cov replicative organelle formation, the least conserved is tm1, which has a multidomain architecture (neuman et al., 2008) . many nidovirus and all coronavirus tm1 proteins contain one or more ubiquitin-like domains which may help to anchor the viral rna to the membranes where replication takes place (hurst et al., 2013) . potentially rna-binding macrodomains (serrano et al., 2007; xu et al., 2009a; wojdyla et al., 2009; saikatendu et al., 2005; tan et al., 2009; chatterjee et al., 2009; johnson et al., 2010) , papain-like proteinases (ratia et al., 2006; wojdyla et al., 2010; van kasteren et al., 2013) , other rna binding domains ) and a well conserved but poorly understood region known only as the y domain (neuman et al., 2008) are also commonly but not ubiquitously found in nidovirus tm1 proteins. all putative tm1 proteins are predicted to contain one or more transmembrane domains, as shown in fig. 1 . the c-terminal region of tm1, from the first transmembrane region to the end of the y domain induces membrane proliferation, which in some ways resembles an autophagy response (angelini et al., 2013) . tm2 and tm3 are recognizable because they contain four or more predicted transmembrane regions, and are encoded immediately before and after the viral main protease (m pro ). bioinformatics generally predicts an even number of transmembrane spans in these proteins, which would be necessary to localize m pro on the same side of the membrane as all of its predicted upstream and downstream cleavage sites. however there are additional hydrophobic regions that are strongly predicted to span the membrane, but which do not for several viruses, including most coronaviruses (kanjanahaluethai et al., 2007; oostra et al., 2007 oostra et al., , 2008 . tm2 contains two potential conserved domains located between the first and second transmembrane domains in coronavirus, and after the final transmembrane domain in most nidoviruses. mutations in the first non-hydrophobic domain of tm2, which is the largest part of the coronavirus replicase to localize on the luminal face of the membrane, have been demonstrated to disrupt rna replication and may cause defects in membrane pairing (gadlage et al., 2010) . deletion of the latter conserved domain of tm2, which has been structurally solved xu et al., 2009b) , was surprisingly well tolerated sparks et al., 2007) . tm2 localizes to membranes, but does not induce any recognizable change to intracellular membranes in the absence of other viral proteins (angelini et al., 2013) . however, co-expression of tm2 with full-length tm1 results in extensive pairing of perinuclear membranes in both coronavirus (angelini et al., 2013) and arterivirus (snijder et al., 2001; posthuma et al., 2008) . additionally, it has recently been shown that co-expression of a fragment of mhv tm1 including the transmembrane region and the c-terminus with tm2 induced er membrane zippering and curvature similar to the phenotype observed after sars-cov tm1 and tm2 co-expression (hagemeijer et al., 2014) . in that report tm1 and tm2 were demonstrated to interact via protein loops on the luminal face of the membrane. the maze-like paired-membrane structures that resulted from coexpression of sars-cov tm1 and tm2 have not ever been reported in coronavirus-infected cells, suggesting that this should be interpreted as a conditional, or perhaps partial phenotype, that is not observed when the full viral replicase polyprotein is expressed. this suggests that membrane pairing is caused by heterotypic interactions between tm1 and tm2 on opposing membranes, but that the final architecture of the paired membranes is dependent on additional viral proteins. tm3 largely consists of transmembrane regions, without the hallmarks of amino acid conservation or predicted structural conservation that would be expected for an enzyme. overexpression of tm3 alone disturbs intracellular membrane trafficking (cottam et al., 2011 (cottam et al., , 2014 , resulting in an accumulation of singlemembrane vesicles around the microtubule organization complex (angelini et al., 2013) . however, quantitative electron microscopy revealed that expression of tm2 with tm3 prevents the membrane disruption seen with tm3 expression alone (angelini et al., 2013) . when sars-cov tm1, tm2 and tm3 are coexpressed, membranecontaining bodies which resembled authentic sars-cov replicative organelles were formed. however, in each of the cell sections where dmv-like membranes were observed, the membrane proliferation phenotype of tm1, the paired membrane phenotype of tm1 + tm2 and the single membrane vesicle accumulation from tm3 were each visible, suggesting that these proteins do not always colocalize efficiently when expressed from plasmids in different parts of the cell instead of being expressed in the natural form as a polyprotein (bwn, personal communication) . this suggests that while tm3 is not necessary for membrane pairing, tm3 may be necessary to induce the formation of the double-membrane vesicles (dmvs) that are characteristic of coronavirus replicative organelles. the formation of large intracellular structures such as the mazelike tm1 + tm2 bodies and dmv-like tm1 + tm2 + tm3 bodies suggests that nsp3, nsp4 and nsp6 may interact both homotypically and heterotypically. sars-cov nsp3-nsp3 interactions have been detected in cells by yeast two-hybridization (pan et al., 2008) and gst pulldown (imbert et al., 2008) , and in purified protein by perfluorooctanoic acid polyacrylamide gel electrophoresis (neuman et al., 2008) . while sars-cov nsp4-nsp4 interactions were not found in yeast-two hybrid or mammalian two-hybrid screens (pan et al., 2008; von brunn et al., 2007) studies with another coronavirus did detect nsp4-nsp4 interactions by venus reporter fluorescence (hagemeijer et al., 2011) . to date, homotypic interactions have not been demonstrated for nsp6 despite several attempts (pan et al., 2008; imbert et al., 2008; von brunn et al., 2007) . heterotypic interactions between coronavirus tm1-3 proteins have been demonstrated biochemically: a tm1-tm2 interaction was detected by mammalian two-hybridization (pan et al., 2008) and weakly detected by venus reporter fluorescence (hagemeijer et al., 2011) . a tm2-3 interaction has been demonstrated by venus reporter fluorescence (hagemeijer et al., 2011) , though it did not appear in other hybridization studies. a one-way interaction between the amino-terminal 192 amino acid domain of tm1 and tm3 detected by yeast two-hybridization (imbert et al., 2008) has also been reported. however, the apparent independence of tm1 and tm3 phenotypes after coexpression, coupled with the abrupt change in both phenotypes in the presence of tm2 suggests that interactions between these proteins may be largely mediated by tm2 (angelini et al., 2013) . molecular interactions between host and viral factors are observed in virtually every step of the viral life cycle. viruses rely on and manipulate established cellular pathways to accommodate their needs during replication and to counteract host innate immune signaling. replication of coronaviruses is no exception; while some host factors have been described in the context of viral rna replication and transcription (zhong et al., 2012) , few studies have looked closely at the complex interplay of host pathways in the establishment of virus-induced membrane-bound replication complexes. the best available evidence suggests that most coronavirus dmo structures derive from er membranes (knoops et al., 2008 (knoops et al., , 2012 maier et al., 2013a) , but the precise mechanism of membrane rearrangement remains elusive. dmo membranes were initially suggested to derive from the early secretory pathway, although the absence of conventional er, ergic and golgi protein markers on viral replicative membranes argues against this hypothesis (verheije et al., 2008; knoops et al., 2010) . since dmvs are reminiscent of the double-membranes of autophagosomes, several lines of controversial evidence hypothesized a diversion of atg (autophagyrelated) proteins and autophagosome function during coronavirus replication, as it is the case for other +rna viruses (prentice et al., 2004; snijder et al., 2006; zhao et al., 2007; maier and britton, 2012; richards and jackson, 2013) . the involvement of autophagy was recently investigated in the context of the avian cov infectious bronchitis virus (ibv) infections (cottam et al., 2011) . the authors conclude that the presence of exogenous, individually expressed ibv nsp6, which localizes to the er, induces the formation of autophagosomes in contrast to other ibv replicase proteins. additionally, although autophagosomes induced by ibv nsp6 or ibv infection appeared smaller than conventional autophagosomes observed after starvation of cells, they were similar in size to dmvs (cottam et al., 2014) . however, the data reported here do not appear to support the assumption that there is a functional link between ibv nsp6 and autophagosomes, and a role of the autophagy in the formation of ibv replicative structures can hereby not be demonstrated. moreover, neither induction nor inhibition of autophagy seems to affect ibv replication (maier et al., 2013b) . it also has to be noted that the induction of autophagy by virus infection is cell type and possibly species dependent, as exemplified by the absence of lc3 puncta accumulation in ibv-infected primary chick kidney cells (maier et al., 2013b) . new evidence concerning the source of membranes for covinduced dmos was proposed, in which mouse hepatitis virus (mhv) probably co-opts a cellular degradation pathway of erassociated degradation (erad) regulators, known as the erad tuning pathway . the erad pathway is responsible for the turnover of folding-defective polypeptides in the er and is modulated by stress-inducible positive regulators of erad-mediated protein disposal such as edem1 (er degradationenhancing alpha mannosidase-like 1) and os-9 (osteosarcoma amplified 9). the latter assist in transporting misfolded proteins into the cytosol for subsequent degradation by the proteasomal system. under physiological conditions, however, low concentrations of edem1 and os-9 are maintained in the er lumen in order to avoid premature degradation of proteins that are undergoing folding programs (calì et al., 2008) . in this case, edem1 and os-9 are selectively confined by interacting with the transmembraneanchored cargo receptor sel1l (suppressor of lin-12-like protein 1) and later released from the er lumen in small short-lived vesicles, called edemosomes, which rapidly fuse with the endolysosomal compartments (bernasconi et al., 2012) . this steady-state disposal of edem1 and os-9 is known as erad tuning pathway. while not relying on the coat protein complex ii (copii) or atg7, it critically depends on the non-lipidated form of lc3 (lc3-i), which is recruited to edemosomes. however, the specific autophagosomal marker gfp-lc3 does not associate with edemosomes, which are therefore distinct structures (noack et al., 2014) . the coronavirus mhv is hypothesized to divert the erad tuning machinery for the generation of dmos. similarly to edemosomes, colocalization of edem1, os-9, sel1l, lc3-i and double-stranded (ds) rna is observed upon mhv infection. moreover, replication of mhv, which does not require an intact autophagy pathway, is impaired upon knockdown of lc3 or sel1l (bernasconi et al., 2012) . dmos furthermore lack conventional er markers and do not associate with gfp-lc3 . altogether, the evidence from this study may suggest that mhv exploits the erad-tuning machinery to establish dmos for replication. in order to learn whether this mechanism might be common to other nidoviruses, other viruses that use a similar replication strategy to mhv were examined. one of these, the arterivirus equine arteritis virus (eav) has been shown to require the same subset of erad tuning factors as mhv to ensure replication (monastyrska et al., 2013) . recently, investigations of the even more distantlyrelated japanese encephalitis virus (jev), which belongs to the flaviviridae family, revealed that it may usurp the same components of the erad-tuning pathway as well (sharma et al., 2014) . consistent with this hypothesis, both viruses were shown to replicate independently of a functional autophagy pathway. the non-lipidated lc3 marker protein, which is essential for the replication of eav and jev, associated with their replication complexes together with edem1 whereas gfp-lc3 did not label these structures. these observations parallel the ones seen for mhv but raise further questions whether this feature is even more widespread amongst +rna viruses. despite the resemblance of mhv, eav and jev in the requirement of host factors for efficient replication, diversion of the erad tuning pathway cannot be considered as a generic way of inducing replicative membranes by these viral families. probable variations within families have to be kept in mind as exemplified by the comparison of dmos from two different coronavirus genus members. indeed, ibv's recently described spherules derived from paired er membranes significantly differ from the dmo structures observed upon alpha-and beta-coronaviruses infections (maier et al., 2013a; neuman, 2013) and their generation might require a different set of factors. furthermore, the morphology of dmos induced by flaviviruses such as hepatitis c virus, dengue virus or west nile virus is highly heterogeneous and the identification of a common, conserved membrane diversion strategy seems unlikely (romero-brey and bartenschlager, 2014). however, it is possible that the diversion of one pathway could lead to the generation of the different arrangements of membrane that we collectively refer to as the dmo. importantly, it has been shown that, in contrast to what is observed during eav infection, endogenous lc3 does not colocalize with membrane puncta induced by expression of eav nsp2 and nsp3, and the membrane modifications induced by the latter are not affected by lc3 knockdown (monastyrska et al., 2013) . similarly, lc3 and edem1 were not recruited to rearranged membranes induced by co-expression of mhv tm1 and tm2 (hagemeijer et al., 2014) . while this still has to be proven in the context of cov tm1, tm2 and tm3 expression, it raises the questions whether lc3 participates to the biological function of dmvs rather than its generation. a novel hypothesis has been recently suggested for poliovirus, according to which the virus might not only co-opt a host pathway, but also divert the functional network of individual proteins (belov and sztul, 2014) . host factors could therefore have a proviral function during infection, distinct from the function for which they have been initially described. accordingly, this is reminiscent with novel functions attributed to lc3 during cellular homeostasis, cytoprotection against invading pathogens or during chlamydia trachomatis' intracellular life cycle (bestebroer et al., 2013) . the dmos of the model coronavirus mhv take the form of perinuclear dmvs which appear either singly, or grouped around and interconnected with a region of paired, convoluted membrane (cm;). a recent study examined dmv formation by wild-type mhvinf-1 (wt) and five temperature-sensitive (ts) mhv mutants, each of which differed from wt by a single amino acid substitution. the panel of ts viruses chosen contained mutations in an interdomain linker of nsp3 (tm1), m pro , the viral rna polymerase, cap n-methyltransferase and cap o-methyltransferase, respectively (stokes et al., 2010; al-mulla et al., 2014; sawicki et al., 2005) . with the exception of the polymerase mutant, which was attenuated tenfold, these viruses produced the same amount of infectious progeny as wt (al-mulla et al., 2014) . all of the mutants produced significantly smaller dmvs than wt virus, varying from almost wt size to 17% smaller (table 2 ). in two of the mutants that produced normal amounts of infectious progeny, not only were the dmvs smaller, there were only about half as many dmvs per visibly infected cell compared to wt (table 2) . examination of the size and number of intracellular virus particles from the same samples did not reveal corresponding changes, suggesting that the observed dmv phenotypes were not an artifact of sample preparation. the number of cms remained in a constant ratio to the number of dmvs present, suggesting that the mutations affected production of the entire dmo. the dmos of human coronavirus 229e (hcov-229e) include dmvs similar to those observed after mhv infection (lundin et al., 2014) . in testing a new antiviral called k22, it was observed that infectivity, viral rna, and dmv formation were all blocked by treatment with 4 m k22. a time of addition study revealed that k22 did not block viral entry, and had the greatest antiviral effects after virus entry during the first few hours of infection, leading to the interpretation that k22 inhibits a cellular or viral component involved in a post-entry, early stage of viral replication. after serial passage of the virus in the presence of k22, resistant mutants were selected. surprisingly, two independently isolated resistance mutations mapped to opposite ends of transmembrane helices in tm3 (nsp6) at positions h121l and m159v. the resistant viruses released similar amounts of new progeny compared to wt, but produced only about half as many dmvs per infected cell. in addition, the dmvs induced by resistance mutants appeared structurally impaired. similarly to mhv nsp4 mutants (gadlage et al., 2010) , k22 escape mutants induced dmv with partially collapsed inner membranes, even when k22 was not present. however, it is important to bear in mind that the fixation, staining and imaging conditions may have an influence on the appearance of membrane structures (knoops et al., 2012) . moreover, the specific infectivity of those newly released virions was about tenfold lower for tm3 mutants than for wt. this suggested that the mutations in nsp6 conferred resistance to k22 at a cost of impairing an early intracellular step in the establishment of infection. from these experiments it was clear that hcov-229e viruses with k22 resistance mutations in tm3 incurred a steep fitness cost, in the form of decreased specific infectivity. there were also indications of a similar decrease in efficiency in the mhv nsp3 mutant brts31, which produced significantly more intracellular rna than wt, but without a corresponding increase in infectious progeny. to find out if the mhv mutants also incurred a fitness cost associated with producing smaller and fewer dmvs, competitive fitness assays were carried out. to do this, equal infectivities of two viruses were added to the same flask at a temperature where both viruses could grow normally. after 24 h in direct competition, the amount of each virus was quantified either by sequencing to look for the ts mutation, or by phenotypically screening for ts and non-ts virus. none of the mhv mutants tested was significantly less fit than wt in continuous or primary fibroblasts, and two mutants were significantly fitter than wt under the assay conditions. one of the viruses with increased fitness compared to wild-type was the n-methyltransferase mutant brts105, which produced only half as many dmvs as wt. these results demonstrated that at least under these experimental conditions, producing larger or more numerous dmvs did not confer a corresponding fitness advantage. when interpreting these findings, it is important to consider that none of the hcov-229e or mhv mutants tested to date has been able to replicate entirely without dmos. and while some of these tests were carried out in primary cells, work in animal models was not possible because of the lack of a small animal model for hcov-229e, and because the mutations restricted the growth of mhv mutants at physiological temperatures. these two studies do not disprove the fundamental connectedness between coronavirus rna replication and dmo formation, but together, they reveal an unexpected plasticity in the size and number of dmvs that are needed to carry out wild-type amounts of rna synthesis. for these reasons, along with the observation that rna replication is detectable before the first appearance of organelles , we favor an interpretation in which the organelles are a late manifestation of accumulated viral proteins resulting from abundant rna expression. in this interpretation, dmos could still play an obligate role in viral replication under specific conditions or in specific cell types, but the primary role for dmos would be to increase the efficiency of either rna production, delivery of newly synthesized rna to sites where it could be translated or packaged, and/or shielding abundantly synthesized viral rna from host cell innate immune sensing pathways. these studies also suggest that at least half of the dmvs present in infected cells may be in excess of what is strictly needed to sustain normal levels of rna synthesis, given that both mhv and hcov-229e mutants replicated normally despite producing only half the normal complement of dmvs. before these studies, very little was known about the potential for natural and induced variation in intracellular membrane rearrangement. the viruses described in these studies all produced normal amounts of progeny virus particles, and were all selected for analysis for reasons unrelated to dmo formation. these represent only a handful of the available nidovirus replicase mutants that have been published. from this work we can hypothesize that other mhv ts mutants, or k22-resistant hcov-229e mutants with replicase defects would probably make either smaller or fewer dmvs, and a larger collection of such mutants will like be highly informative to further our understanding on the pivotal role(s) of dmos in the coronavirus life cycle. hopefully the unique insight provided by these results, together with the relative ease of analysis will make quantitative electron microscopy a routine part of the characterization of new virus mutants. in addition, the accumulated knowledge on the nature of coronavirus dmos and the possibility to experimentally interfere with dmo formation by using small compound inhibitors, such as k22, will allow us to dissect similarities and differences between viral dmos and related cellular organelles. the study of viral replicative organelles remains an area of active research where there are more questions than answers. for the coronavirus dmo, there are still fundamental questions that need to be answered including the roles of specific parts of the dmo in replication, the cellular pathways involved in dmo formation, and whether there are host 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lou, yongliang; tan, wenjie title: discovery of a novel canine respiratory coronavirus support genetic recombination among betacoronavirus1 date: 2017-06-02 journal: virus res doi: 10.1016/j.virusres.2017.05.006 sha: doc_id: 347270 cord_uid: x05tfkgx although canine respiratory coronavirus (crcov) is an important respiratory pathogen that is prevalent in many countries, only one complete genome sequence of crcov (south korea strain k37) has been obtained to date. genome-wide analyses and recombination have rarely been conducted, as small numbers of samples and limited genomic characterization have previously prevented further analyses. herein, we report a unique crcov strain, denoted strain bj232, derived from a crcov-positive dog with a mild respiratory infection. phylogenetic analysis based on complete genome of all available coronaviruses consistently show that crcov bj232 is most closely related to human coronavirus oc43 (hcov-oc43) and bcov, forming a separate clade that split off early from other betacoronavirus 1. based on the phylogenetic and simplot analysis we propose that crcov-k37 was derived from genetic recombination between crcov-bj232 and bcov. in detail, spike (s) gene of crcov-k37 clustered with crcov-bj232. however orf1ab, membrane (m) and nucleocapsid (n) genes were more related to bovine coronavirus (bcov) than crcov-b232. molecular epidemic analysis confirmed the prevalence of crcov-bj232 lineage around the world for a long time. recombinant events among betacoronavirus 1 may have implications for crcov transmissibility. all these findings provide further information regarding the origin of crcov. canine respiratory coronavirus (crcov), an enveloped positivestranded rna virus, belongs to the betacoronavirus genus, subgroup 2a erles et al., 2003) . unlike the enteric canine coronavirus (ccov) belonging to the alphacoronavirus genus, crcov is associated with mild to severe respiratory signs and proposed as an etiological agent of canine infectious respiratory disease (cird) (buonavoglia and martella, 2007) . crcov was first detected in 2003 by rt-pcr in the uk from dogs suffering from severe respiratory disease (erles et al., 2003) . later, the crcov-4182 strain was successfully isolated from a dog with respiratory signs and was cultured in the hrt-18 cell line . to date, data have been made publicly available for only one complete genomic sequence (crcov-k37 strain), which was isolated from a south korea dog (an et al., 2010; lim et al., 2013) . crcov was included in species betacoronavirus 1, which also contain bcov, equine coronavirus, hcov-oc43, porcine hemagglutinating encephalomyelitisvirus (phev), human enteric coronavirus (hecov) and crcov (king, 2011) . in contract, canine coronavirus (ccov) is a member of alphacoronavirus, which include feline coronavirus and porcine transmissible gastroenteritis among others. the virus was distinct from ccov and only showed 69% nucleotide identity in polymerase and 21% amino acid identity in spike protein (buonavoglia and martella, 2007; erles and brownlie, 2008) . the evolutionary origin of crcov remains uncertain, although successful experimental infection of puppies with bcov has suggested that crcov is of bovine origin and transmitted to dogs from cattle (kaneshima et al., 2007) . meanwhile, hcov-oc43 may have emerged after viral transmission from cattle to people (vijgen et al., 2005) . the high nucleotide identity between crcov and bcov, hcov-oc43 and hecov indicated that the four viruses have a common ancestor (erles and brownlie, 2008) , and demonstrated the occurrence of repeated host-species shifts (vijgen et al., 2006) . although crcov strains are frequently circulating in farmed and pet dogs worldwide (erles and brownlie, 2005; kaneshima et al., 2006; priestnall et al., 2006; priestnall et al., 2007; yachi and mochizuki, 2006) , causing respiratory disease, little is known about their genomic evolution and recombination. in the present study, we sequenced and analyzed the first complete genome of crcov derived from china (crcov-bj232 strain) and discuss its role in crcov evolution. this research was reviewed and approved by the ethics committee of the national institute for viral disease control and prevention of the chinese cdc. all animals were treated strictly according to the guidelines for the laboratory animal use and care from the chinese cdc. from december 2013-march 2014, 246 swab specimens (nasopharyngeal and anal swabs) were collected from diseased dogs at the animal hospital of china agricultural university. most of the dogs showed symptoms of gastrointestinal and/or respiratory tract infections, including coughing, lacrimation, vomiting and diarrhea. all the swab samples were collected by professional veterinarians and were stored at −80°c. for molecular detection, total rna was extracted from swab specimens using a qiaamp viral rna mini kit (qiagen, germany) according to the manufacturer's instructions. the rna was used as a template for reverse transcription-pcr (rt-pcr), and the partial sequences of the s, rna-dependent rna polymerase (rdrp), and n genes were amplified using gene-specific primers (table s1 ). in detail, reverse transcription was performed by using a superscript iii kit (invitrogen, san diego, ca). the pcr mixture (25 μl) contain 1 μl cdna, 1 μl forward primer(10 μm), 1 μl reverse primer (10 μm), 12.5 μl q5 ® high-fidelity 2x master mix (new england biolabs) and 9.5 μl nuclease-free water; the mixtures were amplified with 30 cycles at 98°c for 10s, 52°c for 30s, and 72°c for 1 min and final extension step at 72°c for 2 min in an automated thermal cycler (bio-rad, t100 thermal cycler). to better characterize the crcov discovered here, the crcovpositive samples were pretreated and used for random amplification as described previously (wang et al., 2016) ; the amplified dna was used as a template for illumina hiseq2500 sequencing (2 × 125 bp, pairedend reads). the sequencing procedure was conducted by beijing berry genomic company. the full-length genome of crcov was assembled using soap de novo and was annotated based on the sole annotation of crcov k37 strain (jx860640.1). to determine the evolutionary relationships between the crcov discovered here and those identified previously, we collected most of the complete sequences of the betacoronavirus 1 and conducted nucleotide acid sequence alignments using the mafft algorithm. the phylogenetic analyses were conducted based on the nucleotide acid sequences of the orf1ab, s, m, and n genes and complete genome of crcov. all the phylogenetic trees were constructed in the phyml software using the maximum likelihood method, with 500 bootstrap replicates. the following betacoronavirus 1 genome sequences from the genbank database were used for the phylogenetic analysis as follows: bcov reference strain (nc_003045), hecov (nc_012950), hcov-oc43 (nc_005147), crcov-37 (jx860640), and bcov strain quebec (af220295). to confirm the potential recombination events, similarity plots and bootscanning analyses were conducted using the simplot software; a sliding window of 1000 nucleotides and 100-nucleotide steps were used as the default settings. in addition, a schematic diagram of the complete crcov genome was examined to determine the recombination sites. the complete genome of chinabj232 was deposited in the genbank database under accession no. kx432213. other sequences generated in this study, including the partial s, rdrp and complete n gene sequences, were also assigned accession numbers: kx432214-kx432217. a total of 246 swab samples were collected in beijing from december 2013 to march 2014.rt-pcr was performed to detect crcov, and 16 (6.5%) out of 246 swabs originating from chinese dogs tested positive for crcov. in addition, five crcov-positive samples with high titers were initially processed for complete genome sequencing using deep sequencing technology. due to the limited specimen size and low viral load, five clinical samples were used for deep sequencing, and only one complete genomic sequence was eventually obtained. the genomic data, formally referred to as "canine respiratory coronavirus strain bj232" and in this report as "crcov-bj232," were deposited in genbank (kx432213). the crcov-bj232 genome isolated in china was found to have 30,868 nucleotides and showed a genomic organization that was highly similar to the betacoronavirus 1species: 5′-utr(nt 1-143),orf1ab (nt 144-21427), orf2 (nt 21437-22273), he gene (nt 22285-23559), s gene (nt 23574-27665),e gene (nt 28355-28609), m gene (nt 28624-29316), n gene (nt 29326-30672), and 3′-utr (nt 30673-30868). between the s and e gene, three small orfs were identified: orf4a (nt 27655-27789), orf4b (nt 27825-27902) and orf4c (nt 28039-28293), encoding 4.9-, 2.7-and 12.8-kda nonstructural proteins, respectively, which were analogous to those noted in strain crcov-k37. meanwhile, conventional pcr and sanger sequencing were conducted to verify the complete genome sequence of crcov-bj232 with 100% nucleotide identity. thus far, there are only two crcov strains with full-length genomic information submitted to genbank; the previously reported crcov-k37 and our current strain, crcov-bj232. unfortunately, attempts to isolate crcov-bj232 from the five positive samples were unsuccessful; future studies to isolate the virus from more canine samples will allow characterization of its pathogenicity and transmissibility. based on the analysis of whole-genome phylogeny, crcov-bj232 was classified into betacoronavirus 1 species of betacoronavirus group 2a (fig. 1a) and shares a common origin with bcov, crcov-k37, hecov and hcov-oc43. moreover, the latter four viruses clustered together with high branch bootstrap values (fig. 1b) . to investigate the genetic relationships among the betacoronavirus 1 species, we generated phylogenetic trees based on the sequences oforf1ab, s, m and n genes (fig. 2 ). phylogenetic analysis based on different genes consistently show that crcov bj232 is most closely related to hcov-oc43, forming a separate clade of crcov that split off early from other betacoronavirus 1. however, crcov-k37 strain closely clustered with crcov-bj232 strain in s gene, and was more related to bcov strain quebec (af220295.1) in the region of orf1ab, m and n genes. the major genes of the crcov-bj232 strain showed a consistent trend in the evolution, while the phylogenetic topology conflicted in the subregions of crcov strain k37, especially in the s and orf1ab genes. these findings are consistent with recombination, a phenomenon not uncommon in coronaviruses, implying that the crcov k37 strain may have arisen from genetic recombination. similarity plots and bootscanning analyses (fig. 3) were performed to confirm the recombination between crcov and bcov. the previously reported crcov k37 strain was used as a query sequence, and was compared with hcov-oc43 (nc.005147), bcov (bcov-refnc_003045, bcov-db2 dq811784, bcov-quebec af220295), he-cov (hecov-ref nc_012950, hecov-4408 fj415324), and crcov (crcov-bj232 kx432213). in the s gene, strain k37 possessed the highest similarity with the bj232 strain between position 23,842 and 29,072, as expected. however, in the orf1ab, he, m and n genes, strain k37 was apparently related to bcov, which further confirmed the occurrence of genetic recombination between crcov and bcov. the consistency in the results of bootscanning and phylogenetic analyses also supported the possibility of recombination in the s gene. we collected all available crcov sequences from the genbank database and conducted comprehensive phylogenetic analyses separately based on he, s, m and n genes to understand the epidemiology distribution of the recombinant strain(south korea strain k37). the bj232, bj221, bj34 and bj202 strains were collected from china in this study, while the k37, k39 and k9 strains came from south korea, and the 240/05 and 4182 strains were derived from italy and the united kingdom, respectively. as shown in fig. 4 , the crcov strains from china, including the bj232, bj221, bj34 and bj202 strains, clustered with strains prevalent in italy and the united kingdom (the 240/05 and 4182 strains), and the crcov strains detected in south korea (k37, k39 and k9 strains) were all derived from recombinant lineage. crcov is a causative agent for cird and a novel pathogen detected in respiratory samples from dogs (erles and brownlie, 2008) . a serological survey of crcov conducted worldwide revealed that antibodies against crcov were common in the canine population (erles et al., 2004; isaacs et al., 1983; pratelli et al., 2003; priestnall fig. 1 . phylogenetic analysis of all available coronaviruses with reference sequence based on complete genome. a, the current taxonomy of the family coronaviridae; b, the evolution of betacoronavirus 1 based on complete genomes. strainbj232 (accession no. kx432213, indicated by the red dot) was the first crcov with complete genome derived from china. the previously reported crcov-k37 strain, which was detected in south korea, was the sole other crcov strain with complete genome sequence available. some other representative strains of the betacoronavirus genus, subgroup 2a, were also analyzed in phyml program using the maximum likelihood method, with 500 bootstrap replicates. the numbers at the nodes represent the bootstrap support. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) et al., 2006) . in contrast, corresponding genome-wide analyses have rarely been conducted (an et al., 2010; lorusso et al., 2009 ). in the present study, we characterized the first complete genome of crcov (named strain bj232) derived from china, and phylogenetic analyses showed that crcov-bj232occupied a deep branch at the root of members of betacoronavirus 1, being distinct from hcov-oc43 and bcov. another previously described crcov strain, k37, was identified to have arisen from recombination between crcov-bj232 and bcov. in detail, the crcov-k37 strain was related to crcov-bj232 in the s gene, while in the orf1ab, he, m, and n genes, crcov-k37 was noted to possess a close relationship with bcov. recombination is a common phenomenon in coronaviruses and thought to contribute to the emergence of new pathotypes (gorbalenya, 2008; wang et al., 2015) . thus far, most of the recombination events of coronaviruses have been reported between species of the same group (herrewegh et al., 1998; keck et al., 1988) , such as among the batassociated covs (corman et al., 2014) and 229e-related covs (corman et al., 2015) , and the major recombination breakpoint has mainly been within the s gene. genetic recombinant among alphacoronavirus 1 species frequently occurred, such as ferret coronaviruses (lamers et al., 2016; minami et al., 2016) . however, few researches about genetic recombination of betacoronavirus 1 species have been conducted. the discovery of genetic recombination between crcov and bcov confirms the phenomenon, and intraspecies recombination therefore exists for betacoronavirus 1 species. phylogenetic analyses based on all available crcov genes showed that the two clusters of crcov, including recombinant strain (south korea k37 strain) and the chinese bj232 strain, have been prevalent worldwide for many years. crcov and bcov were closely related in betacoronavirus 1, located on the outer leaves of phylogenetic tree, and form an independent branch. it was difficult to determine the evolution orientation (transmission from cattle to canine or both from murine), although the latest research supports the murine origins of betacoronavirus 1 (lau et al., 2011 (lau et al., , 2015 . more crcov genome sequences are needed to understand the evolutionary relationship between crcov and bcov. regarding the pathogenicity and transmissibility of crcov, the chinese bj232 strain and south korea k37 strain were both derived from diseased dogs with respiratory infections. genomic analyses showed that the s genes, which mediate coronavirus attachment, were highly similar in these strains. because recombination may be a driving force for the formation of pathogenic viruses from less pathogenic virus (forni et al., 2017) , recombinant may play an important role in the pathogenicity and transmissibility of crcov. limitation of this study was that the isolation of crcov-bj232 failed on cell culture. more crcov strains were needed to be isolated or rescued using reverse genetic technique to perform experimental inoculation of dogs. in conclusion, herein, we reported the first complete genome sequence of a crcov derived from a diseased dog in china, and confirm that intraspecies recombinant exists for betacoronavirus 1 species. the results may have implications for the origin and evolution of crcov. this work was supported by grants from the national key plan for scientific research and development of china (2016yfd0500301) and megaproject for infectious disease research of china (2014zx10004001,2013zx10004601). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the authors have declared that no competing interests exist. fig. 4 . phylogenetic analyses of all available crcov strains based on he, m, n and s genes. the bj232, bj221, bj34 and bj202 strains come from china in this research,k37, k39 and k9 strains were collected from south korea, and the 240/05 and 4182 strains were derived from italy and the united kingdom, respectively. all the genes were analyzed by phyml program using the maximum likelihood method, with 500 bootstrap replicates. genetic analysis of canine group 2 coronavirus in korean dogs canine respiratory viruses rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat evidence for an ancestral association of human coronavirus 229e with bats investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex detection of a group 2 coronavirus in dogs with canine infectious respiratory disease longitudinal 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natural recombination discovery of a novel coronavirus, china rattus coronavirus hku24, from norway rats supports the murine origin of betacoronavirus 1 and has implications for the ancestor of betacoronavirus lineage a complete genome analysis of canine respiratory coronavirus molecular characterization of a canine respiratory coronavirus strain detected in italy detection of novel ferret coronaviruses and evidence of recombination among ferret coronaviruses genetic diversity of a canine coronavirus detected in pups with diarrhoea in italy serological prevalence of canine respiratory coronavirus serological prevalence of canine respiratory coronavirus in southern italy and epidemiological relationship with canine enteric coronavirus complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event evolutionary history of the closely related group 2 coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc43 origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis metagenomic analysis of viral genetic diversity in respiratory samples from children with severe acute respiratory infection in china survey of dogs in japan for group 2 canine coronavirus infection supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2017.05.006. key: cord-347924-w06ho8sn authors: wen, jin-sheng; jiang, li-fang; zhou, jun-mei; yan, hui-jun; fang, dan-yun title: computational prediction and identification of dengue virus-specific cd4(+) t-cell epitopes date: 2007-12-03 journal: virus res doi: 10.1016/j.virusres.2007.10.010 sha: doc_id: 347924 cord_uid: w06ho8sn in this study, we tried to identify dengue virus-specific cd4(+) t-cell epitopes, which can induce pbmc (peripheral blood mononuclear cells) isolated from df convalescent patients (dengue virus type 1 infection) to secrete ifn-γ. pbmc of df convalescent patients were stimulated in vitro with dengue virus-derived peptides, which were prepared based on the prediction of dengue virus-specific cd4(+) t-cell epitopes by using rankpep online software. subsequently, the frequency of ifn-γ producing t cells and percentage of ifn-γ(+) cd4(+) t cells were measured by using elispot assay and ics assay (intracellular cytokine straining), respectively. the positive response of pbmc by elispot showed that the numbers of sfc (spots forming cells) ranged from 50 to 310 sfc/1 × 10(6) pbmc. the positive response of pbmc by ics assay showed that the percentage of ifn-γ(+) cd4(+) t cells ranged from 0.03 to 0.27%. as a result, c(45-57) (klvmafiaflrfl), e(396-408) (ssigkmfeatarg), ns3(23-35) (yrilqrgllgrsq), and ns3(141-155) (nregkivglygngvv) were identified as dengue virus-specific cd4(+) t-cell epitopes. dengue virus (den) is a single-stranded positive rna virus of the family flaviviridae comprised of four distinct serotypes (den-1, 2, 3, 4) within the flavivirus genus. infection with any of the four serotypes of dengue virus can result in an asymptomatic infection, a self-limited febrile disease (dengue fever, df) or a severe, life-threatening illness (dengue hemorrhagic fever, dhf) (halstead, 1988) . den infections are a serious cause of morbidity and mortality in most tropical and subtropical areas of the world (gubler, 1998; gibbons and vaughn, 2002) . if untreated, the mortality rate of dhf is around 30% but can be reduced to 0.2-5% by fluid replacement. even though previous study suggested that antibodydependent enhancement (ade) take part in the pathogenesis of dhf, the pathogenesis of dhf cannot entirely be explained by antibody, however. currently, many studies suggested that dengue-specific t-cell immune response are hypothesized to play an important role in the immunopathogenesis of dhf during a secondary den virus infection, and induction of immunopathology by t lymphocytes may occur by various mechanisms, including cell-mediated cytotoxicity and/or cytokine production (miskovsky et al., 1994; vergelli et al., 1997) . in addition, other study showed that dhf is associated with high levels of t-cell cytokines such as ifn-␥ and tnf-␣, and evidence of marked t-cell activation (libraty et al., 2002) . recently, immunopathogenesis in dhf has been proposed. the evidence is that both dengue-specific cd8 + and cd4 + lymphocytes have been implicated in pathogenesis and natural immunity to dengue viruses lei et al., 2001) . how t cells contribute to this process is incompletely defined, however, since only a few studies have directly examined t-cell responses during acute disease and few den-specific t-cell epitopes have been identified (green et al., 1999; mongkolsapaya et al., 2003) . therefore, the establishment of a library of defined t-cell epitopes from dengue viruses will help to explore the role of t cells in immunity and disease pathogenesis. in the present study, a combination of bioinformatics tools (epitope-prediction programs rankpep) and in vitro assays (enzyme-linked immunospot assay (elispot) and intracel-lular cytokine staining assay (ics)) was used to screen and select antigen sequences as potential den-specific cd4 + t-cell epitopes, then the selected sequences are tested for biological function by their activation of t cells of den infected populations. a total of 10 df convalescent patients were enrolled in this study. they are early-convalescent-phase den infection cases (den-1) recruited from the eighth people's hospital of guangzhou, china. we also included three flavivirusnaïve guangdong residues. den-1 infection was confirmed by virus isolation in c6/36 cell culture and subsequent serotype identification with den serotype-specific rt-pcr during the viremia-phase (lanciotti et al., 1992) . in addition, den infection was also confirmed by using commercial capture-immunoglobulin m (igm) and igg enzyme-linked immunosorbent assay (elisa) during the convalescent-phase as described previously (kuno et al., 1985) . after obtaining informed consent, peripheral blood samples were drawn for preparation of peripheral blood mononuclear cells (pbmc). meanwhile, the ethics committees of the authors' institutions approved this study. pbmc were isolated from the peripheral whole blood by ficoll-hypaque density gradient centrifugation (400 × g, 30 min, 24 • c) and used directly in elispot assay and ics assay. rankpep online software (http://www.mif.dfci.harvard. edu/tools/rankpep.html) was used for the prediction of denspecific cd4 + t-cell epitopes. the den strains used in the prediction process included den-1 strains, den-2 strains, den-3 strains and den-4 strains ( table 1) . the following criteria were used to select the candidate denspecific cd4 + t-cell epitopes: 1, having a higher ranking scores on rankpep prediction; 2, predicted epitopes should be highly conserved in these selected den strains. four higher-ranking candidate cd4 + t-cell epitopes (c 45-57 , e 396-408 , ns3 23-35 and ns3 141-155 ) were synthesized using 9-fluorenylmethoxy carbonyl-based solid-phase chemistry on a multiple peptide synthesizer (purity >95% pure; huachen company, xi'an, china) and the coincidence of the actual molecular mass with the theoretical molecular mass was confirmed by mass spectrography analysis. peptides were dissolved in phosphate-buffered saline (pbs) and diluted to 2 mg/ml in pbs, and stored at −80 • c for subsequent elispot assay and ics assay. the sequences and other information of these peptides are detailed in table 2 . assay was performed using human ifn-␥ elispot kit (r&d systems minneapolis, mn), following the protocol provided by the manufacturer. briefly, 96-well polyvinylidene difluoride-backed plates, precoated with anti-ifn-␥ monoclonal antibody, were blocked with 200 l of sterile rpmi-1640 medium (containing 2 mm l-glutamine, 100 u/ml penicillin, 100 g/ml streptomycin and 10% heat-inactivated fetal calf serum) for 20 min at room temperature. a total of 1 × 10 5 pbmc were added in 100 l rpmi-1640 medium per well, and individual peptides were subsequently added to a final concentration of 10 g/ml. unstimulated and phytohemagglutinin (pha, 10 g/ml) stimulated cells served as negative and positive controls, respectively. in addition, sars-cov peptide (severe acute respiratory syndrome-coronary virus, shvplatsrtla) used as irrelevant peptide control. after an overnight incubation at 37 • c and 5% co 2 in air, plates were washed with pbs containing 0.05% tween-20. next, the biotinylated anti-ifn-␥ antibody was added. after an overnight incubation at 4 • c, plates were washed, and then incubated for 2 h at room temperature with the diluted streptavidinalkaline phosphatase. after four final washes, plates were developed with 100 l/well of alkaline phosphatase substrate 5bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium substrates (bcip/nbt) chromogen for 1 h at room temperature (protecting from light). color development was monitored and stopped by washing with water. after dried overnight at room temperature, sfc (spots forming cells) per well were counted using an elispot reader (beijing sagecreation science co., ltd., china). the deep-browncolored spots with decreasing density radiating from the center were counted and expressed as responding cells in 1 × 10 6 pbmc. the assay was considered valid only if positive control wells showed >100 spots/1 × 10 6 pbmc and if negative control wells showed <10 spots/1 × 10 6 pbmc. a positive response to a peptide was defined as having ≥50 spots/1 × 10 6 pbmc after subtraction of background as described previously (simmons et al., 2005) . pbmc were resuspended in rpmi-1640 medium and used directly in ifn-␥ ics assay as previously described (mothe et al., 2002) . briefly, each test contained 0.7 × 10 6 pbmc and was incubated with either peptides (sars-cov peptide as irrelevant peptide control) at 10 g/ml or not stimulated (no peptide control) for 6 h at 37 • c and 5% co 2 . meanwhile, brefeldin a (bfa, 10 g/ml, biolegend inc. ca) was added. cells were washed using pbs and then stained using pe-labelled anti-cd3, apc-labelled anti-cd4 and pe-cy5labelled anti-cd8 antibodies (biolegend inc. ca) for 40 min at 4 • c. subsequently, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% saponin, and then stained using fluorescein isothiocyanate (fitc)-labelled anti-ifn-␥ antibody (biolegend inc. ca). data acquisition was performed on a facs calibur flow cytometer (bd bioscience) using cellquest software. the number of events collected for each sample varied between 100,000 and 300,000, depending on the donors. cd4 + /cd8 + cells were gated and analyzed for cytokine expression. peptide-specific cells were defined as ifn-␥-producing cd4 + /cd8 + t cells. between october 2006 and january 2007, we recruited 10 convalescent patients having dengue virus type 1 infection. the mean age was 37 years (ranging from 18 to 64 years) and the mean time (from discharge to drawing blood sample) was 58 days (ranging from 14 to 120 days). the characteristics of the study population are detailed in table 3 . the positive control assay showed that the numbers of sfc ranged from 600 to 1320 sfc/1 × 10 6 pbmc while the negative control (unstimulated pbmc) showed that the numbers of sfc was 0. in 10 df convalescent patients, we identified one or more individual peptides that reproducibly induced an ifn-␥ response in the elispot assay that exceeded 50 sfu/1 × 10 6 pbmc. in total, t cells from 10 df convalescent patients recognized four different peptides. the magnitude of responses to individual peptides ranged from 50 to 310 sfu/million pbmc. the breadth of the response in individual patients ranged from 1 to 4 peptides. in addition, the mean numbers of spots obtained for den-1-seronegative individuals were 0 for c 45-57 , ns3 23-35 and irrelevant peptide, and 10 for e 396-408 and ns3 141-155 . in total, these four peptides ) are den-specific t-cell epitopes. a summary of the individual peptides that reproducibly induced responses in ifn-␥ elispot assay is provided in fig. 1. the levels of circulating cd4 + t cells of 10 patients ranged from 10.89 to 21.46% while the levels of circulating cd8 + t cells ranged from 6.66 to 21.03%. the cd4/cd8 ratios were above one (varying from 1.01 to 1.89) ( table 4 ). for any subject, there were no ifn-␥ + cd4 + t cells in no peptide control and irrelevant peptide control. we identified individual epitopes that producibly evoked ifn-␥ + cd4 + t cells in the ics assay that exceeded 0.01% (ifn-␥ + cells less than or equal to 0.01% of cd4 + t cells were considered as negative). the positive response of pbmc showed that the percentages of ifn-␥ + cd4 + t cells ranged from 0.03 to 0.27% (see fig. 2 ) while the percentages of ifn-␥ + cd8 + t cells were 0 (data not shown). in addition, no responses to these peptides were seen in pbmc of the flavivirus-naïve donor (data not shown). as a result, c 45-57 , e 396-408 , ns3 23-35 and ns3 141-155 are den-specific cd4 + t-cell epitopes (see fig. 2 ). initially, the viral t-cell epitopes were identified either by sequencing of eluted peptides bound to mhc molecules (major histocompability complex) or by screening panels of overlapping peptides derived from the viral protein. however, these two methods have their shortcomings. the former is cumbersome and needs sophisticated equipments (such as mass spectrometer or peptide sequencer). the latter needs synthesis of a series of the overlapping peptides and massive experiments to identify which peptide is epitope, making it an extensive and time-consuming process (drabner et al., 2002) . at present, a novel strategies that utilize computer-based t-cell epitopes prediction methods combined with in vitro/in vivo studies was established to predict potential t-cell epitopes and are becoming increasingly popular as the amino acid sequences of pathogenic proteins are available in database (huang et al., 2004; turcanová and höllsberg, 2004) . epitope-prediction program rankpep was developed by the "molecular immunology foundation" by p.a. reche et al., which uses position specific scoring matrics (pssm), cleavage model and predicts mhc binding peptide from an input protein based on their similarity to a set of peptides known to bind to a given mhc molecule (reche et al., 2004) . among epitopes-prediction programs, rankpep prediction algorithm has higher sensitivity and specificity and represents a quick and effective way to identify t-cell epitopes (drabner et al., 2002; gomez-nunez et al., 2006; reche et al., 2002 reche et al., , 2004 . recently, elispot assay and ics assay were extensively used to measure the frequencies of memory t cells. compared with other in vitro methods, elispot assay and ics assay own the higher sensitivity and the latter have the advantage of defining the phenotype of the cytokine secreting cells (waldrop et al., 1997; butz and bevan, 1998; pala et al., 2000) . therefore, the combination of elispot assay and ics assay is necessary to identify t-cell epitopes. up to date, studies identified some den-specific cd4 + t-cell epitopes, which are mainly located in ns3 protein or c protein fig. 2 . frequencies of ifn-␥ + cd4 + t cells in pbmc of subjects. numbers on the upper right hand corner of each panel indicate ifn-␥ + cd4 + t cells as percentage of cd4 + t cell. data acquisition was performed on a facs calibur flow cytometer (bd bioscience) using cellquest software. the number of events collected for each sample varied between 100,000 and 300,000, depending on the donors. cd4 + /cd8 + cells were gated and analyzed for cytokine expression. (kurane et al., 1995 (kurane et al., , 1998 gagnon et al., 1996; mangada et al., 2004) . however, most of these epitopes are only conserved in den-4 or den-3 or den-2, 3, 4 and the epitope which is highly conserved in den-1 had not been identified. furthermore, the low sensitive methods/technologies (the limiting dilution method and cytotoxic assay) limited general laboratories to identify t-cell epitopes. in this study, we measured a higher frequencies of den peptide-specific ifn-␥ responding cd4 + t cells by using elispot assay and ics assay and then identified four den-specific cd4 + t-cell epitopes. more importantly, among the 10 df convalescent patients, as for each peptide, the breadth of the positive response ranged from 7 to 8 subjects. this suggested that these epitopes could be the immunodominant epitopes and restricted by the high-frequency hla alleles of human population. of course, future experiments would be performed to determine the hla restriction of these epitopes. among these four epitopes, c 45-57 and ns3 141-155 contains the sequence that was previously characterized as den-specific cd4 + tcell epitopes (c 47-55 and ns3 146-154 , respectively) (kurane et al., 1995; gagnon et al., 1996) . therefore, our results indicated that c 47-55 and ns3 146-154 could be the minimal sequence recognized by mhc-ii molecule. interestingly, ns3 146-154 was previously identified as hla-dr15-restricted epitope (kurane et al., 1995) while the putative hla allele of ns3 141-155 by rankpep is hla-dr15. in addition, c 47-55 was hla-dpw4restricted epitope (gagnon et al., 1996) while the restricting hla alleles of c 45-57 by rankpep include dpw4. therefore, these information suggested that the restricting hla alleles of c 45-57 and ns3 141-155 could be closely related with dpw4 and dr15, respectively. in this study, e 396-408 and ns3 23-35 are two novel identified cd4 + t-cell epitopes. since e protein is the important structural protein in den infection and immunity, more researchers aimed to identified t-cell epitopes located in this protein and defined some t-cell epitopes simmons et al., 2005; bashyam et al., 2006) . however, to our knowledge, e 396-408 is the first cd4 + t-cell epitopes identified in human population. importantly, e 396-408 is only highly conserved in den-1 strains. therefore, the identification of e 396-408 would help to explore the role of t-cell immune response induced by e protein in den infection or immunity. previous study shown that ns3 protein appears to be the most immunogenic antigen for cellular responses to den and there exist many t-cell epitopes in ns3 200-324 (mathew et al., 1996) . consequently, most research focused on identifying t-cell epitopes located in this region (kurane et al., 1995 (kurane et al., , 1998 . in this study, the identification of ns3 23-35 suggested that the other region of ns3 could also contribute to t-cell immune response. in conclusion, this study indicated a good correlation between experimentally observed and computationally predicted peptides. therefore, the combination of bioinformatics tools and in vitro assays is a better strategy for the identification of t-cell epitopes. dengue virus-reactive cd8 + t cells display quantitative and qualitative differences in their response to variant epitopes of heterologous viral serotypes massive expansion of antigen-specific cd8 + t cells during an acute virus infection identification of t helper cell-recognized epitopes in the chitinase of the filarial nematode onchocerca volvulus identification of two epitopes on the dengue 4 virus capsid protein recognized by a serotypespecific and a panel of serotype-cross-reactive human cd4 + cytotoxic tlymphocyte clones dengue: an escalating problem peptide binding motif predictive algorithms correspond with experimental binding of leukemia vaccine candidate peptides to hla-a*0201 molecules early cd69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever dengue and dengue hemorrhagic fever pathogenesis of dengue: challenges to molecular biology identification of novel hla-a*0201-restricted cd8+t-cell epitopes on hepatitis delta virus antigen capture elisa for the identification of dengue viruses flavivirus-cross-reactive hla-dr15-restricted epitope on ns3 recognized by human cd4 + cd8 − cytotoxic t lymphocyte clones definition of an epitope on ns3 recognized by human cd4 + cytotoxic t lymphocyte clones crossreactive for dengue virus types 2, 3, and 4 rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction immunopathogenesis of dengue virus infection differing influences of viral burden and immune activation on disease severity in secondary dengue 3 virus infections quantitation of dengue virus specific cd4 + t cells by intracellular cytokine staining dominant recognition by human cd8 + cytotoxic t lymphocytes of dengue virus nonstructural proteins ns3 and ns1.2a studies of the mechanism of cytolysis by hiv-1-specific cd4 + human ctl clones induced by candidate aids vaccines original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever characterization of the peptide-binding specificity of mamu-b*17 and identification of mamu-b*17-restricted epitopes derived from simian immunodeficiency virus proteins flow cytometric measurement of intracellular cytokines prediction of mhc class i binding peptides using profile motifs enhancement to the rankpep resource for the prediction of peptide binding to mhc molecules using profiles multiple specificities in the murine cd4 + and cd8 + t-cell response to dengue virus immunopathogenesis of dengue hemorrhagic fever early t-cell responses to dengue virus epitopes in vietnamese adults with secondary dengue virus infections sustained cd8 + t-cell immune response to a novel immunodominant hla-b*0702-associated epitope derived from an epstein-barr virus helicase-primase-associated protein human autoreactive cd4 + t cell clones use perforin-or fas/fas ligand-mediated pathways for target cell lysis determination of antigen-specific memory/effector cd4 + t cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in hiv-associated immunodeficiency the work was supported by grants from the team project of nature science foundation of guangdong province, china (5200638). key: cord-281101-gv1sgbk1 authors: shin, gu-choul; chung, yoon-seok; kim, in-soo; cho, hae-wol; kang, chun title: preparation and characterization of a novel monoclonal antibody specific to severe acute respiratory syndrome-coronavirus nucleocapsid protein date: 2006-08-30 journal: virus res doi: 10.1016/j.virusres.2006.07.004 sha: doc_id: 281101 cord_uid: gv1sgbk1 severe acute respiratory syndrome-coronavirus nucleocapsid (sars-cov n) protein has been found to be important to the processes related to viral pathogenesis, such as virus replication, interference of the cell process and modulation of host immune response; detection of the antigen has been used for the early diagnosis of infection. we have used recombinant n protein expressed in insect cells to generate 17 mabs directed against this protein. we selected five mabs that could be used in various diagnostic assays, and all of these mabs recognized linear epitopes. three igg(2b) mabs were recognized within the n-terminus of n protein, whereas the epitope of two igg(1) mabs localized within the c-terminus. these mabs were found to have significant reactivity with both non-phosphorylated and phosphorylated n proteins, which resulted in high reactivity with native n protein in virus-infected cells; however, they did not show cross-reactivity with human coronavirus. therefore, these results suggested that these mabs would be useful in the development of various diagnostic kits and in future studies of sars-cov pathology. severe acute respiratory syndrome (sars), which is caused by the sars coronavirus (sars-cov), is a newly emerging disease. sars-cov presented with high virulence and mortality, and affected 30 countries, with more than 8000 cases and over 750 deaths. indeed, as the clinical symptoms of sars are nonspecific compared to those of other respiratory viruses, diagnosis relies largely on laboratory tests . thus, the development of diagnostic laboratory tests for specific and early detection of sars-cov infection is of great importance for both rapid treatment of patients and control of sars outbreaks. thus far, virus isolation methods have generally been performed to determine the presence of infectious virus in samples; this process is relatively time-consuming and inefficient (keyaerts et al., 2005; yamashita et al., 2005) . rt-pcr and real time-pcr for direct detection of sars-cov rna is expen-sive and labor-intensive, relies on the availability of expertise, and may produce false-positive results due to contamination (keightley et al., 2005; huang et al., 2005) . serological methods, such as immunofluorescence assay and enzyme-linked immunosorbent assay (elisa), are not adequate for the early diagnosis of sars, because the median time to seroconversion in sars patients is 17-20 days after the onset of symptoms . therefore, the generation of monoclonal antibodies (mabs) against sars-cov antigens may provide possible diagnostic tools for early diagnosis of sars, because sars-cov can be specifically detected in the respiratory specimens, blood and stool much earlier than antibodies can be used for detection (lau et al., 2004; di et al., 2005) . the sars-cov nucleocapsid (sars-n) protein is a phosphoprotein of 48 kda, and performs multiple functions in viral pathogenesis, such as providing a nuclear-import signal, interfering in the cell process, participating in virus replication and packaging rna (egloff et al., 2004; yan et al., 2004; surjit et al., 2004 surjit et al., , 2005 . thus, this protein may have important roles in the pathogenesis of sars. furthermore, this protein is the most abundantly expressed structural protein during infection and is highly detectable in sars patients lau et al., 2004; di et al., 2005) . therefore, this protein may serve as one of the immunodominant antigens in the early diagnosis of infection. furthermore, some researchers have suggested that antibody against the n protein could modulate cytokine responses such as il 11; non-neutralizing antibodies against n protein were found to protect mice against lethal infection (nakanaga et al., 1986; cheng et al., 2005) . therefore, the development of mabs against sars-n protein may be critical in the development of drugs to treat sars-cov infection, and for further study of the pathogenesis of sars, as well as early diagnosis. here, we report the production of 17 mabs against sars-n and the properties of the mabs, which were determined by isotyping, affinity assay, epitope mapping and reactivity with various isoforms of sars-n protein. we also suggested the applicability of the mabs in various analytical methods, such as ifa, immunoblot and antigen-capture elisa, for diagnosis and functional study of sars-cov n protein. sp2/0 myeloma cells were kindly provided by metabolab inc. (seoul, republic of korea), and mrc-5 cells (atcc, ccl-171) were obtained from american type culture collection. sars-cov stock was provided from the center for disease control and prevention, and this virus was maintained in biosafety level-3 (bsl-3) containment laboratories in the national institute of health, korea center for disease control and prevention. the sars-cov titer was determined to be 1 × 10 4 50% tissue culture infectious doses/ml (tcid 50 /ml). the virus culture supernatants were inactivated by heating at 56 • c for 60 min prior to use. human coronavirus oc43 (atcc, vr-1558; hcov oc43), which was tested to determine cross-reactivity with sars-n mab, was prepared from mrc-5 cells. replication of these viruses was confirmed by rt-pcr and immunofluorescence assay using mab against nucleocapsid protein of human coronavirus oc43 (hcov mab). the hcov oc43 titer was determined by hemagglutination assay (ha) using human red blood cells (rbcs). the complete coding sequence for the n protein (urbani strain, genbank accession no. ay278741, 28,120-29,388 bp) was amplified from the sars-cov genomic rna using pcr. the amplified product was digested with ecori and bamhi, and then inserted into his-tagged pentr bhrnx vector (neurogenex, seoul, republic of korea) to create pentr np7. recombinant baculovirus was generated by co-transfection of sf21 cells with pentr np7 and linearized baculovirus dna using the baculogoldtm system (bd biosciences, san jose, ca). recombinant baculoviruses were harvested from sf21 cell culture medium 48 h post-transfection and broken by three cycles of freezing-thawing. the 6× histidine-tagged recombinant n protein (brsars-n) was purified by metal-chelating affinity chromatography (merck bioscience, darmstadt, germany). the purified protein was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page), as described previously (laemmli, 1970) . to examine the reactivity of sars-n mabs against the phosphorylated protein and non-phosphorylated protein, rsars-n protein expressed in escherichia coli (ersars-n) was purchased as nonphosphorylated protein from biovendor laboratory medicine, inc. (heidelberg, germany). balb/c mice (samtako inc., republic of korea; 9 weeks) were intraperitoneally injected with a mixture containing 50 g purified brsars-n proteins in 100 l phosphate buffered saline (pbs) and an equal volume of freund's complete adjuvant. a boost injection with the same amount of antigen in freund's incomplete adjuvant was administered at 2-week intervals. hybridoma fusion was performed using a method similar to that originally described (kohler and milstein, 1975) , with the following modifications. in brief, the splenocytes were harvested from immunized mice, mixed with sp2/0 cells at a 5:1 ratio, and fusion was carried out with 40% polyethylene glycol-1500 (roche, indianapolis, in). the fused cells were collected by centrifugation at 800 × g for 5 min and the cell pellet was resuspended in dmem (invitrogen, carlsbad, ca) containing 20% fbs and hat supplement (sigma-aldrich korea co., seoul, republic of korea). the cells were seeded in 96-well plates at 200 l/well (2 × 10 5 cells/well) and cultured in a co 2 incubator. antibody produced in medium was measured by indirect enzyme-linked immunosorbent assay (indirect elisa) as described below. a limiting dilution of hybridoma was carried out from putative positive individual wells, and the screening was repeated until hybridoma clones producing a strongly reactive sars-n mab were observed. selected hybridoma clones were maintained in dmem containing 10% fbs, 1× ht supplement (sigma-aldrich korea) and exchanged with fresh media once every 3 or 4 days. the sars-n mab of a selected hybridoma was purified using the immunopure (g) igg purification kit (pierce biotechnology inc., rochford, il) and isotyped with an immunopure monoclonal antibody isotyping kit ii (pierce biotechnology inc.), used according to the instructions of the manufacturer. the indirect elisa was carried out on a maxisorp plate (nalgen nunc international, rochester, ny), which had been coated with 1 g of recombinant sars-ns diluted in 50 mm carbonate buffer (ph 9.6) and incubated overnight at room temperature. non-specific protein binding sites were blocked with 1% bovine serum albumin (bsa) in pbs for 1 h at 37 • c. plates were washed with pbs containing 0.05% tween 20 (pbst). hybridoma supernatants, sars-n mab and anti-sars serum obtained from mice immunized with heat-inactivated sars-cov, or normal mouse serum as negative serum, were then added and incubated for 60 min at 37 • c. after washing with pbst, a 1:1000 dilution of alkaline phosphatase (ap)-conjugated goat anti-mouse igg+ iga+ igm antibody (abcam) in pbst containing 1% bsa was added to all wells and incubated for 60 min at 37 • c. after a final wash, p-nitrophenyl phosphate disodium salt (pnpp, pierce biotechnology inc.) solution was added, and the plates were further incubated for 30 min at room temperature. the color intensity was measured as the absorbance value at 405 nm (od 405 ) in an el340 elisa reader (bio-tek intstruments inc., winooski, vt). isotyping was performed using immunopure monoclonal antibody isotyping kit ii (pierce biotechnology inc.), used according to the instructions of the manufacturer. to examine whether sars-n mab recognizes the linear epitope of sars-n protein, we performed an immunoblot assay in denaturing conditions of the sars-n protein. the purified rsars-n protein were resolved by 10% sds-page, and were then electrotransferred onto a pvdf membrane (bio-rad, hercules, ca) and blocked with 1% bsa in pbst. membrane was incubated with sars-n mabs (1 g/ml) or anti-his tag mab (1:1000 dilution, abcam), as positive control, for 1 h, and non-specific adsorption was washed away by pbst. the bound antibodies were detected by horseradish peroxidaseconjugated goat anti-mouse igg+ iga+ igm secondary antibody (1:2000 dilution, abcam), followed by dab substrate solution (sigma-aldrich korea). a total of 19 linear peptides ranging in size from 11 to 26 amino acid residues were synthesized on the basis of the fulllength nucleocapsid protein sequences of sars-cov urbani strain, genebank aap13445 (cosmo co., seoul, republic of korea). the synthesized peptides were characterized by hplc and mass spectrometry. to identify the epitopes in sars-cov n protein that are targeted by sars-n mabs, competition elisa was performed as described previously (chiang et al., 2005) . the sars-n mabs (10 g/ml) were incubated with the competitor peptide or brsars-n protein (10 g/ml) for 1 h at 4 • c. the mabs were then transferred into the wells of a maxisorp plate that had been coated with 0.5 g/well of sars-n protein. the following steps were then accomplished as described in the indirect elisa procedure. affinity analysis was performed by non-competitive elisa as described previously (huang et al., 2005) , with the following modifications. in brief, maxisorp plate was coated overnight with 1 g/well of brsars-n, as described in the indirect elisa procedure. the plates were blocked with 1% bsa, followed by incubation of sars-n mab with serial dilution (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 and 0.3 nm). after washing with pbst, ap-conjugated goat anti-mouse igg+ iga+ igm antibody (abcam) in pbst containing 1% bsa was added to all wells and incubated for 30 min at 37 • c. the following steps were accomplished in the same manner as that used for indirect elisa, described above. the affinity constants were presented as the concentrations (nm) of mab at 1.000 of od 405. reactivity of sars-n mabs with sars-cov infected cells was determined by immunofluorescence assay, performed according to the instructions of the manufacturer (euroimmun, germany). the sars-n mab (10 g/ml), anti-sars serum (1:100 dilution) and negative serum were added to each well, and the slides were incubated at 37 • c for 30 min. after washing three times with pbs, fluorescein isothiocyanate-conjugated goat anti-mouse igg+ iga+ igm antibody (icn biomedical, 1:100 dilution) was added, and the slides were then incubated at 37 • c for 30 min. the slides were then washed three times with pbs and one time with ultrapure water and observed by fluorescence microscopy. to prepare detector mabs, the purified mabs were labeled with biotin using the ez-link sulfo-nhs-lc-biotinylation kit (pierce biotechnology inc.) according to the instructions of the manufacturer. the purified mab for the antigen-capture was immobilized on the hi-bind microplate (costar, coring incorporated, ny) by incubating 5 g/ml antibody in 50 mm carbonate buffer (ph 9.6) at 4 • c overnight. the wells were then washed twice with pbs, followed by blocking with 2% bsa in pbs, ph 7.4, for 4 h at room temperature. after removing the blocking reagent, the wells were dried and stored at 4 • c prior to use. virus culture supernatant or recombinant n protein diluted in 1% bsa in pbst or virus culture medium was added to the wells (100 l/well) and incubated for 2 h at 37 • c. after washing, the wells were incubated for 1 h at 37 • c with 100 l per well of biotin-conjugated detector mab (diluted 1/200 in pbst with 1% bsa or virus culture medium). after washing, the wells were incubated for 1 h at 37 • c with 100 l per well of ap-conjugated goat anti-biotin antibody (diluted 1/500 in pbst with 1% bsa or virus culture medium, abcam). after washing, 100 l of pnpp was added to each well. the color reaction was stopped after 30 min with 100 l of 1n naoh to each well, and the plates were examined at 405 nm in an el340 elisa reader (bio-tek instruments inc.). the experiments involving the use of the inactivated sars-cov were performed in a bsl-2 laboratory. for the immunofluorescence assay to assess the crossreactivity of mabs with human coronavirus, spot slides were prepared by applying a suspension of cells infected with uvc-irradiated human coronavirus oc43 or a suspension of uninfected mrc-5 cells to the wells of teflon-coated slides. slides were allowed to air-dry before they were fixed in methanol. slides were stored at −70 • c until used for indirect fluorescence assay. the hcov mab (1:100 dilution, chemicon international inc., temecula, ca) as positive control, normal mouse serum as negative control, and sars-n mabs (100 g/ml) were added to the slides, and the slides were incubated at 37 • c for 30 min. the following steps were accomplished in the same manner as that described above for sars-cov. for antigen-capture elisa, human coronavirus oc43 (256 ha unit) supernatant that had been cultured from mrc-5 cells was heat-inactivated for 30 min at 56 • c and serially diluted two-fold. the protein samples were then extracted by three cycles of freezing-thawing before centrifugation at 14,000 rpm for 10 min and dilution in pbs with 1% bsa. the preceding steps were performed in the same manner as that used for sars-cov, described above. full-length sars-n protein was produced in a recombinant baculovirus system and used in the immunization of mice. two weeks after each antigen boost dose, immunized mice were screened for sars-n specific antibody response by indirect elisa. splenocytes isolated from the mice were fused with sp2/0 myeloma cells, resulting in ∼1000 proliferating hybridomas. subsequent screening of these hybridomas and single cell cloning yielded 17 positive clones that constitutively secreted mabs that reacted to sars-n protein by indirect elisa (data not shown). further isotyping of these mabs were determined in order to facilitate future utilization. for the heavy chain subclasses, most of the mabs were found to belong to the igm subtype, two of the mabs belonged to igg 1 , and three of the mabs belonged to igg 2b . the light chain of all of these mabs was of the kappa isotype (table 1) . five of the 17 mabs determined to be of the igg subclass were characterized by further analysis, because these mabs may be suitable for use as diagnostic mabs. immunoblotting was performed with brsars-n protein in order to analyze the reactivity of sars-n mabs against sars-n protein under denaturing conditions and to determine whether these mabs recognize the conformational epitope of sars-n table 1 isotypes of the sars-n mabs generated in this study these isoforms were also observed in immunoblot analysis of total proteins extracted from baculovirus-infected cells and sars-cov-infected cells (data not shown). thus, these isoforms may be fragment cleaved by intracellular protease. the fragment of 25-kda that was not observed in originally purified brsars-n protein could be obtained by freezing and thawing procedures of purified protein. thus, this result indicated that the epitopes of all of the sars-n mabs are linear, and the epitopes of igg 2b and igg 1 mabs are located in the n-terminus and c-terminus of sars-n protein, respectively. to more precisely analyze the epitope of sars-n protein recognized by these sars-n mabs, competitive elisa was conducted using the 19 synthetic peptides covering the full-length sars-n protein sequence. among these synthetic peptides, the n135 peptide (ategalntpkdhigtr; at position 135-150 of sars-n protein) and n17 peptide (tfggptd-stdnnqngg; at position 17-32) could effectively compete in the binding of all of the igg 2b mabs with the sars-n protein, and the n117 peptide (gpeaslpygankegiv; at position 117-132) seems to have slightly weak activity, while the other peptides did not exhibit any effect in this assay ( fig. 2a) . the n17 and n135 peptides contain no apparent common epitopes, which revealed that these mabs are mixed antibodies. the n215 peptide (ggetalalllldrlnqleskvsgkg; at position 215-239) resulted in complete inhibition of the binding activity of all of the igg 1 mabs with the sars-n protein, and the n245 peptide (qtvtkksaaeaskkprqkrtatkq; at position 245-268) seems to have slightly weak activity (fig. 2b ). according to the results of the immunoblot and competitive elisa, the epitopes of the igg 2b mabs is located in the nterminal region at aa 17-32 and 117-150, whereas that of the igg 1 mabs is located in the middle region at aa 215-268 (fig. 2c ). to examine the reactivity of the sars-n mabs against sars-cov n protein under non-denaturing conditions, the indirect elisa was performed with brsars-n protein. four of the selected mabs (22-05-03, 07-19-11, 07-19-21 and 21-10-06) bound slightly better to sars-n protein than 21-10-11, as shown in table 2 . to confirm the reactivity of mabs against sars-n protein, as estimated by indirect elisa, the affinity constants of five mabs were measured by non-competitive elisa, as described in section 2. the affinity constants of 22-05-03, 07-19-11, 07-19-21 and 21-10-06 were significantly higher than that of 21-10-11 (table 2) . these results were similar to those of indirect elisa and indicated that the affinity levels paralleled the reactivity estimated by indirect elisa, and all sars-n mabs, except for 21-10-11 mab, could be suitable for the development of sensitive methods for sars-cov diagnosis. since these mabs were generated using recombinant sars-n protein expressed in insect cells that contained phosphorylated n protein (data not shown), indirect elisa was performed to further examine the reactivity of sars-n mabs with the ersars-n expressed in e. coli as non-phosphorylated protein. although sars-n mabs showed slightly higher reactivity with the brsars-n protein than with the ersars-n protein, all of these mabs showed significant reactivity with the ersars-n protein compared with the reaction of negative serum (fig. 3) . this result revealed that all sars-n mabs were effectively bound with both phosphorylated and non-phosphorylated n protein. immunofluorescence assay was performed on sars-cov infected vero cells to further assess whether the sars-n mabs recognize the native-form of endogenously synthesized n protein in sars-cov infected cells. both the negative serum and the five mabs did not show non-specific reactions with uninfected cells. all five sars-n mabs strongly reacted with sars-cov infected cells, whereas negative serum showed no reaction (fig. 4) . however, 21-10-11 mab showed a significantly weak reaction in affinity constants, but reacted strongly in the immunofluorescence assay; the reason for this result is unclear. the fluorescence signals of the mabs were predominantly shown in the cytoplasm of sars-cov infected cells. this indicated that all mabs were able to detect native-form n protein in sars-cov infected cells. in order to establish a sensitive and less time-consuming antigen-capture elisa for the sars-n protein, we tested each of the pairs of mabs from the five selected mabs; this allowed us to determine the highest detection sensitivity for recombinant n protein and sars-cov culture supernatant. we found that the immobilization of a mixture of 21-10-06 and 07-19-11 mab as capture antibody on the elisa plate, followed by the detection with biotin-conjugated 22-05-03 mab, gave the best result (data not shown). to determine the sensitivity of antigencapture elisa, a serial dilution of the recombinant n protein was used to determine a standard curve (fig. 5) . normal vero cell culture media were used to determine the baseline for antigencapture elisa at an optical density of 0.230 at 405 nm (od 405 ). therefore, the cut-off value for detection of viruses in cell culture was set to be 0.300, which is equal to the mean + 3s.d. of the od 405 for normal cell culture media. according to the cutfig. 4 . detection of sars-n protein in sars-cov-infected cells by immunofluorescence assay. the immunofluorescence assay was performed using the sars ifa kit according to the instructions of the manufacturer. anti-sars indicated positive serum obtained from mice immunized with heatinactivated sars-cov and negative indicated normal mouse serum. these mabs reacted with sars-n in virus-infected vero cells, whereas they did not react with uninfected cells. off threshold (0.300), a 10 −3 dilution of recombinant n protein and a 10 −2 dilution of the virus culture supernatant were considered positive (fig. 5) . thus, it was deduced that as little as 100 pg of recombinant n protein and 10 tcid 50 of virus culture supernatant could be detected. this result revealed that the sars-n mabs could be useful for detecting the n protein in virus culture supernatant and respiratory specimens from sars patients. to determine the specificity of the five sars-n mabs, immunofluorescence assays were performed in human coronavirus-infected mrc-5 cells. all five mabs showed no cross-reactivity with the antigens of the human coronavirus (fig. 6a) ; uninfected mrc-5 cells also showed no crossreactivity (data not shown). to further assess the specificity of the mabs, antigen-capture elisa was performed with human coronavirus-infected cell lysates and brsars-n protein as positive control (fig. 6b) . all of the mabs reacted only with sars-n protein, and did not react with human coronavirus, as shown by the results of immunofluorescence assays. this revealed that all of the sars-n mabs could specifically recognize the n protein of sars-cov. sars-cov is an etiological agent that causes severe acute respiratory syndrome, an infectious disease that has only recently emerged . therefore, there is an intense need for the development of sensitive and specific detection methods for sars-cov infection. many methods have been employed recently for the detection of sars-cov infection (keyaerts et al., 2005; yamashita et al., 2005; keightley et al., 2005; huang et al., 2005) . of these diagnostic methods, rt-pcr and real time-pcr have been the most widely used. however, these methods possess some general problems, as they are time-consuming and labor-intensive, require sophisticated instruments, and have high rates of false positivity. on fig. 6 . cross-reactivity of sars-n mabs with human coronavirus antigens. (a) the immunofluorescence assay was performed by using sars-n mabs on human coronavirus oc43-infected mrc-5 cells. as a positive control, anti-hcov was used as the mab against human coronavirus oc43 n protein, and normal mouse serum was used as the negative control. fm and lm indicate fluorescence and light imagery. none of these mabs showed cross-reactivity with human coronavirus. (b) cross-reactivity of sars-n mabs was examined by antigen-capture elisa using human coronavirus oc43 lysates (256 ha unit), brsars-n protein (500 ng/well) and pbst buffer with 1% bsa as control. the other hand, laboratory methods detecting viral antigen by mabs, including antigen-capture elisa, provide more rapid, less labor-intensive, and more convenient alternatives (lau et al., 2004; di et al., 2005) . in this study, we generated five positive clones secreting specific and highly reactive antibodies against sars-cov n protein in order to develop diagnostic methods. these mabs were available for use in detecting sars-cov n protein by various diagnostic methods, such as immunoblot assay, immunofluorescence assay and antigen-capture elisa (table 3) . we also revealed the availability of these mabs in the quantification of sars-n protein by antigen-capture elisa. the detection limit of this test is 100 pg of recombinant protein and 10 tcid 50 of sars-cov. this sensitivity is consistent with previous studies of sensitivity in other antigen-capture elisas (che et al., 2004; di et al., 2005) . therefore, these five mabs may be employed in the construction of various diagnostic methods for the detection of sars-cov and in quantitative analysis of viral antigen and virus titer. the major antigens of sars-cov structure proteins are the spike (s) protein and n protein (lau et al., 2004; di et al., 2005; lu et al., 2004 lu et al., , 2005 . however, recent reports have demonstrated that, because the s protein is expressed at very low levels in vivo and in cultured cells (zeng et al., 2004; , it is difficult to directly detect the soluble s protein from sars patients. thus, the s protein may not be suitable for use as a practical diagnostic antigen. in contrast, the n protein can be detected at significant levels in patient serum, as well as in respiratory tract samples at early stages of sars-cov infection (lau et al., 2004; di et al., 2005) . these previous reports support that the development of mabs against sars-n protein is an adequate approach for the diagnosis of sars. therefore, we generated mabs directed against sars-cov n protein and demonstrated that these sars-n mabs can successfully detect the n protein in sars-cov-infected cells; this is very useful in diagnosing sars patients. sars n proteins exist as phosphorylated forms in mature viral particles, whereas, in host cells, this protein exists in both the dephosphorylated form and the phosphorylated form (kalicharran and dales, 1995; surjit et al., 2005) . therefore, the mabs available for developing sensitive diagnostic methods have to recognize the non-phosphorylated protein as well as phosphorylated protein. a previous report suggested that, because of conformational differences between proteins, the mabs recognizing a protein expressed in insect cells cannot recognize the protein of same cdna constructs expressed in e. coli (vapalahti et al., 1996) . therefore, the sars-n mabs obtained in the present study were generated using recombinant sars-n protein expressed in insect cells; these mabs may not recognize the n protein expressed in e. coli. however, all of the sars-n mabs reacted significantly with the ersars-n expressed in e. coli, as well as the phosphorylated form of the brsars-n protein. these results demonstrate that these mabs can effectively detect the non-phosphorylated n protein that exists in host cells during viral replication, as well as the phosphorylated n protein in host cells and viral particles. all of these mabs could successfully detect native-form n protein in infected cells, as well as in viral particles. thus, these mabs may be useful in the development of sensitive methods used for the diagnosis of sars. epitope mapping studies of the sars-n mabs demonstrated that one of the three epitopes that were originally reported to be located in the highly immunodominant region (chen et al., 2004; he et al., 2004) is located at the middle region of sars-n protein (aa 215-239; igg 1 subclass mabs). the others were newly identified at the n-terminus, which is shared with an rna binding domain (aa 17-32 and 135-150; igg 2b subclass mabs) that is the minor b cell epitope (he et al., 2004) . furthermore, all of these sars-n mabs were reactive in immunoblotting, which suggests that they recognized linear epitopes in the n protein. a recent report demonstrates that the n protein is easily degraded into various isoforms in the lysates of sars-cov-infected cells (zeng et al., 2004) . we can also suggest, as previously described, that various isoforms existed in sars-n protein expressed in insect cells and could be detected by immunoblot assay using these mabs. therefore, the blend of two mabs against the different epitopes can be used to detect various fragments from sars-n protein and enhance the sensitivity of diagnostic tools. although the sars-n protein shares low homology (approximately 20-30%) with n proteins of other hcovs, a previous report has described that the sars-n protein has strong crossreactivity with sera against hcovs (sun and meng, 2004) . hence, anti-sera against sars-cov may be cross-reactive with other hcovs. however, previous reports support the idea that the sars-n mabs did not recognize the n proteins of other hcovs (che et al., 2004) . therefore, the issues of cross-reactivity during the detection of sars-n protein with polyclonal anti-sera can potentially be overcome by the use of a specific mab against sars-cov. in the present study, the sars-n mabs did not show cross-reactivity with n proteins of hcov, as determined by immunofluorescence assay and antigen-capture elisa. therefore, all of these sars-n mabs will be useful as mabs for the development of specific diagnostic methods to discriminate sars-cov infection from hcovs infection. sars-n protein is currently assumed to play an important role in the pathogenesis of sars, as well as in viral transcription and replication. for example, the n protein can modulate numerous intracellular mechanisms involved in apoptotic signal transduction pathways, cell cycle regulatory pathways and cellular immune response and inflammation (egloff et al., 2004; yan et al., 2004 yan et al., , 2006 liao et al., 2005; surjit et al., 2004 surjit et al., , 2006 surjit et al., , 2005 chang et al., 2006) . furthermore, sars-n protein is released as a soluble antigen in infected cells, and as high levels of n protein circulating in the blood vessels (che et al., 2004; di et al., 2005) . thus, sars-n protein in its soluble form may play an important role in extracellular pathogenesis of sars. however, it is totally unclear which of the intracellular and extracellular mechanisms are involved in viral replication and pathogenesis, and how the intracellular mechanisms are regulated by the n protein. therefore, these sars-n mabs may be extremely useful in providing further insight into the mechanisms of the n protein involved in the pathogenesis of sars. in conclusion, the sars-n mabs generated in the present study will be useful in providing reliable, sensitive, specific and convenient diagnostic kits for sars-cov detection; these diagnostic methods may include the immunoblot assay, immunofluorescence assay and antigen-capture elisa. furthermore, these mabs will be very useful in future studies concerning the contribution of sars-n protein in sars-cov pathology. modular organization of sars coronavirus nucleocapsid protein nucleocapsid protein as early diagnostic marker for sars antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein crossreactivity of antibody against sars-coronavirus nucleocapsid protein with il-11 characterization of a monoclonal antibody specific to the gag protein of porcine endogenous retrovirus and its application in detecting the virus infection monoclonal antibody-based antigen capture enzyme-linked 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b and ccaat/enhancer binding protein proteomic analysis of sars associated coronavirus using two-dimensional liquid chromatography mass spectrometry and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by mass spectrometric analysis we gratefully acknowledge w. bellini from the centers for disease control and prevention for kindly providing of the sars coronavirus. we also thank neurogenex co. for the technical assistance in the production of recombinant baculovirus and cosmo inc. for the technical assistance in the production of the sars mabs.this work was supported by the intramural research fund from the korea centers for disease control and prevention. key: cord-317061-0bx704ao authors: wu, andong; wang, yi; zeng, cong; huang, xingyu; xu, shan; su, ceyang; wang, min; chen, yu; guo, deyin title: prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus date: 2015-10-02 journal: virus res doi: 10.1016/j.virusres.2015.05.018 sha: doc_id: 317061 cord_uid: 0bx704ao coronavirus 3c-like protease (3clpro) is responsible for the cleavage of coronaviral polyprotein 1a/1ab (pp1a/1ab) to produce the mature non-structural proteins (nsps) of nsp4–16. the nsp5 of the newly emerging middle east respiratory syndrome coronavirus (mers-cov) was identified as 3clpro and its canonical cleavage sites (between nsps) were predicted based on sequence alignment, but the cleavability of these cleavage sites remains to be experimentally confirmed and putative non-canonical cleavage sites (inside one nsp) within the pp1a/1ab awaits further analysis. here, we proposed a method for predicting coronaviral 3clpro cleavage sites which balances the prediction accuracy and false positive outcomes. by applying this method to mers-cov, the 11 canonical cleavage sites were readily identified and verified by the biochemical assays. the michaelis constant of the canonical cleavage sites of mers-cov showed that the substrate specificity of mers-cov 3clpro is relatively conserved. interestingly, nine putative non-canonical cleavage sites were predicted and three of them could be cleaved by mers-cov nsp5. these results pave the way for identification and functional characterization of new nsp products of coronaviruses. middle east respiratory syndrome coronavirus (mers-cov) is an enveloped virus carrying a genome of positive-sense rna (+ssrna). it was identified as the pathogen of a new viral respiratory disease outbreak in saudi arabia in june 2012, named as middle east respiratory syndrome (mers). mers-cov emerged ten years after severe acute respiratory syndrome coronavirus (sars-cov) and quickly spread to several countries in middle east and europe (assiri et al., 2013; tashani et al., 2014) . soon after the first report, the mers-cov genome was sequenced and its genomic organization has been elucidated . this new coronavirus is classified in the lineage c of beta coronavirus, and is close to bat coronavirus hku4 and hku5 (de groot et al., 2013; lau et al., 2013) . like other coronaviruses (hussain et al., 2005; zuniga et al., 2004) , mers-cov contains a 3 coterminal, nested set of seven subgenomic rnas (sgrnas), enabling translation of at least nine open reading frames (orfs). the 5 -terminal two thirds of mers-cov genome contains a large open reading frame orf1ab, which encodes polyprotein 1a (pp1a, 4391 amino acids) and polyprotein 1ab (pp1ab, 7078 amino acids), the latter being translated via a −1 ribosomal frameshifting at the end of orf1a. these two polyproteins were predicted to be subsequently processed into 16 non-structural proteins (nsps) by nsp3, a papain-like protease (plpro), and nsp5, a 3c-like protease (3clpro) (kilianski et al., 2013; van boheemen et al., 2012) . protease plays a key role during virus life cycle. it is essential for viral replication by mediating the maturation of viral replicases and thus becomes the target of potential antiviral drugs (thiel et al., 2003; ziebuhr et al., 2000) . investigating the cleavage sites of coronavirus proteases and the processing of polyproteins pp1a/1ab will benefit to identify the viral proteins and their potential function for viral replication. some cleavage sites have been identified and confirmed by previous studies, including three cleavage sites of plpros of human coronavirus 229e (hcov 229e), mouse hepatitis virus (mhv), sars-cov, mers-cov and infectious bronchitis virus (ibv), whose cleavages release the first 3 non-structural proteins (bonilla et al., 1995; kilianski et al., 2013; lim and liu, 1998; ziebuhr et al., 2007) . the canonical cleavage sites of 3clpros, the sites between the recognized nsps, have also been characterized, including all sites of mhv, ibv, sars-cov and a fraction of sites of hcov 229e which release the non-structural proteins from nsp4 to nsp16 (deming et al., 2007; grotzinger et al., 1996; liu et al., 1994 liu et al., , 1997 lu et al., 1995) . for 3clpro of mers-cov, two cleavage sites releasing nsp4 to nsp6 have been identified (kilianski et al., 2013) . however, other cleavage sites remain to be characterized. furthermore, efforts have been taken to predict these cleavages sites by sequence comparison. gorbalenya et al. (1989) made the first systematical prediction on ibv pp1a/1ab according to the substrate specificity of 3c protease of picornaviruses. however, two of their predicted cleavage sites within nsp6 of ibv were proved uncleavable (liu et al., 1997; ng and liu, 2000) . gao et al. (2003) developed a software (zcurve cov) to predict the nsps as well as gene-encoded orfs of coronaviruses more accurately based on previous studies of 3clpros cleavage sites of ibv, mhv and hcov 229e. later on, non-orthogonal decision trees were used to mine the coronavirus protease cleavage data and to improve the sensitivity and accuracy of prediction (yang, 2005) . however, while these methods focus on the prediction of the canonical cleavage sites and target more and more on prediction accuracy to avoid false positives, potential non-canonical cleavage sites might be neglected. for example, a cleavage site between nsp7 and 8 of mhv strain a59 is not predicted by above methods, but proved to be physiologically important since it produces a shorter nsp7 that can support the growth of mhv carrying a mutation on nsp7-8 cleavage site (deming et al., 2007) . therefore, the substrate specificities of coronaviruses 3clpros are complicated. a 3clpro substrate library of four coronaviruses (hcov-nl63, hcov-oc43, sars-cov and ibv) containing 19 amino acids × 8 positions variants was constructed by making single amino acid (aa) substitution at each position from p5 to p3 , and their cleavage efficiencies were measured and analyzed to find out the most preferred residues at each position (chuck et al., 2011) . however, the non-canonical cleavage site with less preferred residues of 3clpro is adopted by coronaviruses (deming et al., 2007) . thus we speculate that other potential 3clpro cleavage sites may still exist in coronaviruses. in order to set up a more moderate and balanced criteria for protease cleavage site identification, we compared six scanning conditions with different stringency to systematically predict the 3clpro cleavage sites on pp1a/1ab of five coronaviruses including mers-cov. as a representative, the cleavability of the predicted cleavage sites of mers-cov 3clpro was analyzed by the recombinant luciferase cleavage assay and the fluorescence resonance energy transfer (fret) assay. the results showed that all 11 canonical cleavage sites of mers-cov pp1a/1ab were cleavable in our experiments and three of nine predicted non-canonical cleavage sites appeared to be cleavable. our study points out a new direction regarding the prediction and identification of cleavage sites of proteases and contributes to understanding the mechanism of coronaviral polyprotein processing. the genome sequences of 28 coronaviruses were downloaded from genebank database and the sequences of the 3clpro cleavage sites were collected from p4 to p2 (tables s1-s4 ). the substrate profiles of each coronavirus group and the whole coronavirinae were summarized (table s5) . the coding sequence of mers-cov nsp5 (nc 019843) was synthesized chemically by genscript and cloned into vectors pet28a and pgex-6p-1, respectively. the catalytic residue mutation c148a was generated by over lapping pcr with mutagenic primers (table s6 ). all the clones and mutations were confirmed by dna sequencing. the expression vectors were transformed into escherichia coli strain bl21 (de3). the cells were grown at 37 • c in lysogeny broth (lb) medium with antibiotics and induced with 0.2 mm isopropylbd-thiogalactopyranoside (iptg) at 16 • c for 12 h. the cells were harvested and resuspended in lysis buffer (50 mm tris-hcl, ph 7.5, 150 mm nacl, 1 mm edta, 0.05% np40, 0.1 mg/ml lysozyme and 1 mm pmsf) at 4 • c. after incubation for 30 min on ice, 10 mm mgcl 2 and 10 g/ml dnase i (sigma) were added to digest the genomic dna. the supernatant of cell lysate was applied to affinity chromatography column after centrifugation. the recombinant protein with his-tag was bound with nickel-nitrilotriacetic acid (ni-nta) resin (genscript) and washed with buffer a (50 mm tris-hcl, ph 7.5, 150 mm nacl), buffer b (50 mm tris-hcl, ph 7.5, 150 mm nacl, 20 mm imidazole) and buffer c (50 mm tris, ph 7.5, 150 mm nacl, 50 mm imidazole). proteins were eluted with buffer d (50 mm tris, ph 7.5, 150 mm nacl, 250 mm imidazole). gst-tagged protein was bound with gst resin (genscript), washed with buffer a and eluted with buffer a supplemented with 10 mm reduced glutathione (gsh). the purified proteins were desalted and concentrated by ultrafiltration using 30 kda amicon ultra 0.5-ml centrifugal filter (millipore). all the cleavage sites (eight residues, ranging from p5 to p3 ) were inserted into glo-sensor 10f linear vector. comparing to the wild type firefly luciferase (550 aa), glo-sensor luciferase has short truncations at both termini with c-and n-part reversed, resulting in the new 234-aa n-and 233-aa c-terminal region respectively. the inserted sequence and the reversed arrangement of the nand c-terminal regions reduce the luciferase activity dramatically. after the recognition sequence was cut off by nsp5, the luciferase recover its activity and luminescence in the presence of luciferase substrate. a back to front recombinant firefly luciferase inserted with different cleavage sites was expressed when the recombinant plasmids were co-incubated with a cell-free protein expression system extracted from wheat germ (promega). after incubation for 2 h at 25 • c, nsp5 was added into the system and the whole system was incubated at 30 • c for 1 h. then, the reaction system was diluted 20 times and mixed thoroughly with equal volume of luciferase substrate. luciferase luminescence was measured by a luminometer (promega) after incubation for 5 min at room temperature. all the 11 conserved putative recognition sites were designed from p12 to p8 , synthesized and modified with a typical shorter wavelength fret pair, n-terminal dabcyl and c-terminal glu-edans by gl biochem (shanghai). the peptides were completely dissolved in dmso and the final concentration of dmso in the reaction system was 1%. 180 m substrate peptide and 16.3 m tagged nsp5 were mixed in the solution of 50 mm tris, ph 7.5, 1 mm edta, 50 m dtt and incubated at 37 • c for 2 h. to calculate kcat/km, different amounts (7.2-180 m) of substrate peptides were co-incubated with 16.3 m nsp5. the reaction system was placed in giernor black plate and the fluorescence was detected by a microplate reader (molecular devices) with ex/em (nm/nm) = 340/490. relative fluorescence unit (rfu) was collected every 30 s for 2 h. the initial slope (slope a = rfu/min) was generated from the linear interval of the rising stage. then, a linear equation was generated using the rfu at plateau (rfu max ) vs. the concentration of substrate. the slope (slope b = rfu/[s]) indicates the rfu change at per unit change of [s] . the initial reaction velocity (v 0 = [s]/min) was calculated through dividing slope a by slope b. the michaelis-menten kinetic constants were generated by lineweaver-burk plot. the coronavirus 3clpros and their cleavage sites are evolutionarily conserved among different genera. to study the genetic diversity and evolution of 3clpro cleavage sites of coronaviruses pp1a/1ab, 308 primary sequences of 3clpro cleavage sites (ranging from p4 to p2 ) of 28 species of coronaviruses were collected and listed in tables s1-s4, including the predicted and verified cleavage sites. 11 canonical cleavage sites of each coronavirus were joined end to end to produce a spliced sequence which was then used to produce a phylogenetic tree (fig. 1a ). in addition, the sequences of all coronavirus 3clpro were used to generate another phylogenetic tree (fig. 1b) . the analyses showed that the phylogenetic distances and taxonomic positions of each virus, in both phylogenetic trees, were mostly consistent with that classified by the international committee on taxonomy of viruses (ictv) (http://www.ictvonline. org/virustaxonomy.asp). these results implied that the cleavage sites of coronaviral 3clpros might co-evolve with 3clpros, and the genetic diversity of both 3clpro and its cleavage sites are relatively conserved between different genera of coronaviruses. however, on the phylogenetic tree generated with 3clpro cleavage sites (fig. 1a) , the members of the genus gammacoronavirus, although clustered closely, is split into alphacoronaviruses and deltacoronaviruses, suggesting that the cleavage sites of gammacoronaviruses may have undergone recombination events during evolution. in order to develop an optimized method for cleavage site prediction that can cover all possible cleavage sites with fewer false positives, we have set three levels of criteria (stringent, moderate and mild) for cleavage site prediction. in the stringent rules, 3clpro cleavage sites only comprise the most preferred residues at each position based on previous description (chuck et al., 2011) . in moderate rules, 3clpro cleavage sites comprise residues which ever appeared in the cleavage sequences of congeneric coronaviruses at each particular position. as for mild rules, the cleavage sites could comprise any residues ever found in the cleavage sequences of all coronaviruses at each particular position. because the substrate preference at p4 and p2 is not strong, we decided to adopt two different lengths of cleavage sequences for prediction, one containing six residues from position p4 to p2 , and the other containing four residues from position p3 to p1 . these two lengths of cleavage sequences, combining with the three different criteria, made up a total of six search conditions for cleavage site predication with decreasing degree of stringency. the canonical cleavage sites of 3clpro for these seven groups of coronaviruses were summarized in tables s1-s4 and used to set conditions iii to vi. possible residues at each particular position of 3clpro cleavage sites were predicted based on all six conditions to make the cleavage site profile of coronaviruses 3clpro (table s5 ). in principle, when condition i was employed, the least number of possible cleavage sites were identified in a scanned sequence, while condition vi predicted the largest number of possible cleavage sites in a scanned sequence. to the applicability, we applied all the six conditions on five representative coronaviruses, including hcov 229e from alphacoronavirus, mhv from betacoronavirus lineage a, sars-cov from beta coronavirus lineage b, mers-cov from betacoronavirus lineage c and ibv from gammacoronavirus. all possible cleavage sites predicted based on each condition were scanned on pp1a/1ab of five representative coronaviruses and the results were summarized in table 1 . as shown in table 1 , increasing numbers of cleavages sites were found for each coronavirus when conditions from i to vi were applied. the results showed that condition i and ii were too strict to cover all 11 canonical cleavages sites; condition v and vi were too loose so as to produce two to three times more than 11 cleavages sites; condition iii could only cover the canonical cleavage sites for sars cov; only condition iv generates an appropriate number of cleavage sites for all five coronavirus. therefore, search condition iv was chosen for further analysis of the cleavage sites of mers-cov. by applying the search condition iv, 9 putative cleavage sites (pss) as well as 11 canonical cleavage sites (css) were predicted (table 2) . although the canonical cleavage sites of mers-cov 3clpro have been predicted by sequence alignment with other coronavirus , our results suggested that the additional cleavage might occur in the process of mers-cov pp1a/1ab processing. to verify the activity of mers-cov 3clpro and cleavability of the predicted cleavage sites, the biochemical assay systems of mers-cov 3clpro were established. as shown in fig. 2a and b, we first expressed and purified mers-cov 3clpro (nsp5) with different tags and mutation: n-terminally gst-tagged nsp5 (gnsp5, 60.4 kda), n-terminally his-tagged (34 extra amino acids with 6× his tag and linker provided by vector pet-28a) nsp5 (hnsp5, 36.9 kda), hnsp5 with catalytic residue mutation c148a (hnsp5m, 36.9 kda) (kilianski et al., 2013) and gst tag-gvlq-nsp5 with c148a mutation and 6× his tag (gnsp5mh, 61.6 kda), in which the sequence motif gvlq represents the last four residues of mers-cov nsp4, mimicking the cleavage site of mers-cov nsp4/nsp5. in the biochemical assays, the gnsp5mh with catalytic residue mutation c148a could not undergo self-cleavage at the cleavage site to release gst in incubation for 16 h (fig. 2c) , indicating that the 3clpro activity of mers-cov nsp5 in gnsp5mh was inactivated by the mutation c148a. thus, gnsp5mh was used as protease substrate in the following biochemical assays. to verify the 3clpro activity of recombinant nsp5s, gnsp5 and hnsp5 were incubated with substrate gnsp5mh for 5 min to 16 h and analyzed by sds-page (fig. 2d) and western blotting, respectively (fig. 2e) . both gnsp5 and hnsp5 showed the proteolysis activity to cleave the substrate gnsp5mh into two parts: gst (26.0 kda) and nsp5mh (34.1 kda), which were confirmed by the correlation of their molecular weight (fig. 2d and e) . however, the 3clpro activity of gnsp5 was obviously weaker than that of hnsp5, which could entirely cleave the substrate gnsp5mh 2 h post treatment ( fig. 2d and e) . these results could be explained by that the larger fusion tag at the n terminus of mers-cov 3clpro significantly reduced the proteolysis activity of 3clpro, which was consistent with the previous observation (xue et al., 2007) . in the biochemical assays, the the tree was generated by the sequence of nsp5 and the method is the same as described above. the number of cleavage sites in pp1ab of 5 representative coronaviruses predicted by using 6 search conditions. condition iii 11 4 11 5 11 0 11 2 11 3 condition iv 11 10 11 14 11 4 11 9 11 5 condition v 11 9 11 17 11 11 11 12 11 11 condition vi 11 15 11 23 11 19 11 19 11 13 a canonical cleavage sites, which are located between recognized nsps. b putative cleavage sites, which are located inside various nsps. c six search conditions are designed: conditions i, iii and v cover six residues from p4 to p2 ; conditions ii, iv and vi cover four residues from p3 to p1 . conditions i and ii are set to comprise the most preferred residues at each position; conditions iii and iv comprise residues appeared in the cleavage sites of congeneric coronaviruses; conditions v and vi comprise residues appeared in the cleavage sequences of any coronaviruses. relatively lower proteolysis activity of 3clpro will benefit to observe the influence of different substrates. therefore, both recombinant gnsp5 and hnsp5 were used as mers-cov 3clpro in the following studies. to rapidly evaluate the proteolysis activity of mers-cov 3clpro toward the predicted cleavage sites of different substrates, a sensitive luciferase-based biosensor assay was adopted. as shown in fig. 3a , the canonical cleavage sites (cs) of mers-cov nsp4/nsp5 (cs4/5) and nsp5/nsp6 (cs5/6), which were experimentally confirmed in a previous study (kilianski et al., 2013) , were inserted into the inverted and circularly permuted luciferase construct pglo-10f, in which the n-terminal and c-terminal halves of luciferase gene are separated. the resulting luciferase in translation system in vitro was inactive and could convert into an active luciferase when cleaved by recombinant viral protease at the engineered cleavage sites (such as cs4/5 and cs5/6). in this system, the luciferase signals were detected when incubated with both gnsp5 and hnsp5, respectively (fig. 3b) . in contrast, the mutated nsp5 (hnsp5m) could not convert the inactive luciferase into active form (fig. 3b ). this result indicated that the luciferase-based biosensor assay could be used to evaluate the proteolysis activity of mers-cov 3clpro. then, the other nine canonical cleavage sites and nine putative cleavage sites composed with 8 aa from mers-cov pp1a/1ab were inserted into the luciferase construct pglo-10f, and the luciferase-based biosensor assays were performed using hnsp5 and hnsp5m, respectively. as shown in fig. 3c , all the 11 canonical cleavage sites of mers-cov 3clpro generated luciferase signal by hnsp5 at least 6.6 times higher than by the inactive hnsp5m, indicating that all these canonical sites could be cleaved by mers-cov 3clpro. these results experimentally verified the existence of the 11 predicted canonical cleavage sites. interestingly, among the nine putative cleavage sites, the luciferase signals of ps1-1, ps3-1 and ps3-3 remarkably increased more than 70 folds when incubated with hnsp5, indicating that the putative cleavage sites, located inside nsp1 and nsp3 of mers-cov respectively, might be cleavable (fig. 3d) . the other 6 predicted putative sites (ps3-2, ps5-1, ps6-1, ps12-1, ps13-1, and ps16-1) showed less than 2.5 folds increase of luciferase signal when they were treated by hnsp5 comparing with those treated by hnsp5m (fig. 3c and d) . due to high sensitivity of the luciferase-based biosensor assay and the fact that the confirmed verification of the recombinant luciferase assays. inactive luciferase was synthesized in the cell-free translation system and the reaction mixture incubated at 25 • c for 2 h. after that, the protein mixture was divided into four parts and incubated with 1.63 m gnsp5, hnsp5, hnsp5m or h2o, respectively. after incubation for 1 h at 30 • c, the reaction product was diluted 20 times and mixed with equal amount of luciferase substrate. after incubation at room temperature for 5 min, the luciferase luminescence was measured. luciferase activation fold was calculated through dividing the signal value of the reaction system treated with active hnsp5 by the one treated with the inactive nsp5 mutant hnsp5m. (c) the luciferase cleavage assay of predicted 11 canonical cleavage sites and (d) 9 putative cleavage sites. the luciferase expression vector inserted with cleavage sites were added to the wheat germ protein translation mix and incubated at 25 • c for 2 h, and the reaction mixture was divided and treated with hnsp5 and hnsp5m, respectively. the dashed line indicates the lowest fold increase of luciferase signal by cleavage of previously confirmed 3clpro cleavage sites. the data presented here are the mean values ± sd derived from three independent experiments. canonical cleavage sites generated at least 6.6 times increase of luciferase signal, the cleavage signal of these six sites may represent the background level, indicating that they are likely uncleavable per se. these results suggest that previously unrecognized 3clpro cleavage sites may exist inside the nsps, which were regarded as non-canonical cleavage sites. the substrate specificity of coronaviruses 3clpro is determined by the residues from p4 to p2 positions of cleavage sites, especially depending on the p1, p2 and p1 positions, which would benefit the prediction of cleavage site and design the broadspectrum inhibitors of coronaviruses 3clpro (chuck et al., 2011; hegyi and ziebuhr, 2002) . previous studies demonstrated that different canonical cleavage sites of some representative coronaviruses are not equally susceptible to proteolysis by recombinant 3clpro (fan et al., 2004; hegyi and ziebuhr, 2002) . to define the susceptibility of the canonical cleavage sites and substrate specificity of mers-cov 3clpro, 20-mer synthetic peptides representing corresponding canonical cleavage sites of mers-cov 3clpro were synthesized and modified with n-terminal dabcyl and c-terminal glu-edans (fig. 4a) . the fluorophore edans and quencher dabcyl are widely used in the biochemical assays based on the fluorescence resonance energy transfer (fret). as shown in fig. 4b , the peptides represented cleavage sites cs4/5 and cs5/6 were tested to optimize the fret assay, and the relative fluorescence unit (rfu) folds of both sites significantly increased when incubated with gnsp5 and hnsp5. although the fret assay system is more costly and less sensitive than the luciferase-based biosensor assay (figs. 3b and 4b), it provides continuous read signals during the process of reaction, which could measure the kinetic characteristic of protease toward different substrates. the initial reaction rate (rfu/min) of all 11 canonical cleavage sites of mers-cov were measured and shown in fig. 4c . the michaelis constants including kcat, km, kcat/km and relative kcat/km were then calculated (table 3) . as shown in table 3 , the substrate specificity of mers-cov 3clpro is relatively conserved with other coronaviruses as previously reported (fan et al., 2004; hegyi and ziebuhr, 2002; ziebuhr and siddell, 1999) . the relative kcat/km values of cs4/5 and cs5/6 indicated that the cleavage sites flanking mers-cov 3clpro are converted significantly faster than other sites. the efficient proteolysis at the sites flanking nsp5 implies that the nsp5 (3clpro) might be released from the polyprotein 1a/1ab at the very early stage of the maturation of viral nsps, which is similar with the hcov, tgev, sars-cov and mhv (fan et al., 2004; hegyi and ziebuhr, 2002) . however, the relative kcat/km value of cs4/5 is lower than that of cs5/6 (table 3) , which is different from that of the coronaviruses (fan et al., 2004; hegyi and ziebuhr, 2002) . this could be explained by that the residue gly (g) at the p4 of cleavage site between nsp4 and nsp5 of mres-cov reduces the protease activity of 3clpro comparing with the residues ser (s), ala (a) and thr (t) of other coronaviruses (tables s1-s4) as previous described (chuck et al., 2011) . whether such disparity plays any role in the replication and pathogenesis of mers-cov is unknown. the processing of viral polyprotein by 3clpro is essential for the replication of coronaviruses. besides the 11 canonical cleavage sites of coronaviruses, some additional cleavage sites inside nsps, so-called non-canonical cleavage sites, have also been identified (deming et al., 2007) . therefore, more non-canonical 3clpro cleavage sites are to be identified in different coronaviruses. in this study, we designed six search conditions for predicting 3clpro cleavage sites, among which, the search condition iv provides a feasible way to reveal the potential cleavage sites of 3clpro within coronaviruses. based on the genetic diversity of different coronavirus genera (fig. 1) , the scanning condition iv adopted the residues of 3clpro cleavage sites, which ever appeared in the cleavage sequences of congeneric coronaviruses at position p3 to p1 . in contrast, conditions i, ii, iii, v and vi were either too restrictive or generated too many false positive outcomes (table 1 ). in the suggested condition iv, 4 residues from position p3 to p1 were applied to the prediction of 3clpro cleavage site. by measuring the relative protease activities of 3clpro from different coronavirus genera against 19 amino acids × 8 positions of substrate variants, it is shown that the substrate specificity of position p5, p2 and p3 are significantly lower than other positions (chuck et al., 2011) . therefore, the consideration of six or more residues is unnecessary, which could lead to leave-out of potential cleavage sites (table 1) . comparing with the previous researches on the prediction and identification of 3clpro cleavage sites, the scanning condition iv showed its advantages. for example, the two nonexistent putative cleavage sites predicted within nsp6 of ibv (gorbalenya et al., 1989; liu et al., 1997; ng and liu, 1998) were avoided in our prediction method (data not shown). notably, the noncanonical cleavage site at the end of mhv nsp7 identified by deming et al. could be predicted using scanning condition iv. by using the search condition iv, 9 putative cleavage sites were predicted in mers-cov pp1ab in addition to the 11 canonical cleavage sites. the luciferase signal of cs10/12 increased 6.6 fold when treated with nsp5 in the recombinant luciferase cleavage assays, which is the lowest among the 11 canonical cleavage sites (fig. 3c) . therefore, the 6.6 fold increase of luciferase signal was used arbitrarily as a threshold for judging positive and negative. among the nine predicted putative cleavage sites, three sites (ps1-1, ps3-1 and ps3-3) showed obviously increasing signals at least 70 times above the background (fig. 3d ) and therefore were regarded as cleavable sites. the increase of signals of other six predicted putative cleavage sites was less than 2.5 times (fig. 3d) . therefore, they were regarded as non-cleavable sites and thus as false positives from the prediction. interestingly, the homologous sequence of ps1-1 and ps3-1 are conserved in lineage c of betacoronavirus including mers-cov, batcov hku4 and batcov hku5 (fig. 5a and b) . however, ps3-3 is mers-cov unique sequence (fig. 5c) . moreover, the cleavability of a cleavage site in biochemical assays is a necessary but not sufficient condition for its physiological existence in the viral infection. a predicted cleavage site may or may not be accessible by a protease. the 3d structure model of mers-cov adpribose-1-monophosphatase (adrp) domain built by comparative protein modeling and papain like protease (plpro) domain (bailey-elkin et al., 2014) showed that both ps3-1 and ps3-3 are located at the surface of adrp and plpro domain, opposite to the enzymatic active centers ( fig. 5d and e) , suggesting that these two sites are like approachable by the proteinase. most recently, the crystal structure of mers-cov 3clpro was determined (needle et al., 2015) . although ps5-1 is also located at the surface of mers-cov 3clpro, the self-cleavage of mers-cov nsp5 was not observed in this study (fig. 2) . therefore, the threshold we proposed in the luciferase-based biosensor system to exclude the false positive prediction results is reasonable (fig. 3d) . however, further studies are needed to identify the predicted cleavage products from the cells infected by mers-cov. currently, such work with live mers-cov is limited in our research facilities due to the biosafety rules, but it can be addressed in collaboration in the future. notably, the outcomes of the two cleavage assay systems were different. the signal fold change of highly sensitive luciferasebased biosensor assay is dependent on the accumulation of active luciferase cleaved by nsp5 during 1 h (section 2), while the outcome of the fret assay is instant relative fluorescence unit (rfu) signal. the rfu/min is the initial speed of the reaction, which reflects but not equals to the efficiency of the cleavage. these differences may be caused by the steric hindrance of the luciferase subunits, the distance between fluorophore and quencher of substrates for fret assay and substrate solubility. therefore, the activity observed in the two different systems cannot be compared directly. based on the characteristic of the two cleavage assay systems, the highly sensitive luciferase-based biosensor assay might be more suitable to high throughput screen the predicted putative cleavage site of protease while the fret assay better for cleavage kinetic analysis. according to the michaelis constants of mers-cov, the substrate specificity of mers-cov 3clpro is relatively conserved with other coronaviruses (fan et al., 2004; hegyi and ziebuhr, 2002) . notably, the pro (p) has been selected as result of evolution at position p2 of cleavage site between nsp10 and nsp12 (cs10/12) of lineage c betacoronavirus, which is not preferred by the 3clpro based on the previous study (chuck et al., 2011) . however, the relative kcat/km value of mers-cov cs10/12 is 0.053, which is 26.5 fold higher than that of sars-cov (fan et al., 2004) . this indicated that the substrate preferences of some cleavage sites could still be varied among different genera of coronaviruses and the proposed scanning condition iv regarding the residues ever appearing in the cleavage sequences of congeneric coronaviruses is reasonable. in summary, we proposed an optimized search condition for predicting cleavage sites of coronavirus 3clpro. we verified the 11 canonical cleavage sites of pp1ab in biochemical assays. we further identified three non-canonical cleavage sites in the nsps of mers-cov. the results provide clues for possible identification of novel cleavage products of coronavirus nsps and will benefit the studies of the mechanisms of coronavirus replication. processing of polyprotein 1a/1ab by 3clpro is essential in coronavirus life cycle. the 3clpro cleavage site prediction methods established by previous studies are focus on the accuracy, while some noncanonical cleavage sites were missed. in this study, we built a moderate prediction method to balance the accuracy and false positive outcomes. using this method, 9 putative cleavage sites, in addition to the 11 canonical sites, were predicted in mers-cov pp1ab and the cleavability of 3 of them was experimentally confirmed. interestingly, all these 3 non-canonical cleavage sites are located upstream to nsp4, which is in contrast with previous understanding that the coronavirus 3cl protease only cleaves from nsp4 to nsp16. this suggests a novel role of 3clpro in coronavirus pp1a/1ab processing. however, the cleavability of these putative cleavage sites needs to be further verified in the viral proteins of mers-cov-infected cells. finally, the catalytic constants of the 11 canonical cleavage sites of mers-cov 3clpro showed its conservation with the cousins in coronaviridae. hospital outbreak of middle east respiratory syndrome coronavirus crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to 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specificities among coronavirus main proteases identification of novel subgenomic rnas and noncanonical transcription initiation signals of severe acute respiratory syndrome coronavirus assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus characterisation of a papain-like proteinase domain encoded by orf1a of the coronavirus ibv and determination of the c-terminal cleavage site of an 87 kda protein a 100-kilodalton polypeptide encoded by open reading frame (orf) 1b of the coronavirus infectious bronchitis virus is processed by orf 1a products proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a59 structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity 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 site australian hajj pilgrims' knowledge about mers-cov and other respiratory infections mechanisms and enzymes involved in sars coronavirus genome expression genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans production of authentic sars-cov m(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction mining sars-cov protease cleavage data using non-orthogonal decision trees: a novel method for decisive template selection isolation of a novel coronavirus from a man with pneumonia in saudi arabia human coronavirus 229e papain-like proteases have overlapping specificities but distinct functions in viral replication processing of the human coronavirus 229e replicase polyproteins by the virus-encoded 3c-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab virus-encoded proteinases and proteolytic processing in the nidovirales sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2015.05. 018 key: cord-346104-18x8u2oe authors: black, wendy; troyer, ryan m.; coutu, jesse; wong, karsten; wolff, peregrine; gilbert, martin; yuan, junfa; wise, annabel g.; wang, sunny; xu, dan; kiupel, matti; maes, roger k.; bildfell, rob; jin, ling title: identification of gammaherpesvirus infection in free-ranging black bears (ursus americanus) date: 2019-01-02 journal: virus res doi: 10.1016/j.virusres.2018.10.016 sha: doc_id: 346104 cord_uid: 18x8u2oe herpesvirus infection was investigated in black bears (ursus americanus) with neurological signs and brain lesions of nonsuppurative encephalitis of unknown cause. visible cytopathic effects (cpe) could only be observed on days 3–5 post-infection in hrt-18g cell line inoculated with bear tissue extracts. the observed cpe in hrt-18g cells included syncytia, intranuclear inclusions, and cell detachments seen in herpesvirus infection in vitro. herpesvirus-like particles were observed in viral culture supernatant under the electron microscope, however, capsids ranging from 60 nm to 100 nm in size were often observed in viral cultures within the first two passages of propagation. herpesvirus infection in the bear tissues and tissue cultures were detected by pcr using degenerate primers specific to the dna polymerase gene (dpol) and glycoprotein b gene (gb). dna sequencing of the amplicon revealed that the detected herpesvirus has 94–95% identity to ursid gammaherpesvirus 1 (urhv-1) dna sequences of dpol. phylogenetic analysis of dpol sequences indicates that black bear herpesviruses and urhv-1 are closely related and have small distances to members of rhadinovirus. interestingly, black bear herpesvirus infections were also found in bears without neurological signs. the dpol dna sequence of black bear herpesviruses detected in neurological bears were similar to the those detected in the non-neurological bears. however, the gb dna sequence detected from the neurological bear is different from non-neurological bear and has only 64.5%–70% identity to each other. it is possible that at least two different types of gammaherpesviruses are present in the u. americanus population or several gammaherpesviruses exist in ursine species. gammaherpesviruses are common infections in a wide variety of mammalian species. the subfamily of gammaherpesvirinae includes four characterized genera: lymphocryptovirus, macavirus, pervavirus, rhadinovirus and unassigned genus. lymphocryptoviruses are often associated with lymphoproliferative diseases, and have been identified in at least 9 different species, such as human, macacine, cercopithecine, leporid and ovine. human herpesvirus 4 (hhv4) is one of the prominent lymphocryptoviruses associated with a number of human cancers (grywalska et al., 2013; petersson, 2015) . hhv4, also known as epstein-barr virus (ebv), prevalent in 90 to 95% of the human population, is also clinically associated with various neurological diseases such as primary central nervous system lymphoma, multiple sclerosis, alzheimer's disease, cerebellar ataxia, and encephalitis (ali and lawthom, 2013; corssmit et al., 1997; davies et al., 2016; fallo et al., 2005) . human herpesvirus 8 (hhv8), also known as kaposi's sarcomaassociated herpesvirus (kshv), and murine gammaherpesvirus-68 (mhv-68) are most studied rhadinoviruses. hhv8 or khsv is mainly linked to the development of kaposi's sarcoma and a rare b cell lym-neurotropic and can infect neuronal cell lines, sh-sy5y and ntera2 cells, as well as human primary neurons (cho and song, 2014) . mhv-68, also called murid gammaherpesvirus 4, was originally isolated from a bank vole by passage through mouse brain (terry et al., 2000) . mhv-68 is known to persist in the brain after cerebral infection and can causes cerebral infection with inflammation in 50% of the infected animals (hausler et al., 2005) . rhesus macaque rhadinovirus, another member of the rhadinovirus genus, has been shown to cause encephalomyelitis and diseases similar to multiple sclerosis in human (axthelm et al., 2011) . members of percavirus, such as equine gammaherpesvirus 2 and mustelid gammaherpesvirus 1, are not linked to a specific disease, except equine gammaherpesvirus 5 (ehv-5), which is linked to equine multinodular pulmonary fibrosis (williams et al., 2007) . members of the macavirus can be either non-pathogenic, such as bhv-6, or pathogenic, such as malignant catarrhal fever virus (mcfv) and caprine herpesvirus 2 (chv-2). bhv-6 is ubiquitous in healthy cattle populations (koptopoulos et al., 1988) , but mcfv and chv-2 can cause fatal multisystemic disease in sheep and buffalo (brenner et al., 2002; li et al., 2005 li et al., , 1999 loken et al., 2009) . herpesvirus infection in bears has rarely been documented. recovery of viral sequences corresponding to the alphaherpesviruses, such as equine herpesvirus 1 (ehv1) and equine herpesvirus 9 (ehv9), have been published in case reports involving captive ursid mortalities (dombrowski et al., 2016 ). an investigation of squamous cell carcinomas revealed the presence of a gammaherpesvirus in multiple captive sun bears (heloarctos malayanus) (lam et al., 2013) . however, successful culture of a gammaherpesvirus of ursine origin has not been demonstrated. this report documents the detection and partial characterization of gammaherpesvirus from multiple black bears (ursus americanus) with and without neurological diseases from nevada, california and oregon, usa. bear 1(b1) and bear 2 (b2) were two of four black bears showing neurological diseases that had been euthanized by the nevada department of fish and wildlife. b1, an approximately 2 year old male, was unable to rise, but could drag itself by his front legs for a short distance. b2, a 1 year old female, was not afraid of people and exhibited slight unsteadiness or tremor. bears nv17a-17c were bears that either died from vehicular trauma or were urbanized animals euthanized as a result of their habituation to humans. bears z1 and z3 were urbanized bears around 1-1.5 years old euthanized by california department of fish & wildlife. bear o1 was juvenile, male american black bear and was euthanized by oregon department of fish & wildlife due to unsuitability for rehabilitation and release. following necropsy examination of these animals, tissues were submitted to the oregon veterinary diagnostic laboratory for diagnostic testing. the human rectal tumor (hrt-18g) cells, marbin darby canine kidney (mdck), vero maru (vero), crandell feline kidney (crfk cells), porcine kidney 15 (pk-15) and rabbit kidney-13b (rk-13b) cells were maintained in dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum (seradigm), penicillin (100u/ml) and streptomycin (100 μg/ml) (sigma-aldrich, inc.) at 37°c with 5% co2 in a humidified incubator. the viral isolate was propagated in hrt-18g cells in f-15 media supplemented with 2.5 μg trypsin and 2.5 μg/ml pancreatin (1xtpc) (gibco). the pooled tissue preparation from each bear was inoculated to hrt-18g cells, absorbed for 1 h on a rocker, washed three times with serum free f-15 media containing 1xtpc following absorption, and cultured in serum free f-15 media containing penicillin (100u/ml) and streptomycin (100 μg/ml) (sigma-aldrich, inc.) at 37°c with 5% co2 in a humidified incubator. virus particles purified by ultracentrifugation of infected cell supernatants at 29 k rpm, were adsorbed to formvar-coated carbon-stabilized copper grids by floating grids on a drop of 10 μl sample mixed with 10 μl of 2% phosphotungstic acid (pta) (ph 6.9) in water on the parafilm. the grids were then blotted dry with whatman filter paper by side-blotting and allowed to air dry before viewing under the electron microscope. images were obtained with tei titan 80-200 transmission electron microscope. total tissue dna was isolated from approximately 100 mg of each tissue using ezna tissue dna extraction kit (omega bio-tek). for each tissue sample, about 0.5 μg of total dna was used as templates in either degenerate pcr or pcr with urhv-1 specific primers. viral dna from tissue culture was isolated from cell pellet of 5 ml total tissue lysates using ezna tissue dna extraction kit (omega bio-tek). nested degenerate (or panherpesvirus) pcr primers, poldegf1 (gayttysmiagyytitaycc) and poldegr1 (ttickiacsaritciacic-cytt), poldegf2 (atiatimwrgcicayaayytitg) and poldegr2 (aaiaiiswrtcigtrtciccrta) targeting conserved regions of the catalytic subunit of the dna polymerase of herpesviruses were used to detect herpesvirus as previously described (pozo et al., 2016) . the nested panherpesvirus pcr was optimized with purified viral dna of human herpesvirus type 1 (hhv-1), leporid herpesvirus type 4 (lhv-4) and koi herpesvirus (khv) ( supplementary fig. 1 ). the primers ug341f (5′-acctacacaggcaccctcac-3′) and ug341r (5′-aatga aggcccttgacctct-3′) specific to ursid herpesvirus 1 were selected based on the dna sequence deposited in genbank (accession no. jx220982.1). in addition, another sets of nested herpesvirus degenerate (or consensus) primers, dfa (5′-gayt tygcnagyytntaycc-3′), ilk (5′-tcctggacaagcagcarnys gcnmtnaa-3′), and kg1 (5′-gtctt gctcacc agntcnacnccytt-3′); tgv (5′-tgtaactcggtgtaygg-nttyacnggngt-3′) and iyg (5′-cacagagtccgtrtcnccrta-dat-3′) targeting the dna polymerase gene, were also used to detect herpesvirus as previously described (vandevanter et al., 1996) . in addition, genus-specific glycoprotein b gene (gb) primers were used to detect gb dna as reported previously (ehlers et al., 2008) . pcr with the panherpesvirus oligomers poldegf1 and poldegr1 was performed in a 50-μl solution consisting of 5.0 μl 10 × pcr amplification buffer (gbiosciences), 5.0 μl 360 gc enhancer (applied biosystems) or 5 μl of 50% dmso (invitrogen), 1.0 μl dntps at 10mmeach, 0.8 μm primers of poldegf1and poldegr1, 1.25 u taq dna polymerase (gbiosciences), and 0.2-0.5 μg total tissue dna. the mixture was subjected to 94°c for 2 min, and 40 cycles of 94°c for 30 s, 40°c for 45 s, and 72°c for 45 s, and followed by a 5-min elongation reaction at 72°c after the final cycle. the products of the first-round amplification with primers poldegf1 and poldegr1are then subject to a second-round of pcr with primers poldegf2 and poldegr2. the second-round of pcr or the nested pcr was run at the same conditions as the first round of pcr amplification. pcr with the herpesvirus consensus primers dfa, ilk, kg1, tgv, and iyg were performed as described previously (vandevanter et al., 1996) . pcr with genus-specific glycoprotein b gene (gb) primers were performed as reported previously (ehlers et al., 2008) . pcr with urhv-1 specific primers, ug341f and ug341r, was performed as follows: a 50-μl solution consisting of 5.0 μl 10× amplification buffer (gbiosciences), 1.0 μl dntps at 10 mm each, 0.4 μm primers (ug314f and ug341r), 1.0 u taq dna polymerase (gbiosciences), and 0.25-0.5 μg total tissue dna, was subjected to 94°c for 2 min, and 30 cycles of 94°c for 30 s, 52°c for 45 s, and 72°c for 45 s, followed by a 5-min elongation reaction at 72°c after the final cycle. sequences of pcr products were determined by direct sequencing of pcr products purified by a chargeswitch pcr clean-up kit (invitrogen) or qiaquick gel extraction kit (qiagen). sequencing was performed by the center for gene research and biocomputing (cgrb) at oregon state university and by the research technology support facility genomics core at michigan state university. the cgrb used an abi prism ® 3730 genetic analyzer with a bigdye ® terminator v. 3.1 cycle sequencing kit, employing abi prism®3730 data collection software v. 3.0 and abi prism ® dna sequencing analysis software v. 5.2. the nucleotide sequences were analyzed with the geneious software. to determine whether infectious viruses were present in the tissues collected from nevada (nv) black bears b1 and b2, tissue preparations from each organ were inoculated to mdck, vero, crfk, pk-15 and rk-13b cells. no visible cytopathic effect (cpe) was observed in any of those cells 2 weeks post-infection. however, inoculation of hrt-18g cell with the pooled tissue preparations from b1 brain, kidney, spleen and lymph node, yielded visible cpes, such as syncytia, intranuclear inclusions and cell detachments, at 3-5 day post-infection (dpi) ( fig. 1c and d), although fusion (black arrows) ( fig. 1a and b) was also observed in uninfected controls; this was attributed to trypsin and pancreatin treatment and was different from syncytia (white arrow) seen in the infected cells ( fig. 1c and d). to determine if similar cpe could be produced from tissues collected from black bears without any clinical neurological disease, tissue preparations from oregon (or) bear o1 were also investigated by virus isolation. similar cpes were observed in hrt-18g cells following infection of pooled bear tissue preparations from o1 ( fig. 1e and f). the virions of herpesviruses are enveloped particles and have icosahedral capsids at 100-120 nm in size. to determine if cpe observed in the infected hrt-18g was caused by herpesvirus infection, virions isolated from tissue culture supernatant were subjected to negative staining and examined by transmission electron microscopy. herpesvirus like capsids were seen in the cells infected with tissue preparations from nv bear b1, which had cns clinical signs ( fig. 2a) . herpesvirus like particles were also seen in cells infected from tissue preparation from or bear o1, which had no cns clinical signs (fig. 2b ). in addition, varied sizes of capsids and virions were observed in tissue cultures infected with the fecal samples of ca bear z3 and or bear 1 (fig. 2c-d) , where coronavirus-like particles (open arrow) and capsids over 50 nm (solid arrow) were also observed in the infected tissue cultures. coronaviruses are commonly observed in the intestine tract of animals, such as cervids and cattle. it is possible that coronaviruses are common in ursine species. there are conserved amino acid sequences within the dna polymerase within the herpesvirus family, which can be used to design degenerative primers for detection of herpesviruses. to determine whether the observed viral particles are herpesviruses, tissues collected from the two nv black bears b1 and b2 were examined by nested pcr using panherpesvirus primers (pozo et al., 2016 ) and herpesvirus consensus primers (vandevanter et al., 1996) . to determine if herpesviral genome is present in nv bear b1 and b2, total dna from brain, kidney, liver and lymph nodes was analyzed by the nested panherpesvirus pcr. as shown in fig. 3 , amplicons within the predicted size were detected in all tissue dna samples from both nv bears b1 and b2, when 360 gc enhancer was included in the pcr reactions. dmso is also an enhancer for gc rich samples and when dmso was included in pcr reaction, only correct amplicons were detected in total dna of all b1 tissues, but only one of b2 tissues. therefore, 360 gc enhancer was used in the rest of the panherpesvirus pcr tests. in addition, correct pcr products were also obtained when herpesvirus consensus primers were used (data not shown here). to determine if the herpesviral genome is present in black bears without clinical neurological signs, another four bears (nv17a, nv17b, nv17c and nv14a) from nevada and one bear (z3) from california were tested by nested panherpesvirus pcr primers, which were more sensitive than the herpesvirus consensus primers (unpublished data). as shown in fig. 4 , a product within the predicted size was amplified from total dna of spleen from ca bear z3 and nv bear17a. however, the positive amplicon was not detected in all the tested tissues and tested bears (fig. 4b, nv17b and nv17c). to ensure the panherpesvirus pcr reactions worked properly, hhv-1 (h), lhv-4 (l) and khv (k) genomic dna were always included as controls when bear tissue dna were analyzed. their products were also sequenced to confirm the reactions worked correctly. to confirm the panherpesvirus pcr amplicons are indeed herpesviral dna, amplicons of lymph node total dna from nv bear b1, spleen total dna from ca bear (z3) and nv bear nv17a were sequenced and analyzed by geneious software. as shown in fig. 5 , the dna sequence alignments of amplicons from b1, nv17a, and z3 total dna are almost identical except the reads near the end, and they have > 94% identity to ursid gammaherpesvirus 1 (urhv-1). it suggests the herpesviral dna detected from nv bear and ca bear are very similar. dna sequencing of the pcr product amplified by the herpesvirus consensus primer of vandevanter et al. (vandevanter et al., 1996) also confirmed the detection of herpesviruses sequences from bears b1 and b2. blast (altschul et al., 1990) analysis of these sequences revealed nucleotide similarities ranging from 90% to 96% to corresponding dna polymerase sequences of gammaherpesviruses from diverse species of seals (data not shown). to investigate phylogenetic relatedness, the panherpesvirus pcr amplicon dna sequences from bears b1, z3, and n17a were compared to dna sequences from representative members of the gammaherpesvirinae using jukes cantor, neighbor-joining analysis. as . panherpesvirus pcr analysis of tissue total dna isolated from b1 and b2 bears diagnosed with neurological diseases. the pcr reaction was performed similarly as described in supplemental fig. 1 with either dmso or 360 gc enhancer. the predicted product is > 400 bp in size. template dna was as follows: 1&5: brain, 2&6: kidney; 3&7: liver; 4&8: lymph node; p: hhv-1; mw: 1 kb-plus dna ladder (invitrogen). shown in fig. 6 , the amplicons from bear tissues and urhv-1 branch out together and have smaller distance to members of rhadinvirus, such as hhv-8 and rhadinovirus 1 (rhv-1). the sequences of amplicons by the herpesvirus consensus primers are more close to phocid herpesvirus 2 (phv-2) and harp seal herpesvirus isolate (hshv), which have not been assigned genus (data not shown here). both phv-2 and hshv branched together with member of macavirus, such as bhv-6, which used to be member of rhadinavirus in older classifications. these data suggest that urhv-1 and the bear herpesviruses under investigation are phylogenetically close to each other and have dna sequences similarity to members of rhadinavirus within the subfamily of gammaherpesvirinae. the glycoprotein b gene (gb) encodes a structural protein on the surface of the envelope, which plays an important role in viral entry to specific host cells{petrovic, 2017 #376}. it does not have high homology between different groups of herpesviruses. genus-specific (gs) glycoprotein b gene (gb) primers have been used successfully in characterization of several novel gammaherpesviruses {ehlers, 2008 #374; lozano, 2015 #375}. here, using the gs gb primers, gb like dna sequence were amplified from dna samples from both neurological bear b1 and non-neurological bears. interestingly, gb dna sequences from neurological bear b1 had only 64.5% identity to the gb dna amplified from non-neurological bear o1. however, the gb dna sequence amplified from b1 dna has higher identity (92.4%) to gb of harp seal herpesvirus isolate fmv04-1493874, while the gb dna sequences from bear o1 had 68.3% identity to gb dna sequence of sea otter herpesvirus clone herpes_23 (kx024493.1). it is possible that different types of gammaherpesviruses exist in the black bear population. phylogenetic analysis revealed that both gb dna from neurological bear b1 and non-neurological bear o1 branch out together with harp seal herpesvirus and sea otter herpesvirus (fig. 7) , and are distant to gb dna of ovine herpesvirus type 2 (ohv-2) of macavirus, equine herpesvirus 2 (ehv-2) from percavirus. phocid herpesvirus 2; ehv-2: equid gammaherpesvirus 2; hhv-8: human herpesvirus type 8; rhv-1: gorilla rhadinovirus 1 isolate; urhv-1: ursid gammaherpesvirus 1; b1: amplicon from nevada bear b1lymph node; nv17a: amplicon from nevada bear nv17a spleen; z3: amplicon from california bear z3 spleen. the accession numbers used for dna polymerase coding sequence analysis were given following each virus name. to confirm that the herpesvirus like particles observed under em were indeed urhv-1 like herpesvirus virions, both hrt-18g cell line passages 1 and 2 from bears b1 and o1 were tested by pcr using urhv-1 specific primers, ug341f and ug341r. when the total dna from tissue cultures passage 1 (p1) and passage 2 (p2) were tested directly by urhv-1 specific pcr, no amplicons were observed (data not shown). however, when the total dna of p1 and p2 were tested by hybrid nested pcr, with the first round of pcr reaction using panherpesvirus primers poldegf1 and poldegr1, and the second round of pcr reaction using urhv-1 specific primers ug341 and ug341r, visible amplicons were observed in urhv-1 specific pcr reactions (fig. 8) . uninfected cell cultures yielded no amplicons. dna sequence alignment shows amplicons of total dna from p1 and p2 have > 90% identity to urhv-1 (fig. 9 ). to determine how common this urhv-1 like virus is in black bear population, various tissues from 15 bears collected from nevada, california, and oregon were analyzed by this hybrid nested pcr with panherpesvirus primers in primary reaction and urhv-1specific primers in the secondary reactions. as shown in table 1 , 7 out of 10 bears from nevada, 3 out of 4 from california, and the sole bear from oregon tested positive by this technique. this suggests that black bear herpesviruses have dpol dna similar to urhv-1. an investigation of a cluster of cases of black bears in the reno, nevada area exhibiting neurologic clinical signs was undertaken in the winter and spring of 2014. a nonsuppurative encephalitis was identified in these animals via histopathology, but at this time, the causative agent remains unclear. however, during this investigation, herpesviruses were considered a potential cause, as ursine encephalitis cases have been linked to alphaherpesviruses (dombrowski et al., 2016) . investigations using degenerate pcr primers, both at the ovdl and at michigan state university vdl, resulted in amplification of dpol dna sequences with homology to gammaherpesviruses in a variety of tissues and in bears with and without evidence of neurologic disease. however, investigations using genus-specific glycoprotein b gene (gb) primers revealed that the gb dna sequence obtained from neurological bear b1 is different from those amplified from bears without neurologic disease. it is possible that a specific type of gammaherpesvirus is associated with bear neurologic disease. however, if several gammaherpesviruses with similar dpol dna sequences exist in black bears, it will require further study to connect a specific herpesvirus with neurological diseases in bears. tissue culture of an ursid herpesvirus has not been reported before. our initial virus isolation attempts were in cell lines that are commonly used for animal virus isolations. vero, crfk, rk-15, and mdbk are all susceptible to alphaherpesvirus infections, such as bovine herpesvirus type 1, equine herpesvirus type 1, pseudorabies virus, feline herpesvirus type 1, and canine herpesvirus type 1. infections of those alphaherpesviruses produce cpe, such as syncytia, intranuclear inclusions, and cell detachments. no cpe was observed in those cells following inoculation of the bear tissue preparation within the 2 week observation period. hrt-18g cells are normally used for animal coronavirus isolations in the veterinary diagnostic lab. the fecal sample from z3 was suspected for coronavirus and was inoculated to hrt-18g cells. interestingly, cell fusion and cell detachment were observed at 3-5 day post-infection, which was different from the cpe seen in coronavirus infections. cpes from coronavirus infections are characterized with cell death and cell detachment. it is interesting that human rectal epithelial cells are susceptible to this bear herpesvirus infection. under em examination, herpesvirus-like capsids and particles were observed in infected hrt-18 cells. however, the observation of complete herpesvirus virions is rare in infected hrt-18g cells. in addition, capsids of variable size were also observed in tissue culture infected with bear fecal samples, and many of them have morphology similar to rotavirus and coronavirus ( fig. 2c and d) . since hrt-18g cells are susceptible to coronavirus infection, it is not surprising to see coronavirus in cultures infected with fecal samples. it is possible coronaviruses are common in black bears. another interesting finding in this study is that the production of urhv-1like virus in vitro is low. the viral genome can only be detected in direct cell lysates by nested urhv-1 specific pcr. again, it is possible that the black bear herpesvirus replicates poorly in vitro. this correlates with the few cpe plaques observed in infected hrt-18g cells. fig. 7 . phylogenetic tree of the gb dna sequences. the scale bar represents genetic distance (nucleotide substitutions per site), and branch lengths are given above the branches. the abbreviations in the virus names are as follows: hshv: harp seal herpesvirus isolate fmv04-1493874 (kp136799); sohv: sea otter herpesvirus (kx024493.1); ohv-2: ovine herpesvirus 2 (af385442.1); rfhvmnm78114: the macaque homolog of kaposi's sarcoma (ks)-associated herpesvirus (kf703446); ehv-2: equid herpesvirus 2 (hq247756.1); blb-gb (mk089801) : gb dna amplified from non-neurological black bear. b1: gb amplicon from nevada bear b1 lymph node; o1: gb amplicon from oregon bear spleen. fig. 8 . detection of urhv-1 like dna using urhv-1 specific primers. total dna samples were analyzed by pcr using urhv-1 specific primers ug341f and ug341r. the abbreviations of tissues are as follows: p: total dna of lymph node from bear b1; n: total dna of hrt-18g cells; z2: total dna of spleen from z2; z3: total dna of spleen from z3; p1: total dna of viral culture passage 1, p2: total dna of viral culture passage 2. o1: oregon bear without neurological signs, b1: nevada bear with neurological signs. mw, 1 kb-plus dna ladder. panherpesvirus pcr was developed by pozo f. et al to detect bat herpesviruses (pozo et al., 2016) . there were two sets of nested degenerate pcr primers selected to detect the conserved herpesvirus genes, dna polymerase and terminase in the bat herpesvirus study. the selection of those primers was based on hhv-5 reference genome (accession no. nc_006273). here, we find we can use pandpol primer sets, which target the hhv-5 dna polymerase, genome position between 79774 and 78991, 79750-79201, respectively. the anticipated pcr product with poldeg1f/poldeg1r and poldeg2f/poldeg2r are 713-992 bp and 482-758 bp, respectively. the conditions reported by pozo f. et al can amplify the dna polymerase sequence of alphaherpesviruses, such as hhv-1 and lhv-4, but not alloherpesviruses, such koi herpesvirus (khv). however, inclusion of 360 enhancer in the pcr was able to amplify dna polymerase coding sequence of khv dna. another interesting observation in this study is that positive amplification occurred in all the tested tissues from bears b1 and b2 with neurological signs, however, fewer tissues tested positive in bears nv17a-c without clinical signs, and some instances, no tissue tested positive in bears without clinical signs. the lymphoplasmacytic nature of the encephalitis may account for the positive result in brain tissues from neurologic bears. the tissues which tested positive in non-neurological bears were often spleen and lymph nodes (fig. 4) . it is possible that this black bear herpesvirus persists in these lymphoid rich tissues, like many other gammaherpesviruses and this hypothesis was pursued using whole blood edta samples collected from three other bears that had no neurologic signs. positive amplification with urhv-1 specific primers was observed in total dna of white blood cells (wbc) from two black bears from nevada and one from oregon. dna sequence of the amplicon from the wbc total dna is similar to sequences amplified from bear spleen samples from ca z3 and nv17a and lymph node sample of b1 (fig. 10) . these findings are in agreement with the common feature of b cell latency of gammaherpesviruses. dna sequence alignment analysis shows that pcr amplicons of dpol from the black bear tissues have the highest homology to urhv-1 (fig. 5) . urhv-1 is a novel herpesvirus detected by degenerate pcr primers in sun bears (helarctos malayanus) with oral squamous cell carcinoma (lam et al., 2013) . phylogenetic analysis shows that urhv-1 fig. 9 . dna sequence alignments of ug341 pcr product with urhv-1 sequence detected in the sun bear (accession number jx220982). 1: amplicon of p2 from nevada bear b1 virus isolation shown in fig. 8 . 2: amplicon of p2 from oregon bear o1 virus isolation shown in fig. 8 . 3: jx220982, urhv-1 detected in sun bear. fig. 10 . dna sequence alignments of ug341 pcr product amplified from white blood cell (wbc) total dna. 1: amplicon of lymph node total dna from bear b1; 2: amplicon of spleen total dna from bear nv17a. 3: amplicon of wbc total dna from a new oregon bear submitted to ovdl in 2018. 4: amplicon of wbc total dna from a new nevada bear submitted to ovdl in 2018. 5: amplicon of spleen total dna from bear z3. is closest to members of rhadinovirus (fig. 6) . the old classification of gammaherpesvirinae consists of 2 genera, lymphocryptovirus and rhadinovirus, the later includes the macavirus and percavirus. the new classification separates the macavirus and percavirus from rhadinovirus, which consists of viruses appearing to be paraphyletic. it is possible that this black bear herpesvirus is paraphyletic under certain conditions. it is unknown if urhv-1 is oncogenic. we speculate that the virus may merely have been present in lymphocytes associated with the carcinomas. the phylogenetic analysis shown in fig. 6 , suggests that this black bear virus and urhv-1 are close to members of rhadinovirus and macavirus. however, when genus-specific glycoprotein b (gb) gene primers (ehlers et al., 2008) were used, gb dna sequences from neurological bear had lower identity to gb dna sequence amplified from non-neurological bear. since they have less than 70% identity in gb gene, we propose that at least two different black bear herpesviruses exist in free-ranging black bears (ursus americanus) and they may be close related to urhv-1. more dna sequences will be needed for further classification. in summary, new black bear herpesviruses that are phylogenetically close to urhv-1 and harp seal herpesvirus isolate fmv04-1493874 have been detected in free-ranging black bears (ursus americanus) with and without neurological disease. these black bear herpesviruses may have limited replication in hrt-18g cells. further study will be needed to determine if a specific black bear herpesvirus is associated with neurological disease in black bears (ursus americanus). epstein-barr virus-associated cerebellar ataxia basic local alignment search tool japanese macaque encephalomyelitis: a spontaneous multiple sclerosis-like disease in a nonhuman primate an unusual outbreak of malignant catarrhal fever in a beef herd in israel a gammaherpesvirus establishes persistent infection in neuroblastoma cells severe neurological complications in association with epstein-barr virus infection barr humbug: acute cerebellar ataxia due to epstein-barr virus blindness in a wild american black bear cub (ursus americanus) novel mammalian herpesviruses and lineages within the gammaherpesvirinae: cospeciation and interspecies transfer epstein-barr virus associated with primary cns lymphoma and disseminated bcg infection in a child with aids epstein-barr virus-associated lymphoproliferative disorders murine gammaherpesvirus-68 infection of mice: a new model for human cerebral epstein-barr virus infection the epizootiology of caprine herpesvirus (bhv-6) infections in goat populations in greece a novel gammaherpesvirus found in oral squamous cell carcinomas in sun bears (helarctos malayanus) sheep-associated malignant catarrhal fever in a petting zoo transmission of caprine herpesvirus 2 in domestic goats infection with ovine herpesvirus 2 in norwegian herds with a history of previous outbreaks of malignant catarrhal fever primary characterization of a herpesvirus agent associated with kaposi's sarcomae nasopharyngeal carcinoma: a review identification of novel betaherpesviruses in iberian bats reveals parallel evolution murine gammaherpesvirus-68 infection of and persistence in the central nervous system detection and analysis of diverse herpesviral species by consensus primer pcr equine multinodular pulmonary fibrosis: a newly recognized herpesvirusassociated fibrotic lung disease linking kshv to human cancer we thank carlson college of veterinary medicine at oregon state university (osu) and biomedical undergraduate summer research (kw) for funding this study. we thank the supports from oregon department of fish and wildlife and california department of fish and wildlife. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.virusres.2018.10.016. key: cord-302083-9q1i20o6 authors: jung, kwonil; saif, linda j.; wang, qiuhong title: porcine epidemic diarrhea virus (pedv): an update on etiology, transmission, pathogenesis, and prevention and control date: 2020-06-02 journal: virus res doi: 10.1016/j.virusres.2020.198045 sha: doc_id: 302083 cord_uid: 9q1i20o6 porcine epidemic diarrhea virus (pedv), a member of the genus alphacoronavirus in the family coronaviridae, causes acute diarrhea and/or vomiting, dehydration and high mortality in neonatal piglets. two different genogroups of pedv, s indel [pedv variant containing multiple deletions and insertions in the s1 subunit of the spike (s) protein, g1b] and non-s indel (g2b) strains were detected during the diarrheal disease outbreak in us swine in 2013-2014. similar viruses are also circulating globally. continuous improvement and update of biosecurity and vaccine strains and protocols are still needed to control and prevent pedv infections worldwide. although the non-s indel pedv was highly virulent and the s indel pedv caused milder disease, the latter has the capacity to cause illness in a high number of piglets on farms with low biosecurity and herd immunity. the main pedv transmission route is fecal–oral, but airborne transmission via the fecal–nasal route may play a role in pig-to-pig and farm-to-farm spread. pedv infection of neonatal pigs causes fecal virus shedding (alongside frequent detection of pedv rna in the nasal cavity), acute viremia, severe atrophic enteritis (mainly jejunum and ileum), and increased pro-inflammatory and innate immune responses. pedv-specific iga effector and memory b cells in orally primed sows play a critical role in sow lactogenic immunity and passive protection of piglets. this review focuses on the etiology, transmission, pathogenesis, and prevention and control of pedv infection. porcine epidemic diarrhea virus (pedv), a member of the genus alphacoronavirus in the family coronaviridae of the order nidovirales, causes acute diarrhea, vomiting, dehydration and high mortality in neonatal piglets. the disease was reported in the european and asian pig industries over the last 30 years, with the virus first appearing in england (wood, 1977) and j o u r n a l p r e -p r o o f one study reported that four sows exposed via feedback to a g1b s indel pedv at approximately seven months pre-farrowing and then re-exposed to a g2b non-s indel pedv at day 109 of gestation provided long-term (7 months), passive immune protection of piglets against challenge with the same g2b non-s indel pedv (goede et al., 2015) . there was 0% mortality rate of piglets (with 57% reduced incidence of diarrhea), compared with mean 33% mortality and 100% morbidity rates of piglets born to non-immunized sows. however, another study revealed that more than 80% of the piglets inoculated orally with a g1b s indel pedv (iowa106 strain) at 3-4 days of age had diarrhea after challenging with a g2b non-s indel pedv at 24 days of age, whereas none of the piglets inoculated previously and then challenged with the same non-s indel pedv showed diarrhea (annamalai et al., 2017; lin et al., 2015a) . antigenic cross-reactivity or cross-neutralization between pedv and feline infectious peritonitis virus (fipv) was detected by enzyme linked immunosorbent assay (elisa), immunoblotting and immune-precipitation (zhou et al., 1988) , or vn tests (zhao et al., 2019) . despite no serological cross-neutralization detected between pedv and tgev (hofmann and wyler, 1989; pensaert and de bouck, 1978) , investigators found some antigenic cross-activity between pedv and tgev (miller, but not purdue strain) based on at least two conserved epitopes on the n-terminal region of their n proteins, as well as via their m proteins or whole virus particles (gimenez-lirola et al., 2017; lin et al., 2015b; xie et al., 2019) . truncation of the n-terminal region of the n protein or avoidance of m-or purified whole virus-based serologic tests may eliminate pedv and tgev shared epitope(s) or reduce the possible cross-reactivity for serologic j o u r n a l p r e -p r o o f assays (gimenez-lirola et al., 2017; xie et al., 2019) . polyclonal hyperimmune antisera against pdcov did not cross-react with pedv . among n, m and e proteins and whole pedv particles, only the m protein of pedv showed cross-reactivity in 1/12 convalescent sera from pdcov-infected pigs (gimenez-lirola et al., 2017) . however, one study reported antigenic cross-reactivity between us pdcov and pedv strains, possibly due to at least one shared epitope in the n-terminal region of their n proteins . for sads-cov, monoclonal antibody against the n protein did not cross-react with pedv, tgev or pdcov (pan et al., 2017) . the fecal-oral route is the main means of direct transmission of pedv via the feces and/or vomitus of infected pigs (jung and saif, 2015) . indirect contact transmission of pedv is also frequent within and between farms, particularly, with a low biosecurity, via other contaminated fomites including (jung and saif, 2015; kim et al., 2017b) : transport trailers (lowe et al., 2014) , farm workers' hands, boots and clothes (kim et al., 2017b) , feed (bowman et al., 2015a; dee et al., 2014; schumacher et al., 2017) , feed ingredients and additives, such as spray-dried porcine plasma (pasick et al., 2014; perri et al., 2018) , and feed totes used for transporting bulk feed or feed ingredients (anon., 2015; scott et al., 2016) . pedv remained infectious on tote material for 35 days at room temperature (scott et al., 2016) . pedv cross-contamination also occurred during feed manufacturing (schumacher et al., 2018) . j o u r n a l p r e -p r o o f 8 the fecal-nasal route is another route of pig-to-pig, or farm-to-farm (up to 10 miles away) airborne transmission of pedv via aerosolized pedv particles that are infectious in nursing pigs (alonso et al., 2014; alonso et al., 2015; beam et al., 2015; gallien et al., 2018a; . airborne pedv transmission occurs within farrowing herds where newborn piglets, highly susceptible to the virus, are raised (alonso et al., 2015; niederwerder et al., 2016) . the nasal cavity of naïve pigs housed at a distance from clinical pigs was frequently positive for pedv rna (niederwerder et al., 2016) . aerosolized pedv does not infect only the intestine of pigs (alonso et al., 2015) , but it also infects the epithelium lining the nasal cavity . reported that dendritic cells in the lamina propria of the nasal mucosa or lymphoid tissue carry and transfer pedv to cd3 + t cells. pedv-loaded t cells may reach the intestine via unknown specialized endothelial venules during blood circulation. the virus loaded onto t cells had the capacity to infect intestinal epithelial cells via cell-to-cell contact and transfer infection . compared with neonatal piglets, however, higher doses of aerosolized pedv may be required to infect weaned and older pigs (niederwerder et al., 2016) . in the study, none of aerosol contact, 28-day-old pigs (0/5 pigs) was infected, although their nasal cavities (5/5 pigs) were positive for pedv rna. the severity of pedv infection and the disease, and the transmissibility of pedv, depends on the overall immunity and health status of the pig population and the levels of biosecurity on farms (pensaert and martelli, 2016) . nevertheless, transmissibility of pedv via direct or aerosol j o u r n a l p r e -p r o o f contact also varies dependent on the pedv genogroup. direct contact or aerosol transmission rates were significantly higher in pigs infected with non-s indel pedv compared with s indelinfected pigs (gallien et al., 2018a) . gallien et al. (2018) revealed that despite the presence of aerosolized s indel pedv, none of aerosol contact pigs (0/10 pigs) was infected, whereas 10/10 aerosol contact pigs were infected by non-s indel pedv (gallien et al., 2018a) . porcine small intestinal villous enterocytes express aminopeptidase n (apn), a 150-kda glycosylated transmembrane protein, which was tentatively identified as the cellular receptor for pedv (li et al., 2007; liu et al., 2015a; nam and lee, 2010) . however, porcine apn may not be the major cell surface receptor for pedv (ji et al., 2018; shirato et al., 2016) . concomitantly, apn knockout pigs were susceptible to infection with pedv, but not with tgev that uses apn as the cellular receptor (whitworth et al., 2019; zhang et al., 2019) . cell membrane cholesterol or two cell surface molecules, such as sialic acids and occludin expressed on the apical surface of secretary (goblet) or absorptive enterocytes, respectively, were involved partially in binding and entry of pedv into enterocytes (deng et al., 2016; jeon and lee, 2017; luo et al., 2017) . the strains of pedv, such as cell culture-adapted vs. wild-type, may be a factor involved in the cellular entry via sialic acids (deng et al., 2016; . intracytoplasmic localization of pedv antigen in goblet cells of infected pigs and porcine intestinal enteroids in vitro (jung and saif, 2017; raises questions on whether or how goblet cells are utilized for pedv replication, or if they only transfer pedv to absorptive enterocytes. j o u r n a l p r e -p r o o f we demonstrated the tissue tropism of pedv to certain intestinal locations (jung et al., 2018) . during acute pedv infection [post-inoculation hour (pih) 12-24] in nursing pigs, pedv initially infected mainly the mid-jejunum and ileum and to a lesser extent, the proximal and distal jejunum and duodenum (jung et al., 2018) (fig. 1) . the pylorus was not the site of acute pedv infection (jung et al., 2018) , whereas the villous enterocytes of the large intestine were frequently infected; however, infected colonic enterocytes did not undergo necrosis (jung et al., 2015a; jung et al., 2014) . unlike colonic enterocytes, pedv-infected small intestinal villous enterocytes undergo acute necrosis and exfoliation from the lamina propria, leading to marked villous atrophy or fusion in the small intestine (jung et al., 2018; jung et al., 2014) . like tgev (kim et al., 2000) and pdcov (jung et al., 2016) , pedv may not induce apoptotic death of intestinal villous enterocytes in vivo (jung and saif, 2015) . however, pedv-infected vero cells in vitro underwent apoptosis (kim and lee, 2014) . during acute pedv infection (pih 12-24) of nursing pigs, early localization of pedv antigen was evident in the villous-crypt interface of the small intestine, rather than the villous tips (jung et al., 2018) (fig. 1) . iimmature enterocytes were the major site of initial pedv infection, although the exact reason remains obscure (jung et al., 2018) . the pedv antigen-positive regions subsequently expanded to the upper and then the entire villous epithelium of the jejunum to ileum (<24 hours after oral inoculation) (jung et al., 2018) . the villous-crypt interface is close to blood vessels in the submucosa. although pedv-related viremia might be a result of diffusion of replicated pedv from the acutely infected intestine to blood (jung et al., 2018) , further studies are j o u r n a l p r e -p r o o f needed to investigate whether pedv or pedv-loaded cd3+ t cells circulating in blood reach and infect the villous-crypt interface or other villous regions, and other types of cells of extra-intestinal origin (jung et al., 2018; . pedv antigens were also frequently detected in the intestinal crypt cells or antigen presenting cells, such as macrophages, in the lamina propria or peyer's patches (debouck et al., 1981; jung et al., 2014; lin et al., 2015a; madson et al., 2016; stevenson et al., 2013; sueyoshi et al., 1995) . pedv remains infectious in dendritic cells for <24 hours in vitro (gao et al., 2015) , but dendritic cells may not be a site of pedv replication . instead, pedv appeared to employ dendritic cells to cross the epithelial barrier of the nasal cavity . lung tissues of oronasally infected pigs were negative for pedv antigen (debouck et al., 1981; jung et al., 2014; stevenson et al., 2013; sueyoshi et al., 1995) . however, the upper respiratory tract may be the site of pedv infection, because the epithelium lining the nasal cavity was positive for pedv antigens . although acute viremia was frequently noted in infected pigs during the acute or incubation stage of infection (jung et al., 2018; jung et al., 2014; , pedv antigens were not detected in other major organs, such as liver and kidneys (debouck et al., 1981; jung et al., 2014; stevenson et al., 2013; sueyoshi et al., 1995) . the reproductive organs of experimentally infected boars, such as cowpers's glands, were also negative for pedv rna, but their semen was transiently positive for pedv rna (gallien et al., 2018b; gallien et al., 2019) . nevertheless, whether the pedv rna-positive semen contained infectious viral particles is unclear. j o u r n a l p r e -p r o o f during the incubation period (<pih 16) in nursing pigs, a few to 50% of the villous epithelial cells positive for pedv antigen were seen in the villous-crypt interface and upper villous epithelium of the small intestine (debouck et al., 1981; (jung et al., 2018) . during this period, infected enterocytes underwent necrosis, but complete villous atrophy was not observed (jung et al., 2018) . when clinical signs were first seen (pih 16-24), moderate to large numbers of infected villous epithelial cells were observed throughout the small intestine (debouck et al., 1981) . during this period, up to 100% of the epithelial cells were positive for pedv antigen in the mid-jejunum to ileum where moderate to severe villous atrophy was observed (jung et al., 2018) . during the middle [post-inoculation day (pid) 1-3] to late (pid 4-5) stages of pedv infection of nursing pigs (5-9-day-old), regardless of the locations (except for the duodenum with milder villous atrophy), all small intestinal segments exhibited similar, extensive villous atrophy (mean 1-2 intestinal villous height to crypt depth ratios, compared with 5-9 of uninfected pigs) (chen et al., 2016a; jung et al., 2015a) . during this period, large numbers of pedv-infected epithelial cells could still be observed (debouck et al., 1981) . diarrhea induced by pedv is a consequence of malabsorption and maldigestion due to massive loss and functional disorders of infected enterocytes, followed by decreased brush border membrane-bound digestive enzymes (coussement et al., 1982; jung et al., 2006) . dehydration is exacerbated by vomiting and decreased appetite (jung and saif, 2015) . serotonin is involved in induction of vomiting, and increased pro-inflammatory cytokine responses in part led to decreased appetite (jung et al., 2018) . pedv-infected piglets also showed hyperkalemia and acidosis due to j o u r n a l p r e -p r o o f the loss of bicarbonate (jung and saif, 2015) . the cardiac contractility is impaired by acidosis and dehydration. in the small intestine of clinically infected pigs, the tight or adherens junctions of the villous enterocytes were destroyed (jung et al., 2015b) . expression of the junction proteins, such as zonula occludin-1, was also decreased or altered, followed by decreased transepithelial resistance (curry et al., 2017; jung et al., 2015b; luo et al., 2017; zong et al., 2019) . during the clinical period, there were remarkably reduced numbers of goblet cells in the small intestine (jung and saif, 2017) , followed by decreased amounts of mucins (curry et al., 2017; jung and saif, 2017) . the damaged gut integrity may lead to uptake of luminal food and bacteria causing allergic reactions and co-infections. the impaired gut integrity or digestive function usually recovered by the second week after infection in survived pigs (curry et al., 2017) . in infected gnotobiotic or conventional nursing piglets (1-10-day-old), a few (<5%) to all of the inoculated pigs began to show diarrhea at pid 1, which mostly coincided with or was detected several hours later than the first detection of viral rna in serum or feces (chen et al., 2016a; gerber et al., 2016; jung et al., 2015a; jung et al., 2018; liu et al., 2015c; madson et al., 2016; thomas et al., 2015a) . fecal viral rna titers shed peaked by pid 1, or occasionally, 1-3 days later, and the titers decreased remarkably during the middle to late stages of infection and then titers remained low (with at least one more recurrent peak, possibly due to ped re-infection of regenerating enterocytes) during the recovery stage of infection (chen et al., 2016a; jung et al., 2015a; lin et al., 2015a; madson et al., 2016) . a one-three-day delay in onset of diarrhea was observed in nursing pigs inoculated with s indel pedv compared with the non-s indel counterparts (gallien et al., 2018a; lin et al., 2015a) , indicating a longer incubation time for s j o u r n a l p r e -p r o o f indel pedv. in infected conventional weaned to feeder pigs (21-56-day-old), a few (<5%) to all of the inoculated pigs began to show diarrhea at pid 1-4, which coincided with or was detected 1-2 days later than the first detection of viral rna in feces (gallien et al., 2018a; gerber et al., 2016; jung et al., 2015a; lohse et al., 2017; madson et al., 2014; niederwerder et al., 2016; thomas et al., 2015a) (table 1) . fecal viral rna titers shed peaked by 1-3 days after diarrhea was first detected (gallien et al., 2018a; jung et al., 2015a; lohse et al., 2017; madson et al., 2014; niederwerder et al., 2016; thomas et al., 2015a) . detailed data from boars or sows are summarized in table 1 . duration of diarrhea in infected nursing pigs varied from 2-8 days, with a higher variability for the us s indel strain (2-7 days) compared with the us non-s indel (7-8 days) (lin et al., 2015a) . prolonged fecal viral rna shedding (at least pid 14) was evident in 20/33 (61%) us non-s indel pedv-infected nursing (10-day-old) pigs (gerber et al., 2016) (table 1) . diarrhea in weaned to feeder pigs (21-56-day-old) inoculated orally with us non-s indel pedv was observed for approximately 6 days niederwerder et al., 2016; thomas et al., 2015a) . prolonged fecal viral rna shedding was evident in non-s indel or s indel pedvinfected weaned or feeder pigs for 15-26 days (gallien et al., 2018a; lohse et al., 2017; madson et al., 2014; niederwerder et al., 2016; thomas et al., 2015a) . duration of fecal shedding was similar between non-s indel-(15-26 days) (gallien et al., 2018a; lohse et al., 2017; niederwerder et al., 2016; thomas et al., 2015a) and s indel-infected weaned or feeder pigs (15-18 days) (gallien et al., 2018a; lohse et al., 2017) . longer fecal viral rna shedding was observed in weaned pigs infected via direct contact (42-46 days) or aerosol contact (48 days) compared with the orally infected counterparts (18-21 days) (crawford et al., 2015; gallien et al., 2018a) . however, infectious pedv was shed in feces only for the initial 2 weeks tested using sentinel naïve pigs (crawford et al., 2015) . the minimum infectious dose [0.56 median tissue culture infectious dose (tcid50)] of non-s indel pedv required to infect 5-day-old nursing pigs was lower than for 3-week-old weaned pigs (56 tcid50) (thomas et al., 2015a) . similarly, 3 plaque-forming unit (pfu)/pig of the non-s indel pc22a strain caused diarrhea in 100% of 4-day-old cesarean-derived, colostrum-deprived (cdcd) piglets (liu et al., 2015c) . compared with nursing pigs that began to show diarrhea, villous atrophy, and fecal virus shedding at pid 1, a longer incubation time of non-s indel pedv was required for weaned pigs to show fecal virus shedding (1 more day), or diarrhea and lesions (2 more days) (jung et al., 2015a) . one factor contributing to the higher susceptibility of nursing pigs to non-s indel pedv infection is immaturity of gut innate immunity, as evident by a functional defect in natural killer cells in the ileum and blood (annamalai et al., 2015) . the lack of diversity and decreased firmicutes to bacteroidetes ratio in the gut microbiota of neonatal piglets contributed to the immature gut innate immunity . compared with grower pigs (gastric ph 2-3), neonatal pigs have a higher gastric ph (4-6) that allows pedv to survive in the stomach (mavronmichalis, 2016). compared with nursing pigs, several factors that influence a shorter recovery of weaned pigs from ped include: 1) the faster regeneration of enterocytes in weaned pigs compared with j o u r n a l p r e -p r o o f nursing pigs (moon et al., 1973) ; and 2) the anatomically and physiologically fully developed large intestine of weaned pigs where water reabsorption is greater (jung et al., 2015a) . there was a lack of lgr5 (leucine-rich repeat-containing g protein-coupled receptor 5, marker for crypt stem cells)-positive crypt base columnar cells (lgr5+ cells) and low proliferation of crypt cells in the small intestine of naïve nursing piglets (9-day-old), causing the slower turnover of enterocytes (jung et al., 2015a) . unlike nursing pigs, naïve weaned pigs (3-week-old) exhibited high proliferation of intestinal crypt cells and large numbers of lgr5+ cells in the crypts, leading to the higher turnover rate of enterocytes (jung et al., 2015a) . after pedv infection, large numbers of lgr5+ cells and high proliferation of crypt cells were maintained, possibly relating to the rapid recovery from ped in weaned pigs. detection of viremia where viral rna in serum ranged from 4.5-8.6 log10 genomic equivalents (ge)/ml was identified in gnotobiotic neonatal (5/5; 100%), or conventional 9-dayold nursing (16/16; 100%) and 26-day-old weaned pigs (11/20; 55%) infected with a us non-s indel pedv strain at pid 1-5 (jung et al., 2015a; jung et al., 2014) . pedv rna was detected in all sera from 20 nursing pigs infected with either one of three different us non-s indel strains or a us s indel strain at pid 3 (chen et al., 2016a) . the classical pedv br1/87 also induced viremia in 5/13 inoculated weaned pigs (38.5%) at pid 2-7 (lohse et al., 2017) . in experimentally infected nursing pigs, the frequency of viremia and serum viral rna titers peaked at pid 1, then decreased progressively, and ceased at pid 14-21 (chen et al., 2016a; jung et al., 2015a; opriessnig et al., 2014) . a significantly higher frequency of viremia in weaned pigs infected via direct or aerosol contact, or orally with non-s indel pedv was identified, compared with the s j o u r n a l p r e -p r o o f indel counterparts (gallien et al., 2018a) . the role of viremia in the pathogenesis of pedv is still obscure, although pedv identified in the serum could be infectious pasick et al., 2014; perri et al., 2018) . during the clinical period, alteration in the abundance or proportion of gut microbiota (dysbiosis) occurred in nursing pigs and their dams koh et al., 2015; liu et al., 2015b; song et al., 2017; tan et al., 2019) . the transfer of a healthy gut microbiota from infected sows to their offspring was impaired . the dysbiosis was characterized by reduced numbers of beneficial bacteria (verrucomicrobia) and increased numbers of harmful bacteria (fusobacterium and proteobacteria) (liu et al., 2015b; tan et al., 2019) , followed by decreased cellular transport and catabolism . a study showed that feeding bacillus subtilis to 2-week-old pigs infected with pedv reduced intestinal pathology (but not clinical disease) (canning et al., 2017) . similarly, live or cell-free supernatants of some lactobacillus plantarum strains had antiviral effects against pedv infection in vero cells (sirichokchatchawan et al., 2017) . however, this feed additive method (1/77 herds surveyed; 1.2 %) was rarely used to mitigate ped on us pig farms during the 2013-2017 epidemic (niederwerder and hesse, 2018) . early studies verified high enteropathogenicity of the g1a cv777 in nursing pigs (coussement et al., 1982; pensaert and martelli, 2016) . the s1 of the s protein and the rest of the genome of the s indel pedv (g1b) was genetically closest to that of g1a and the highly virulent j o u r n a l p r e -p r o o f g2 pedv, respectively (lee, 2015; pensaert and martelli, 2016; vlasova et al., 2014) , the strain was initially thought to be virulent. however, during the initial outbreaks by s indel pedv in the us and europe, the low to zero mortality rates of pigs in the field were distinct from those of us non-s indel infections with up to 100% mortality rates in nursing pigs (boniotti et al., 2016; goede et al., 2015; stevenson et al., 2013; wang et al., 2014b) . nevertheless, difference in virulence of the two types of strains in the field was not often observed (grasland et al., 2015; mesquita et al., 2015; stadler et al., 2015; steinrigl et al., 2015) . a study using a wild-type strain of s indel pedv reproduced as severe ped in infected sows and their piglets as that observed in the field (leidenberger et al., 2017) . other studies showed generally milder pathogenicity of s indel pedv in 3-7-day-old conventional pigs, compared with non-s indel pedv (chen et al., 2016a; lin et al., 2015a; yamamoto et al., 2015) . however, the virulence of s indel pedv was increased in the pedv challenged suckling piglets when the mothers became sick, corresponding to poor immune, nutritional or health conditions (lin et al., 2015a) . the mortality rates of piglets increased from 0% to 75%, and the peak fecal pedv rna titers shed were also as high as those of non-s indel strain. the virulence of pedv was attenuated by: 1) high cell-culture passage (93 rd -160 th ) in the cell line of non-swine origin, vero cells (with trypsin in the cell culture medium) (chen et al., 2015; kweon et al., 1999; lin et al., 2017a; sato et al., 2011; song et al., 2003) ; 2) modified cell . in addition, pedv variants of mild virulence, such as the s1 n-terminal domain (ntd)-del variant, also occurred naturally (su et al., 2018; suzuki et al., 2016; zhang et al., 2018a) . pedv strains fully adapted to vero cells and serially passaged at least 70 times (with trypsin or gcdca in the cell culture medium) tended to replicate less efficiently in the intestine of pigs (chen et al., 2015; kim et al., 2017a; lin et al., 2017a; sato et al., 2011; wu et al., 2019) (table 2 ) and elicited low serum pedv-specific antibody titers (lin et al., 2017a) . twelve-week-old pigs orally inoculated with 8.2 log10 tcid50 of g1a pedv 83p-5 (100 th passage in vero cells) exhibited only intermittent fecal viral rna shedding in 3 of 4 pigs (sato et al., 2011) . none of the 10-dayold pigs orally inoculated with 6 log10 tcid50 of g2b pedv yn (144 th passage in vero cells) showed fecal viral rna shedding; however, pedv antigen was detected in the small intestine (chen et al., 2015) . compared with g2b pedv yn (144 th ) and pc22a (140-160 th passage in vero cells) grown with trypsin, g2b pedv 8aa (70 th -105 th passage in vero cells) grown with gcdca and attenuated at lower cell passage numbers, caused no or little fecal viral rna shedding in 1day-old pigs (6 log10 tcid50/pig) (kim et al., 2017a) . four-day-old cdcd pigs orally inoculated with 2 log10 pfu of pc22a (140 th -160 th ) showed low to moderate fecal viral rna shedding titers, with mild villous atrophy (lin et al., 2017a) (table 2) . six-day-old conventional pigs orally inoculated with 6 log10 tcid50 of g2b pedv ct (120 th passage in vero cells) showed mild diarrhea and moderate fecal viral rna shedding titers, with mild villous atrophy . on the other hand, pregnant sows orally inoculated with g1a pedv dr13 (100 th passage in vero cells) showed no clinical signs but high vn antibody levels in the colostrum (song et al., 2007) . il-17) responses in acutely infected neonatal pigs (annamalai et al., 2015; jung et al., 2018) . this coincided with increased numbers of pedv antigen-positive cells (gao et al., 2015; madson et al., 2016) or inflammatory cells (lymphocytes or neutrophils) in the gut-associated lymphoid tissue (galt) (coussement et al., 1982; de arriba et al., 2002b; debouck et al., 1981; jung and saif, 2015) , and the onset of necrosis and exfoliation of infected enterocytes (jung et al., 2018) . necrosis of enterocytes is in part involved in the innate or pro-inflammatory cytokine responses in infected pigs, as also observed in vitro (koonpaew et al., 2019; lin et al., 2017b) . the increased systemic innate and pro-inflammatory cytokine responses that occur during acute pedv infection are beneficial for differentiation of naïve t cells into cytotoxic or helper t cells, leading to removal of infected cells or production of vn antibodies, respectively (siegrist, 2013) . however, highly increased, prolonged pro-inflammatory cytokines that can cause decreased appetite, etc. are also detrimental (jung et al., 2018; langhans, 2000) . increased systemic ifnα and ifnγ cytokine responses were also observed in pedv-infected gilts at pid 13-17 and pid 6-8, respectively (langel et al., 2019b) . clinically infected pigs responded to a primary pedv infection and seroconverted at pid 7-14 (mean pid 14) (pid 14-21 for serum pedv-specific iga antibodies), with peak serum j o u r n a l p r e -p r o o f pedv-specific igg or iga antibody titers at pid 21 (annamalai et al., 2017; chen et al., 2016b; de arriba et al., 2002a; de arriba et al., 2002c; gallien et al., 2018a; gerber et al., 2016; niederwerder et al., 2016; opriessnig et al., 2014; thomas et al., 2015a) . there were lower serum igg or vn antibodies in s indel-infected pigs compared with the non-s indel counterpart (annamalai et al., 2017; chen et al., 2016b) . serum vn antibodies were detected 7 days earlier than serum pedv-specific igg antibodies (chen et al., 2016b; thomas et al., 2015a) . there were low titers of pedv-specific secretory iga (siga) in the feces of pedv-infected pigs (10-day or 8-week-old) by pid 14 (gerber et al., 2016) . serum pedv-specific iga and igg antibodies in naturally infected sows remained high for 6 months, whereas fecal pedv-specific siga antibodies disappeared 1-2 months after infection (ouyang et al., 2015) . pedv-specific iga or vn antibody titers of sows infected at 3 months pre-farrowing peaked in the colostrum at post-farrowing day (pfd) 1, decreased rapidly in milk by pfd 3, and then declined only gradually during pfd 4-19 . memory b cell responses were first identified by in vitro secondary stimulation in mononuclear cells from pedv cv777-infected nursing pigs at pid 21 (de arriba et al., 2002a) . the number of pedv-specific iga antibody-positive pigs also rose from 30% at the day of reinfection to 70% 3 days later (gerber et al., 2016) . similarly, the number of pedv-specific iga antibody secreting cells of sows infected at 3-4 months pre-farrowing and then after re-exposure to pedv at pfd 3-5, increased in the milk at pfd 8-14, compared with pfd 0 (colostrum) or pfd enhanced biosecurity, such as decontamination and disinfection of transport trailers and other potential contaminated fomites, supply of secure feed or feed additives (gordon et al., 2019; niederwerder and hesse, 2018) , and maintenance of strong hygiene measures on swine farms and farm workers (kim et al., 2017b) , is critical to prevent direct or indirect contact transmission of pedv. transport trailers (19/72 herds surveyed; 26.4%) or feed (21/72 herds surveyed; 29.2%) were thought to be the main contaminated fomites (55.6% in total) in us swine farms during the 2013-2017 epidemic (niederwerder and hesse, 2018) . spillover of pedv from pedv-infected farms to others or wildlife should also be controlled through high quarantine protocols that should include no movement of pigs until the diarrhea is terminated and the disease is controlled (niederwerder and hesse, 2018) . indeed, 28 of 287 fecal samples ( use of 0.5% virkon s and 2.06% clorox that reduced the quantity of pedv rna were the best disinfectants for pedv inactivation in feces (bowman et al., 2015b) . super-oxidized water j o u r n a l p r e -p r o o f (ph 6.0) also effectively inactivated pedv at room temperature after 10-90 min contact . washing trailers with a high pressure power washer, followed by disinfection and drying, was effective for their sanitation and decontamination (baker et al., 2018; thomas et al., 2015b) . accelerated hydrogen peroxide or peroxygen-based disinfectants effectively inactivated pedv on contaminated metal (trailer) surfaces at 20 °c after 30 min contact (holtkamp et al., 2017) and at -10 °c after 40-60 min contact (baker et al., 2017) , or at -10 to 4 °c after 10-30 min contact (baker et al., 2018) , respectively. pedv rna was still detectable after disinfection, but no infectious virus was detected. the levels of pedv-specific siga antibody in the colostrum/milk of dams are key to determine the extent of passive immunity of their piglets against pedv infection (langel et al., 2019a; langel et al., 2019b; langel et al., 2016; langel et al., 2020; saif et al., 2012) . the siga antibody is resistant to proteolytic enzymes and is highly efficient at neutralizing pedv in the intestinal mucosa (langel et al., 2016) . in the mammary glands of female swine, however, there is an absence of lymphoid follicles for antigen processing and iga or igg antibody production ( fig. 2a and b) . the pedv-specific siga antibodies in the colostrum and milk are produced by effector b cells or plasmablasts that migrate from the galt of the pedv-infected intestine to the mammary gland (langel et al., 2016; langel et al., 2020; saif et al., 2012) . this is defined as the gut-mammary-siga axis, that was first described by saif and bohl in studies of immunity to tgev [reviewed in (saif et al., 2012) ]. the details were reviewed previously (langel et al., 2016; langel et al., 2020; saif et al., 2012) . the endothelium in the mammary glands contains specialized j o u r n a l p r e -p r o o f endothelial venules that attract and bind to the specific integrins on the surface of the igaplasmablasts, such as α4β7 and chemokine receptor 9 (ccr9) (bourges et al., 2008; jiang et al., 2016; salmon, 2000) . mucosal addressin cellular adhesion molecule 1 (madcam-1) and chemokine ligand 25 expressed in the specialized venules ( fig. 2d and e) interact with α4β7 and ccr9, respectively. extravasated siga antibody-secreting cells reside in the alveoli-supporting fibrous stroma of the mammary glands ( fig. 2a-c) . the number of pedv-specific siga antibodysecreting cells may positively correlate with the amount and density of these endothelial surface molecules in the mammary glands (salmon, 2000) , although the specific role of each surface molecule in the gut-mammary-siga axis needs to be better characterized. immunization of pregnant sows is important in the control of epidemic ped and to reduce the number of deaths of suckling piglets (jung and saif, 2015; langel et al., 2020) . the most common practice used to initiate herd immunity in us pig farms during the 2013-2017 epidemic when no pedv vaccines were available, was use of whole-herd feedback using a load-closeexpose protocol (59/77 herds surveyed; 76.6%), in which a controlled exposure for gilt acclimation (23/77 herds surveyed; 29.9%) was included (niederwerder and hesse, 2018) , whereas 19/77 herds surveyed (24.7%) used vaccination. there is no standard feedback protocol for any type of pig production systems to have the most consistent and highest efficacy (niederwerder and hesse, 2018) . immunization of pregnant sows, or gilts expected to be bred (gilt acclimation), is undertaken by exposure to virulent autogenous virus, such as minced intestines from infected neonatal piglets negative for other infectious agents (jung and saif, 2015; niederwerder and hesse, 2018) . feedback stimulated lactogenic immunity via the gut-mammary-siga axis in sows (saif et j o u r n a l p r e -p r o o f al., 2012), as well as prolonged, passive immune protection of piglets against pedv infection (goede et al., 2015) . in our study, strong protective immunity (100% survival rate of the piglets challenged with pc22a) was induced in gilts inoculated orally once with virulent non-s indel pedv strain pc22a (10 5 pfu/gilt) during mid-gestation (day 57-59) (langel et al., 2019a ). gilts exposed to the virus earlier (day 19-22) or later (day 96-97) in the gestation period showed less protective immunity, as evident by 87.2% or 55.9% survival rates of their piglets, respectively. sows given feedback or orally inoculated with virulent pedv during mid-gestation also discontinued fecal pedv shedding prior to farrowing (langel et al., 2019a; leidenberger et al., 2017) . whether prolonged pedv shedding post-exposure and during farrowing and lactation may cause infection of piglets, remains unclear. however, the most efficient and safest timing for feedback should be mid-gestation. because of the frequent inefficiency or lack of safety, feedback should be replaced by oral immunization with high, consistent doses of live attenuated pedv that is minimally replicative, but immunogenic in the intestine of pigs (song et al., 2007) . pregnant sows orally inoculated with live attenuated pedv (dr13 strain) vaccine twice at 2 and 4 weeks pre-farrowing exhibited higher pedv-specific iga and vn antibody levels in the colostrum compared with the counterpart sows administered the same vaccine im (song et al., 2007) . although the im route has also been used as an administration option using live pedv vaccine (kweon et al., 1999) , to what extent pedvspecific iga memory b cells are induced in sows initially primed via an im route with live pedv vaccine, has not been confirmed. the efficacy of pedv im boost vaccination is dependent on induction of iga memory b cells in initially field exposed or orally primed sows (gillespie et al., j o u r n a l p r e -p r o o f 2018), similar to the success of tgev oral prime/parenteral boost vaccine strategies (saif et al., 2012) . live attenuated strains of pedv have been isolated from the feces of pigs, suggesting that recombinant or mutated strains of pedv might have emerged (gerdts and zakhartchouk, 2017; guo et al., 2016) . for maintaining or boosting herd immunity initiated through feedback or oral live vaccines in farrowing herds, im administration with either live pedv (sato et al., 2018) or inactivated, adjuvanted whole-virus pedv vaccine (gillespie et al., 2018) once or twice at 4-5 or 2 weeks prefarrowing was effective in boosting lactogenic immunity in sows positive for pedv-specific iga memory b cells in galt. many vaccine candidates or products have been tested and developed in the us and worldwide. the details were reviewed previously gerdts and zakhartchouk, 2017) . im live vaccine booster plus feedback was more effective in reducing the number of deaths of suckling piglets, compared with either im live vaccine alone or feedback alone (sato et al., 2018) . prime and boost im vaccination with inactivated pedv provided incomplete protection of piglets against pedv infection (gerdts and zakhartchouk, 2017; niederwerder and hesse, 2018) , because of poor lactogenic immunity induced in their vaccinated dams (gillespie et al., 2018) . despite the safety issue, therefore, feedback alone, or prime and boost vaccination via oral and/or im routes with live pedv have been frequently used for maternal immunization against pedv infection. j o u r n a l p r e -p r o o f pedv remains a threat to the swine industry in the us and worldwide because no effective vaccines are currently available. regardless of the genogroup, pedv can be deadly in pig populations with no immunity or low immunity or health status. the extent of pedv transmission and ped on farms has a reverse relationship with the levels of biosecurity. high biosecurity to control ped should be practiced prior to seasonal or expected epidemics. concurrently, pregnant sows are immunized by either feedback or vaccination. however, the current feedback or vaccination protocols are frequently inefficient or unsafe, due to: 1) no standardized protocol for feedback; 2) poor capacities of current im vaccines (live or killed) to induce lactogenic immunity; 3) antigenic differences of vaccine vs. epidemic strains; and 4) possibility of continuous reinfection by pedv used for feedback within herds. in addition, current live vaccines, which were generated by conventional cell culture-adaptation methods, may revert to virulent pedv or recombine with field pedv strains to generate new strains after they are applied in the field. therefore, better vaccination protocols and vaccine strains (attenuated oral vaccines) are required for prevention and control of ped. pedv-specific iga memory b cells in orally primed sows play a critical role in boosting lactogenic immunity of sows and passive protection of piglets when piglets are exposed to pedv during the suckling period. feedback can be replaced by oral immunization with live attenuated pedv vaccines that are minimally replicative, but immunogenic in the intestine of pregnant sows, thereby inducing pedv-specific iga memory b cells in the galt or other mucosal and systemic lymphoid tissues and lactogenic immunity. lastly, ideal live attenuated vaccines should not revert to virulent pedv or recombine with field pedv strains to generate new virulent pedv strains, which remains an obstacle in vaccine development (hou and wang, 2019) . none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. annamalai, t., saif, l.j., lu, z., jung, k., 2015. age-dependent variation evidence of infectivity of airborne porcine epidemic 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epidemic diarrhea virus infection on tight junction protein gene expression and morphology of the intestinal mucosa in pigs histologic (a and b) and immunohistochemical (c) localization of mammary igasecreting cells, and expression of mucosal addressin cellular adhesion molecule hematoxylin and eosin-stained mammary gland tissue of a gilt at 4 days post-farrowing, showing fully expanded alveoli, with mostly empty lumens. (b) higher magnification of panel a (inset), showing clusters of plasma cells characterized by the typical peripheral nuclei in the abundant cytoplasm in the interlobular stroma. original magnification, 200 (a) and 400 (b). (c) immunohistochemistry (ihc)-stained mammary gland tissue of a gilt at 5 days post-farrowing, showing a moderate number of iga-positive cells we sincerely thank all the authors cited in our review for their efforts and contributions to understand pedv. we apologize to authors whose contributions to pedv research may have been up to pid 21 9.7-10.2 log10 rna copies/ml at pid 4-6 direct contact pid 4 pid 4-14 up to pid 49 6.6-9.3 log10 rna copies/ml at pid 6-21 indirect aerosol contactno clinical signs in 10 pigs (0/10) loose stool in 2/5 pigs (40%) at pid 1-10 or 15low (with mean peak viral rna shedding titer of 5.00 log10 ge/ml), as tested at pid 1-10 or 15mild villous atrophy at pid 9 (mean vh:cd ratio of 5.2 vs. 6.8 in the control)no detection of pedv antigen at pid 9ct (120 th ) 2b (non-s indel) 6 log10 tcid50 6-day 5-6 0 at pid loose stool in some pigs at pid 4-5, at monitored at pid 1-10moderate (with mean peak viral rna shedding titer of approximate 6-7 log10 ge/ml at pid 4-5), as tested at pid 1-10 mild villous atrophy in a pig at pid 3no or little detection of pedv antigen in a pig at pid 3wu et al., 2019 key: cord-295099-ghc85pf5 authors: sun, zehua; yan, lixin; tang, jiansong; qian, qian; lenberg, jerica; zhu, dandan; liu, wan; wu, kao; wang, yilin; lu, shiqiang title: brief introduction of current technologies in isolation of broadly neutralizing hiv-1 antibodies date: 2018-01-02 journal: virus res doi: 10.1016/j.virusres.2017.10.011 sha: doc_id: 295099 cord_uid: ghc85pf5 hiv/aids has become a worldwide pandemic. before an effective hiv-1 vaccine eliciting broadly neutralizing monoclonal antibodies (bnmabs) is fully developed, passive immunization for prevention and treatment of hiv-1 infection may alleviate the burden caused by the pandemic. among hiv-1 infected individuals, about 20% of them generated cross-reactive neutralizing antibodies two to four years after infection, the details of which could provide knowledge for effective vaccine design. recent progress in techniques for isolation of human broadly neutralizing antibodies has facilitated the study of passive immunization. the isolation and characterization of large panels of potent human broadly neutralizing antibodies has revealed new insights into the principles of antibody-mediated neutralization of hiv. in this paper, we review the current effective techniques in broadly neutralizing antibody isolation. antibody responses to neutralize human immunodeficiency virus-1 (hiv-1) are mediated by direct binding to viral spikes, which are trimers composed of glycoproteins gp120 and gp41 (pincus et al., 2017a; pincus et al., 2017b; blair et al., 2007; morris et al., 2000; micoli et al., 2000; pegu et al., 2017; haynes and mascola, 2017; liao et al., 2004; brodine et al., 2003; ward and wilson, 2017; debnath et al., 1994; moore et al., 1993) . gp120 with the attachment of virus particles to the target cells, and the gp41 engages in virus-cell fusion (durham and chen, 2016; desai et al., 2015; dale et al., 2011; bieniasz et al., 1997; rausch et al., 1990) . while successful in-vitro, recombinant gp120 has failed to generate cross neutralizing antibodies in vivo possibly due to the natural conformation of gp120 (prevost, 2017; wang et al., 2016; doores et al., 2015; doores et al., 2010; zhou et al., 2010; wu et al., 1997; watkins et al., 1996; robert-guroff et al., 1992) . monoclonal antibodies were first generated in mice in 1975 using a hybridoma technique (patke et al., 2017; holzlohner and hanack, 2017; bhatia et al., 2016; pogson et al., 2016; abusneina and gauthier, 2016; milstein, 1999) , and were subsequently accepted globally by manufacturers. now, it is well understood that rodent and murine derived antibodies are immunogenic in humans; when binding to antigens within the human body, the complexes can be recognized as immunogenic agents. to solve this problem, human equivalent forms were generated from rodent or murine antibodies, known as humanized antibodies (pardi et al., 2017; wiehe et al., 2017; rothenberg, 2016; bhowmick et al., 2016; hamanoue et al., 2016) . in recent years, new techniques have evolved to isolate human monoclonal antibodies against hiv-1 directly which offer an advantage of reduced immunogenicity. a panel of hiv-1 specific potent broadly neutralizing antibodies has been isolated in recent decades. the first generation antibodies, 2f5, 2g12 and 4e10, were used in combination for passive immunization (morris et al., 2014; mehandru et al., 2004; armbruster et al., 2004) . a second generation antibody, igg b12, was observed to neutralize numerous virus isolates with ic50 in nm level (1 nm is equal to about 0.15ug/ml) for the first time (li et al., 2017; bunnik et al., 2010; ashish et al., 2010; zwick et al., 2003; sun et al., 2015) . the new generation of potent broadly neutralizing antibodies, including vrc01-class antibodies, nih45-46, pg9/16,10e8, bg18, ioma, 35o22, acs202, can neutralize virus isolates ranging from 73% to 98% with ic50 less than 50 μg/ml, and can neutralize 59%-72% of virus isolates with ic50 less than 1 μg/ml (morales et al., 2016; huang et al., 2012; braibant et al., 2013; thenin et al., 2012; ringe et al., 2012; euler et al., 2011; davenport et al., 2011; pancera et al., 2010; wu et al., 2010; scheid, 2015; scheid et al., 2011) . worth noting, broadly neutralizing antibody 10e8 neutralizes 98% of tested viruses with geometric mean ic50 of 0.22 μg/ml. 10e8 binds to the cell-surface envelope as opposed to phospholipids. the binding of 10e8 and mper reveals a highly conserved gp41-hydrophobic residue and a critical arginine or lysine just before the transmembrane region. the breadth and potency of 10e8 demonstrates a conserved site for hiv neutralization and potential target for hiv vaccine design (huang et al., 2012) . the highly potent vrc01-class broadly neutralizing antibodies (bnabs) individually neutralize up to 90% of hiv-1 isolates. vrc01class antibodies are considered a sub group of bnabs and have already been isolated from several hiv-1 infected patients. they each neutralize different virus isolates through the interaction with hiv gp120 cd4 binding site. vrc01-class bnabs are derived from human vh1-2 gene family, which occupies 2% of the human antibody repertoire. structural studies revealed that these vrc01-class abs interact with gp120 cd4 binding site by mimicking the cd4 molecule; specifically the secondary structure formed by heavy chain structure and cdr3 in light chain. additionally, an important deletion in cdrl1 present in many vrc01class abs can facilitate the interaction with the cd4 binding site and avoid conflict with glycan at asn 276 (n276) on loop d (zhou et al., 2010; georgiev et al., 2014; wu et al., 2015; zhou et al., 2013) . nextgeneration sequencing (ngs)-derived sequencing reveals that vrc01 lineage is comprised of at least six distinct heavy chain clades and five light chain clades. this observation suggests that extraordinary variation in antibody immunity may only occur within a few antibody lineages − or even a single lineage (wu et al., 2015) . generation of an unmutated common ancestor of vrc01-class antibodies could be a potential mechanism for opposing hiv-1. after stimulation by virus and co-evolution with the virus, the unmutant common ancestor has ability to mature to bnabs. human antibodies can be elicited by b cells to eliminate "foreign immunogens" directly or indirectly. some antibodies can be produced to neutralize foreign antigens by various mechanisms, while some nonneutralizing antibodies can perform effector functions such as mediating nk cells via antibody-dependent cellular cytotoxicity (adcc) (liao et al., 2004; lin et al., 1998; thomann et al., 2015; ito et al., 2009; mandi et al., 1982) . serum immunoglobulins produced naturally by human immunity have been applied in passive immunotherapy against many infectious diseases since 1990. but this approach suffers a number of problems. not only have these polyclonal immunoglobulins displayed mixed efficacies, but they also pose the potential threat of bloodborne pathogen transmission. in addition, the resource of serum is limited and expensive. these disadvantages limit the application of polyclonal immunotherapy (berry, 2017; stevens et al., 2016; wang et al., 2007; sili et al., 2003; lang et al., 1993) . recent technology developments have made it possible to isolate human antibodies against hiv-1 from both in vivo and in vitro sources. these improved techniques include display methods, epstein-barr virus (ebv) immortalization, classical hybridoma procedures, and single-cell sorting followed by molecular cloning. 2. techniques to make human monoclonal antibodies in the mid-1970s, hybridoma technology was invented for production of mouse monoclonal antibodies with defined antigen specificities and neutralization activity for application in clinical therapies (kelso et al., 2016; hugwil, 2013; tomita and tsumoto, 2011; martin-lopez et al., 2007; hencsey et al., 1996; honda et al., 1990; köhler and milstein, 1975) . hybridomas can be generated by effective fusion of b cells and partner cells followed by screening of individual antibody producing cells. production of monoclonal antibody in individual hybridoma cells can be easily quantified by surface plasmon resonance imaging (stojanovic et al., 2015) . b cell hybridomas can be an important source for screening of monoclonal antibodies. highthroughput screening is used to characterize mouse igg antibodies; including sub-isotypes, binding activity, and neutralizing activities (liu et al., 2015; szafran et al., 2016) . fluorescent antigen can be used to sort antigen binding hybridoma cells from a mixture as opposed to the traditional way of screening using multi-micro well plate screening and limiting dilution. as an extension of this technology, t cell hybridoma was also generated (krishnan et al., 2015) . however, mouse monoclonal antibodies were shown to be problematic in humans due to their immunogenic properties and low effector function. high immunogenicity prevented their application in humans where prolonged dosing was required. differences in sequence and glycosylation pattern of the fc region make rodent mabs poorly effective in mediating effector functions in human (li et al., 2006) . human b cells proved to be difficult to immortalize using hybridoma technique (li et al., 2006; cafri et al., 2013; park et al., 1997) . the primary issue was that mabs produced by human b cell hybridoma cells could react against selfantigens. in addition, the lack of sufficient maturation outside of the germinal centers and low binding affinities of mabs derived by hybridoma, meant that only 1.5% of screened clones typically reacted to the target antigen. due to these problems, antibody humanization was developed as an alternative to generate human format antibodies from rodent derived antibodies (martin and rees, 2016; choi et al., 2015; olimpieri et al., 2015; safdari et al., 2013; tsurushita et al., 2004; ohtomo et al., 1995) . humanized antibodies are produced by methods of genetic engineering and can potentially reduce the immune response against non-human derived antibodies. this is done by combining the variable (v) region binding domain of a rodent antibody with human antibody constant (c) regions. this kind of chimeric antibody retains the binding specificity with less immunogenicity to humans. in some cases, the chimeric antibodies retain the effects in mediating human complement-dependent cytotoxicity (cdc) or adcc. it is also possible to produce a humanized antibody without creating a chimeric intermediate first. once the precise sequences of the desired cdrs are known, these dna sequences can be directly cloned into antibody expression vectors with human antibody "scaffold". a wide variety of human antibody expressing vectors has been developed using this method. for example, pdr12 vector is a human igg1 antibody expressing vector which was originally used for expression of human hiv-1 potent neutralizing antibody igg b12. antibody vrc01 was cloned into a different cloning vector described previously by tiller et al. (tiller et al., 2008) . another method to immortalize human memory b cells is by epstein-barr virus (ebv) mediated transformation (lee et al., 2011; klein, 1996; straub and zubler, 1989; ohashi et al., 2017; mclaughlin et al., 2017; traggiai et al., 2004) . the c3b, ebv-binding receptor positive b cells can be immortalized to generate antibodies for isolation using single cell sorting, or can be cultured with feeder cells for further screening or cloning. the optimized methodology is to activate b cells using toll-like receptor 9 (tlr9) agonist cpg dna before or during ebv infection, which has been used to isolate human monoclonal antibodies against various pathogenic viruses (katsumura et al., 2012; bass and darke, 2004; sun et al., 2017) . it has also been reported that human b cells can be immortalized by transforming a retrovirus encoding anti-apoptotic factor b cell lymphoma 6 (bcl6) and bcl-xl in the presence of interleukin 21 (il-21) and cd40 ligands (schrader et al., 2012; rydstrom et al., 2010; kusam et al., 2009 ). this process can trigger memory b cells to differentiate into antibody secreting cells. the immortalization of human b cells provides a substantial resource of human b cells for the subsequent screening. after culturing for 1-2 weeks, antibodies can be collected and isolated from the supernatant to perform neutralization against different viruses. immortalized b cells can also be sorted by antigen using flow-based technology, and the heavy and light chain genes of antibodies secreted by immortalized b cells can then be cloned. the famous 2g12, 2f5 and 4e10 antibodies are generated by this method (buchacher et al., 1994) . however, due to the low efficiency of ebv-induced b cell transformation, this method has limitation in application. though optimization methods were studied based on ebv-induced b cell transformation, the efficiency still needs a dramatic improve (sun et al., 2017; lu et al., 2016; kwakkenbos et al., 2016) . some reports show that memory b cells can be cultured short-term, and the supernatant can be used directly for screening of neutralization activity in a high throughput neutralization assay (micro-neutralization). a number of famous neutralizing antibodies have been isolated based on these methods including pg9, pg16, pgt121-128 and 10e8 (huang et al., 2012; ringe et al., 2012; walker et al., 2011; walker et al., 2009; falkowska et al., 2014) . hiv-1 potent neutralizing antibodies pg9 and pg16 are classic antibodies isolated by microneutralization. these were originally isolated from 30,000 activated memory b cells from one clade a infected donor screened from approximately 1800 characterized hiv infected donors. the memory b cells were cultured at clonal density for the purpose of cloning antibody heavy and light chain pair from each culture well. pg9 neutralized 127 out of 162 viral isolates and pg16 neutralized 119 out of 162 viruses with a median potency (ic50) less than 0.2 μg/ml. 10e8 is an anti-gp41 antibody isolated from 16,500 memory b cells, sorted and cultured with il-2, il-21, and cd40-ligand expressing feeder cells. 10e8 neutralized 98% of the tested pseudoviruses with an ic50 below 50 μg/ml, and 72% of the tested viruses with an ic50 values below 1 ug/ml. b cell culture and microneutralization can isolate potent broadly neutralizing hiv-1 antibodies. however, the large scale screening of b cells makes this method labor intensive and costly, in addition to low yield; typically ten antigen-specific clones can be isolated out of ten thousands of b cells ( fig. 1a and b) . in current reports, ebv transformation was optimized with efficiency from 0.1%-2% to 7.8%, which enabled the generation of immortalized b cell libraries. however the efficiency still needs to be improved to enable greater b cell survival (sun et al., 2017) . phage display is a library method which permits screening of antibodies from a large recombinant library (falkowska et al., 2014; boots et al., 1997; chan et al., 1996; aghebati-maleki et al., 2017; rahbarnia et al., 2017; finlay et al., 2017) . antibodies can be displayed in the form of either single chain variable fragments (scfv) or antigen-binding fragments (fab). recent reports show that single domain antibody can also be displayed in phage display system (duarte et al., 2016; kazemi-lomedasht et al., 2016; li et al., 2016; rotman et al., 2015; tang et al., 2013) . many antibodies against different viruses including rabies virus, severe acute respiratory syndrome (sars) virus, ebola virus, yellow fever virus, hepatitis c virus, dengue virus, hepatitis a virus, influenza virus, and hiv, have been successfully isolated using this technology. the principle for phage display technique in isolating antibodies is to construct a phage display recombinant antibody library first. recombinant antibody library can increase the diversity of antibodies in b cell repertoires, which promotes the screening of antibodies with novel properties (graus et al., 1998) . antibody libraries are in general constructed by randomly assembling antibody heavy and light chain variable region, and through gene shuffling of the heavy and light chain to further increase the diversity. in summary, phage display creates greater diversity of antibodies and provides rapid and high throughput way of screening. m43 and m44 are hiv-1 cross-reactive human monoclonal antibodies isolated from a recombinant phage display library by competitive antigen panning (zhang et al., 2012; zhang et al., 2008; zhang et al., 2006; zhang et al., 2004a; zhang et al., 2004b) . m44 is a gp41 specific cross-reactive antibody, and m43 can recognize both gp120 and gp41. b12 is another potent neutralizing antibody isolated by phage display from recombinant antibody libraries, and it is considered one of the most potent neutralizing antibodies isolated by phage display, as it can neutralize about 40% of known hiv-1 isolates (li et al., 2017; mantis et al., 2007; haussner et al., 2017; wu et al., 2009 ). m43, m44 and b12 can be considered standard antibodies isolated by phage display; however the potencies are much lower than antibodies isolated by micro neutralization, such as pg9/16 and/or 10e8. phage display selects antibodies in vitro, and antibodies expressed in phage system may not necessarily represent those in mammalian cells due to the difference in protein folding and post transcriptional modifications. it is difficult to draw direct conclusions and gain an understanding of the natural occurrence of antibody heavy and light chain selection based on phage display alone (zhang et al., 2003) . to avoid these limitations, other display methods have been developed to display antibodies on the surface of yeast or mammalian cells, which can be sorted by flow cytometry according to antigen specificity. this is known as yeast surface display, which has become a powerful engineering tool for displaying recombinant proteins on the surface of saccharomyces cerevisiae via genetic fusion (traxlmayr and shusta, 2017; mei et al., 2017; andreu and del olmo, 2017; sheehan and marasco, 2015; gera et al., 2013; boder et al., 2012) . yeast surface display is a eukaryotic expression system with the capability to induce post translational modifications on recombinant antibodies. each yeast cell expresses fusion recombinant proteins which allows for the application of cell sorting for a specific antigen. yeast display system was not only reported used for isolation of antibodies, but also reported to be used in displaying antigens and other recombinant proteins (srivastava et al., 2013; guo et al., 2015) . there is a panel of yeast display antigen library which has been constructed, including yeast display h1n1 antigen epitope library, and yeast display siv antigen epitope library for epitope mapping. hiv-1 specific antibodies isolated by display techniques are less potent than those isolated by micro neutralization or single b cell sorting and cloning. however in recent studies, phage/yeast display showed great potentials in isolating single domain antibodies from recombinant libraries. single-domain antibodies are considered a separate class of antibodies composed of antibody fragments consisting of a single monomeric antibody domain. single-domain antibodies allow a broad range of applications due to their small molecular mass and size, efficient production, and high affinity. single domain antibodies can be labeled using fluorescent molecules for diagnostic purpose or biotechnological usage. with the conjunction of drug or toxin, single domain antibodies can also be used for therapeutic application. the most well-studied single domain antibodies are antibody heavy-chain variable domains. the dna sequence will be optimized in order to improve the stability. in addition to heavy-chain variable domains, there are other types of single domain antibodies which have been studied, including ch2 antibodies, and light-chain variable domain antibodies, and some other forms of nanobodies (duarte et al., 2016; li et al., 2016; gong et al., 2016; louis et al., 2014; lulf et al., 2014; bouchet et al., 2012; bouchet et al., 2011; boudet et al., 1995) . mammalian cell display is a powerful method for the isolation of antibodies in scfv format or full length igg with high affinity. it has been shown that single chain antibodies can be displayed on the surface of human hek-293t cells and used for affinity maturation (soga et al., 2015; tomimatsu et al., 2013; beerli et al., 2008) . mammalian cell expression system ensures that all of the cellular components will be involved in process of antibody synthesis. isolation of human antibodies by mammalian cell display can benefit from the advantages of mammalian cell expression system.mammalian cell display relies on the transfection of antibody expression vectors. in comparison with the yeast display system, mammalian cell expression system allows multiple recombinant protein/antibody delivering vectors in one cell. thus a further modification, enrichment, and single clone isolation will be needed for panning purposes (fig. 1c ). an important advance in antibody screening and cloning technology is the development of single cell sorting and single cell reverse transcription pcr (ouisse et al., 2017; evans et al., 2015; battye et al., 2000) . human single b cells with different antigen specificities can be sorted by flow cytometer directly, and the original vh and vl pairing of the antibody from single human b cells can be amplified in a high efficient way, with the requirement of relatively few cell numbers. this methodology is quite efficient in obtaining antibody heavy and light chain from extremely rare and highly discrete b cell subpopulations (fig. 1d) . memory b cells and plasma cells are ideal cell types for the purpose of monoclonal antibody cloning due to the specificity of the cell types. this method is always combined with single cell cloning after flow cytometry based single cell sorting to sort the antigen specific b cells, thus accurate probe design for sorting is essential. the gp120 or gp41 proteins are not ideal probes, because they are reactive with many hiv-1 antibodies, including non-neutralizing but strong binding antibodies (wu et al., 2010; wu et al., 2015; zhou et al., 2015) . one successful example of probe design in fishing potent broadly neutralizing hiv-1 monoclonal antibodies is the agents' pair of rsc3 and δrsc3. rsc3 and δrsc3 are computer-assisted designed for sorting of individual b cells expressing cd4bs antibodies. the resurfaced stabilized core 3 (rsc3) is a functional structure core of the cd4 binding site with loop deletion. rsc3 preserved the antigenic structure of the cd4 binding site and eliminated other antigenic regions by substitution with simian immunodeficiency virus (siv) or non-hiv-1 residues. δrsc3 contains one amino acid deletion (at position 371) in rsc3. this single amino acid mutation can knock out the function of cd4 binding site. cd4 binding site antibodies like vrc01 and b12 can bind efficiently with rsc3 but fail to bind with δrsc3. potent broadly neutralizing antibody vrc01 is isolated through the single b cell positive sorting by rsc3 and negative sorting by δrsc3. vrc01 neutralized up to 90% of the tested pseudoviruses with an ic50 below 50 μg/ml, and 70% of the tested viruses with an ic50 values below 1 ug/ml (wu et al., 2010; wu et al., 2015; mccoy and burton, 2017) . bg18, ioma are another two successful bnmabs isolated by reasonable probe design and single cell sorting/cloning (gristick et al., 2016; freund et al., 2017) . n-glycans on the trimeric envelope glycoprotein (env) can be accommodated by broadly neutralizing antibodies. an engineered crystal structures of the hiv-1 env trimer with an exposed native glycan shield of high-mannose and complex-type nz. sun et al. virus research 243 (2018) 75-82 glycans was used to fishing out the bnmabs. ioma, a new cd4-binding site (cd4bs) antibody was thus defined. the heavy chain of ioma derives from vh1-2*02, which is considered the germline gene of vrc01class bnabs, but its light chain lacks the short cdrl3 which defines vrc01-class bnabs. bg18, bg1, and nc37 are three bnabs isolated by single igg+ b cell sorting by using four different fishing agents in four separate sorts: 2cc core, gp140yu2, a 1:1 mixture of gp14092ug37.8 (clade a) + gp140cza79012 (clade c), and bg505 sosip.664 (sok et al., 2014; dey et al., 2009; yang et al., 2000; sanders et al., 2013; kovacs et al., 2012) . antibody bg18 (vh4-4 and vl3-25) targeted the glycan-v3 portion of envelope, can neutralize 64% of viruses in a 118virus panel tested with a geometric mean ic50 of 0.03 mg/ml. antibodies nc37 (vh1-46/1-2, vk3-20) and bg1 showed geometric mean ic50 of 0.3 and 0.67 mg/ml respectively. nc37 can bind to a quaternary trimer epitope, the core of which overlaps with the cd4bs, and bg1 binds to the v1v2 region. antigen baiting and fishing has proven quite successful in isolating hiv-specific human monoclonal igg expressing b cells and igm expressing b cells from infected donors using cleverly engineered antigen probes. flow cytometry based single cell sorting and cloning can also be applied in understanding the natural selection of antibodies by cloning of antibodies in b cells from different infection stage from one donor or b cells in the same stage from different donors. 454 deep sequencing allows for the study of the maturation of a specific antibody population and corresponding b cell subpopulations by the stimulation of virus isolates in progressingstages of infection (morris et al., 2017; banerjee et al., 2017; carter et al., 2015; karlsson hedestam et al., 2017; soto et al., 2016; stramer et al., 2016; zhu et al., 2013; zhu et al., 2012) . the difficulty in eliciting bnabs in immunized animals has been concluded (walker et al., 2011; sanders et al., 2013; sok et al., 2017) . the enormous antigenic diversity of the envelope glycoprotein and the n-linked glycan shield are considered as two major points. recent study shows that success of rapid elicitation of broadly neutralizing antibodies to hiv-1 by immunization in cows. jr-fl gp120 and bg505 sosip are immunized to cows, and strong immune response is observed. and broadly neutralizing antibodies were isolated from peripheral blood mononuclear cells (pbmcs) by sorting with biotinylated bg505 sosip. the broadly neutralizing antibody nc-cow1 displayed a 72% neutralization breadth with a potent median ic50 of 0.028 μg /ml on a 117-virus panel tested.this study gives lights in investigating the generation of antibodies against pathogens that have evolved to avoid human antibody responses in animals. in recent decades, the technology advances have allowed for specific human antibodies to be isolated directly from human b cells and/ or from recombinant libraries. these isolated human monoclonal antibodies can be used in therapy because of their safety, efficiency, specificity and tolerance in humans; thus are a powerful tool in fighting against pathogens. among the developed antibody isolation methods over the last decade, it is difficult to highlight one as the sole preferred method. different methodology has different advantages. display techniques basing on recombinant library can be used for isolating single domain antibodies with enlarged library size. but this technique cannot provide us the information of the original pair of heavy chain and light chain of a specific antibody. single b cell sorting and culturing facilitate the rapid cloning of potent neutralizing antibodies with the requirement of large labor cost. reasonable design of probes and the development of native-like stabilized trimers have greatly improved efficiency in screening of functional envelope-binding b cells. thus it is hard to conclude the best way to generate novel bnabs. combined methods can be used in screening and isolating potent neutralizing antibodies in order to maximize the advantages of different methods. meanwhile, the development of novel methodology of broadly neutralizing antibodies isolation and screening are encouraged to broaden our knowledge and enlarge the recourses of antibody classes. a purposed method basing on single cell sorting and viral neutralization instead of fishing agents should provide a direct and efficient way to minimum the workload. in addition to passive treatment and presentation, another important purpose of isolation and characterization of potent broadly neutralizing antibodies is the functional study of neutralizing epitopes which provides key knowledge for efficient vaccine design. human broadly neutralizing antibodies serve as a potential source of discovering neutralizing epitopes that can be targeted to fight against many infectious diseases and can thus facilitate the design of the vaccines. antibody structure studies can provide new insights into the mechanism of recognition of immune evasion epitopes at the atomic level, and how antibodies mature to capture the virus mutant (morales et al., 2016; wibmer et al., 2017; rujas et al., 2016) . however in recent reports, few immunogen candidates were reported that can elicit potent broadly neutralizing antibodies in several immunization studies. native-like hiv-1 envelope trimers bg505 sosip.664 and b41 sosip.664 are reported to induce neutralizing antibodies against the tier-2 virus in rabbits, even though the potency is much weaker in comparison to the broadly neutralizing antibodies isolated from human long term nonprogressors (sanders et al., 2015; bradley et al., 2016) .pre-stimulation of b cells by heterogenous epitopes or antigens showed improved stimulation of b cells in generating potent neutralizing antibodies (yang et al., 2015) . it has also been reported that pgt121-class germlinetargeting stabilized trimer immunogens primed pgt121-like responses in pgt121 inferred-germline knock in mice, highlighting the hypothesis of prime-boosting and sequential immunization strategy (steichen et al., 2016) . all these observations highlight the neutralizing antibodies' role in hiv-1 prevention. altogether, understanding antibody generation and function mechanisms can help to guide efficient vaccine design and produce effective therapy. not applicable. not applicable. not applicable. no. not applicable. the authors claimed no interest. ammonium ions improve the survival of glutaminestarved hybridoma cells isolation and characterization of anti ror1 single chain fragment variable antibodies using phage display technique development of a new yeast surface display system based on spi1 as an anchor protein passive immunization with the anti-hiv-1 human monoclonal antibody (hmab) 4e10 and the hmab combination 4e10/2f5/2g12 global structure of hiv-1 neutralizing antibody igg1 b12 is asymmetric evaluation of a novel multi-immunogen vaccine strategy for targeting 4e10/10e8 neutralizing epitopes on hiv-1 gp41 membrane proximal external region improved efficiency of ebv transformation of b-lymphocytes single cell sorting and cloning isolation of human monoclonal antibodies by mammalian cell display antibody immunoprophylaxis and immunotherapy for influenza virus infection: utilization of monoclonal or polyclonal antibodies? hybridoma cell-culture and glycan profile dataset at various bioreactor conditions humanized monoclonal antibody alemtuzumab treatment in transplant hiv-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the ccr-5 co-receptor identification and characterization of uk-201844: a novel inhibitor that interferes with human immunodeficiency virus type 1 gp160 processing engineering antibodies by yeast display anti-human immunodeficiency virus type 1 human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries inhibition of the nef regulatory protein of hiv-1 by a singledomain antibody single-domain antibody-sh3 fusions for efficient neutralization of hiv-1 nef functions single peptide and anti-idiotype based immunizations can broaden the antibody response against the variable v3 domain of hiv-1 in mice structural constraints of vaccine-induced tier-2 autologous hiv neutralizing antibodies targeting the receptor-binding site cross-group neutralization of hiv-1 and evidence for conservation of the pg9/pg16 epitopes within divergent groups diverse hiv-1 subtypes and clinical: laboratory and behavioral factors in a recently infected us military cohort generation of human monoclonal antibodies against hiv-1 proteins; electrofusion and epstein-barr virus transformation for peripheral blood lymphocyte immortalization emergence of monoclonal antibody b12-resistant human immunodeficiency virus type 1 variants during natural infection in the absence of humoral or cellular immune pressure production of lacz inducible t cell hybridoma specific for human and mouse hiv-1 neutralizing antibody response and viral genetic diversity characterized with next generation sequencing human recombinant antibodies specific for hepatitis c virus core and envelope e2 peptides from an immune phage display library antibody humanization by structure-based computational protein design cell-to-cell transfer of hiv-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion binding interactions between soluble hiv envelope glycoproteins and quaternary-structure-specific monoclonal antibodies pg9 and pg16 three-dimensional structure-activity analysis of a series of porphyrin derivatives with anti-hiv-1 activity targeted to the v3 loop of the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 fluorescent protein-tagged vpr dissociates from hiv-1 core after viral fusion and rapidly enters the cell nucleus structure-based stabilization of hiv-1 gp120 enhances humoral immune responses to the induced co-receptor binding site a nonself sugar mimic of the hiv glycan shield shows enhanced antigenicity two classes of broadly neutralizing antibodies within a single lineage directed to the high-mannose patch of hiv envelope generation of immunity against pathogens via single-domain antibody-antigen constructs measuring t cell-to-t cell hiv-1 transfer, viral fusion, and infection using flow cytometry activity of broadly neutralizing antibodies: including pg9, pg16, and vrc01, against recently transmitted subtype b hiv-1 variants from early and late in the epidemic assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging broadly neutralizing hiv antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers phage display: a powerful technology for the generation of highspecificity affinity reagents from alternative immune sources coexistence of potent hiv-1 broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller antibodies vrc01 and 10e8 neutralize hiv-1 with high breadth and potency even with ig-framework regions substantially reverted to germline protein selection using yeast surface display specific determination of influenza h7n2 virus based on biotinylated single-domain antibody from a phage-displayed library selection of recombinant anti-hud fab fragments from a phage display antibody library of a lung cancer patient with paraneoplastic encephalomyelitis natively glycosylated hiv-1 env structure reveals new mode for antibody recognition of the cd4-binding site simian immunodeficiency virus infection evades vaccine-elicited antibody responses to v2 region successful treatment with humanized anti-interleukin-6 receptor antibody (tocilizumab) in a case of aa amyloidosis complicated by familial mediterranean fever 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of ebv in diffuse large b-cell lymphoma (dlbcl): comparative analysis between ebv-positive and −negative dlbcl with ebv-positive bystander cells humanization of mouse ons-m21 antibody with the aid of hybrid variable regions tabhu: tools for antibody humanization antigen-specific single b cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies crystal structure of pg16 and chimeric dissection with somatically related pg9: structure-function analysis of two quaternary-specific antibodies that effectively neutralize hiv-1 administration of nucleoside-modified mrna encoding broadly neutralizing antibody protects humanized mice from hiv-1 challenge generation and characterization of a novel fusion partner cell line for the production of human macrophage hybridoma bisfabs: tools for rapidly screening hybridoma iggs for their activities as bispecific antibodies use of broadly neutralizing antibodies for hiv-1 prevention identification of human anti-hiv gp160 monoclonal antibodies that make effective immunotoxins design and in vivo characterization of immunoconjugates targeting hiv gp160 immunogenomic engineering of a plug-and-(dis)play hybridoma platform influence of the envelope gp120 phe 43 cavity on hiv-1 sensitivity to adcc responses evolution of phage display technology: from discovery to application peptides derived from the cdr3-homologous domain of the cd4 molecule are specific inhibitors of hiv-1 and siv infection, virus-induced cell fusion, and postinfection viral transmission in vitro. implications for the design of small peptide anti-hiv therapeutic agents subtle alteration of residues including nlinked glycans in v2 loop modulate hiv-1 neutralization by pg9 and pg16 monoclonal antibodies cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates fusion of higg1-fc to 111in-anti-amyloid single domain antibody fragment vhh-pa2h prolongs blood residential time in app/ps1 mice but does not increase brain uptake structural basis for broad neutralization of hiv-1 through the molecular recognition of 10e8 helical epitope at the membrane interface cd40 is a potential marker of favorable prognosis in patients with diffuse large b-cell lymphoma treated with immunochemotherapy antibody humanization methods − a review and update a next-generation cleaved, soluble hiv-1 env trimer, bg505 sosip.664 gp140, expresses multiple epitopes for broadly neutralizing but not nonneutralizing antibodies hiv-1 vaccines: hiv-1 neutralizing antibodies induced by native-like envelope trimers sequence and structural convergence of broad and potent hiv antibodies that mimic cd4 binding hiv-specific b cell response in patients with broadly neutralizing serum activity global gene expression changes of in vitro stimulated human transformed germinal centre b cells as surrogate for oncogenic pathway activation in individual aggressive b cell lymphomas phage and yeast display large-scale expansion of dendritic cell-primed polyclonal human cytotoxic t-lymphocyte lines using lymphoblastoid cell lines for adoptive immunotherapy mammalian cell surface display as a novel method for developing engineered lectins with novel characteristics recombinant hiv envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex rapid elicitation of broadly neutralizing antibodies to hiv by immunization in cows developmental pathway of the mper-directed hiv-1-neutralizing antibody 10e8 identification of dominant antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin antigen of pandemic h1n1 influenza virus hiv vaccine design to target germline precursors of glycandependent broadly neutralizing antibodies preserved antiviral adaptive immunity following polyclonal antibody immunotherapy for severe murine influenza infection quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging two human immunodeficiency virus type 2 cases in us blood donors including serologic, molecular, and genomic characterization of an epidemiologically unusual case immortalization of ebv-infected b cells is not influenced by exogenous signals acting on b cell proliferation. effects of mutant el-4 thymoma cells and transforming growth factor-beta reconstitution and characterization of antibody repertoires of hiv-1-infected elite neutralizers isolation and characterization of hiv-1 envelope glycoprotein specific b cell from immortalized human naive b cell library use of hca in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns a human single-domain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cell surface naturally occurring substitutions of conserved residues in human immunodeficiency virus type 1 variants of different clades are involved in pg9 and pg16 resistance to neutralization in vitro glycoengineering of igg1 and its effect on fc receptor binding and adcc activity efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning a rapid screening and production method using a novel mammalian cell display to isolate human monoclonal antibodies hybridoma technologies for antibody production an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus directed evolution of protein thermal stability using yeast surface display humanization of a chicken anti-il-12 monoclonal antibody broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target broad neutralization coverage of hiv by multiple highly potent antibodies optimizing adoptive polyclonal t cell immunotherapy of lymphomas: using a chimeric t cell receptor possessing cd28 and cd137 costimulatory domains hiv-1 gp140 epitope recognition is influenced by immunoglobulin dh gene segment sequence the hiv-1 envelope glycoprotein structure: nailing down a moving target resistance of human immunodeficiency virus type 1 to neutralization by natural antisera occurs through single amino acid substitutions that cause changes in antibody binding at multiple sites structure and recognition of a novel hiv-1 gp120-gp41 interface antibody that caused mper exposure through viral escape immunodominance of antibody recognition of the hiv envelope v2 region in ig-humanized mice interaction of chemokine receptor ccr5 with its ligands: multiple domains for hiv-1 gp120 binding and a single domain for chemokine binding mechanism of human immunodeficiency virus type 1 resistance to monoclonal antibody b12 that effectively targets the site of cd4 attachment rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1 maturation and diversity of the vrc01-antibody lineage over 15 years of chronic hiv-1 infection characterization of stable: soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins identification of non-hiv immunogens that bind to germline b12 predecessors and prime for elicitation of cross-clade neutralizing hiv-1 antibodies broadly cross-reactive hiv neutralizing human monoclonal antibody fab selected by sequential antigen panning of a phage display library identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody identification of a novel cd4i human monoclonal antibody fab that neutralizes hiv-1 primary isolates from different clades selection of a novel gp41-specific hiv-1 neutralizing human antibody by competitive antigen panning cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens identification and characterization of a broadly cross-reactive hiv-1 human monoclonal antibody that binds to both gp120 and gp41 structural basis for broad and potent neutralization of hiv-1 by antibody vrc01 multidonor analysis reveals structural elements: genetic determinants, and maturation pathway for hiv-1 neutralization by vrc01-class antibodies structural repertoire of hiv-1-neutralizing antibodies targeting the cd4 supersite in 14 donors somatic populations of pgt135-137 hiv-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatics de novo identification of vrc01 class hiv-1-neutralizing antibodies by next-generation sequencing of b-cell transcripts a novel human antibody against human immunodeficiency virus type 1 gp120 is v1: v2, and v3 loop dependent and helps delimit the epitope of the broadly neutralizing antibody immunoglobulin g1 b12 we wish to thank dr. mei-yun zhang, dr. zhiwei chen (the university of hong kong), and dr. paul zhou (institute pasteur of shanghai cas) for helpful discussion.z. sun et al. virus research 243 (2018) 75-82 key: cord-279849-zzkliu76 authors: dapalma, t.; doonan, b.p.; trager, n.m.; kasman, l.m. title: a systematic approach to virus–virus interactions date: 2010-01-20 journal: virus res doi: 10.1016/j.virusres.2010.01.002 sha: doc_id: 279849 cord_uid: zzkliu76 a virus–virus interaction is a measurable difference in the course of infection of one virus as a result of a concurrent or prior infection by a different species or strain of virus. many such interactions have been discovered by chance, yet they have rarely been studied systematically. increasing evidence suggests that virus–virus interactions are common and may be critical to understanding viral pathogenesis in natural hosts. in this review we propose a system for classifying virus–virus interactions by organizing them into three main categories: (1) direct interactions of viral genes or gene products, (2) indirect interactions that result from alterations in the host environment, and (3) immunological interactions. we have so far identified 15 subtypes of interaction and assigned each to one of these categories. it is anticipated that this framework will provide for a more systematic approach to investigating virus–virus interactions, both at the cellular and organismal levels. by design, viral infections in the laboratory almost always occur in the absence of other viruses. while this may be a logical starting point for virus research, viral infections in nature rarely occur in isolation. polymerase chain reaction (pcr)-based techniques have revealed that persistent viral infections are present in all domains of life. archaea can be hosts to several types of phage viruses (snyder et al., 2003) . most environmental and commensal bacterial isolates are infected with one or more phage viruses (ackermann and kropinski, 2007) . yeasts, including endomyces magnusii (pospisek et al., 1994) and even laboratory strains of saccharomyces cerevisiae harbor multiple types of endogenous rna viruses (fujimura and esteban, 2007) . filamentous fungi are similarly affected (nogawa et al., 1993) . phytoplankton are commonly infected by dsdna viruses (bellec et al., 2010) , while both wild and domesticated vascular plants have been shown to carry numerous persistent viral infections, including badnaviruses (kenyon et al., 2008) . finally, genomic sequencing of animals has shown that chromosomes of both invertebrates and vertebrates are permeated with genetic sequences that resemble integrated retroviruses, in various states of viability (gifford and tristem, 2003) . approximately 8% of human genomic dna, for example, is derived from retroviral genomes (tristem, 2000) . however, persistent infections in animals are not limited to retroviruses. the picorna-like nora virus has been detected in all drosophila melanogaster strains tested, both laboratory and wild caught (habayeb et al., 2006) , and life-long herpesvirus infections are well known in humans (chen and hudnall, 2006; pancharoen et al., 2001) as well as marine bivalves, fish, reptiles, birds, and other mammals from mice to elephants (vandevanter et al., 1996) . therefore in any individual, persistent, sometimes congenital, often asymptomatic viral infections provide a background onto which all future viral infections are superimposed. given the now significant evidence for ubiquitous endogenous viral infections in all domains of living things, virus coinfections would seem to be the rule rather than the exception in nature, if not the laboratory. yet, they have rarely been systematically studied for possible effects of one virus on the other, with most documented interactions having been discovered accidently. it is certainly true that coinfection may not result in interactions for all virus species. however, because many viruses induce profound changes in their host, it seems likely that virus-virus interactions are common and may be critical to understanding viral pathogenesis and evolution. therefore, in this review we identify known and potential types of virus-virus interactions (vvis) and organize them into three categories: (1) direct interactions of viral genes or gene products, (2) indirect interactions that result from alterations in the host environment, and (3) a subset of indirect interactions called immunological interactions, unique to organisms equipped with an adaptive immune system. we have so far identified fifteen subtypes of vvi and assigned each to one of the three major categories (table 1) . this framework provides a systematic approach to investigating vvi, both at the cellular and organismal levels. as a first step, it is necessary to define vvi. we define a vvi as a measurable difference in the course of infection of one virus as a result of a concurrent or prior infection by a different species or of course the simple case of a prior viral infection conferring protective immunity against future infections with an immunologically identical virus is well known. but this interaction follows directly from the role of the adaptive immune system, and is therefore a rather uninteresting sort of interaction. a trivial case also exists with virus-induced generalized immunosuppression, as seen with human immunodeficiency virus (hiv)/aids. generalized immunosuppression, regardless of the cause, results in increased replication and pathology by some viruses that have a benign disease course in immunocompetent persons. however, this effect is not specific to viral pathogens. therefore, the discussion of vvi involving virus-induced immunosuppression will be limited here to those in which there is evidence of other viruses impacting the course of infection of the immunosuppressive virus, or evidence of direct interaction of proteins or nucleic acids of the immunosuppressive virus with proteins or nucleic acids of a coinfecting virus (lisco et al., 2009) . these uninformative examples aside, we have collected reports of vvi involving unexpected direct, environmental, and immunological interactions and used them to create what is intended to be a growing online database of these important phenomena. in the sections below, the current subtypes of vvi are each described and illustrated with a few examples. some categories have many more currently known examples than those described here, which are included in the online database. we do not mean to represent either the examples below nor the online list as a complete collection. instead, our aim is to provide the framework to launch a publicly available collection of vvi which allows continuous updates by and for the scientific community. we define a direct vvi as an occurrence in which nucleic acids or proteins of one virus physically interact with the genes or gene products of a coinfecting virus. this definition encompasses six different subtypes of interaction: helper viruses, pseudotype viruses, superinfection exclusion, genomic recombination, embedded viruses, and heterologous transactivation. direct interactions require coinfection of the same cell to take place, but the infections need not take place close in time. in several cases of documented direct interactions described below, cells latently infected with one virus may be infected by the second virus years, or even millenia later if the initial viral genome is incorporated into the germline of the host. a helper-dependent virus is any virus that is replication defective on its own, and therefore requires the gene products of another virus to produce infectious progeny. engineered recombinant helper virus/helper-dependent virus pairs have been developed as safe viral vector technologies (dorigo et al., 2004; marconi et al., 2008) , but several natural pairs of such viruses are also documented. one of the first helper-dependent viruses described was bacteriophage p4, a bacteria-infecting virus that is able to replicate its own genome, but requires the presence of a coinfecting bacteriophage, such as p2, to provide capsid components and cell lysis (shore et al., 1978; six and klug, 1973) . in plants, the carrot mottle virus of the umbravirus genus has been shown to be dependent upon viruses of the luteoviridae family for encapsidation and transmission by aphids (waterhouse and murant, 2008) . a mammalian helper-dependent virus is adeno-associated virus (aav), a replication-defective parvovirus that generally requires a host cell that is coinfected with an adenovirus or herpesvirus in order for virions to be produced and escape the host cell (buller et al., 1981; goncalves, 2005) . since the original description of aav, it has been discovered that genotoxic stress and other factors may also make a host cell permissive for aav progeny production, indicating that aav is not entirely replication defective (meyers et al., 2000; yalkinoglu et al., 1988) . a more clearly defined case is that of hepatitis d virus (hdv), a human pathogen. originally, discovered in the 1970s as a subtype of hepatitis b virus (hbv), further studies led to the understanding that hdv only occurred in hbv infected individuals. although the small viroid-like hdv is able to reproduce its own rna and ribonucleoprotein capsids, it requires the hbv membrane glycoprotein, hepatitis b surface antigen (hbsag) in order to associate with cell membranes and bud as infectious particles (rizzetto, 2009) . therefore, naturally occurring helper-dependent viruses appear to be dominated by non-enveloped viruses that can replicate their genome autonomously and only require a helper virus for packaging and/or release. it should be noted that the interaction between a helperdependent virus and its respective helper virus is not necessarily unidirectional. both aav and p4 significantly inhibit the replication of their helper viruses (barrett et al., 1973; timpe et al., 2006) . in contrast, hdv coinfection with hbv results in increased activity of both viruses, often resulting in a more severe clinical course than hbv alone (rizzetto, 2009 ). virus pseudotyping occurs when two species of virus coinfect a host cell and progeny virions are produced that contain nucleic acid of one parental virus and some structural proteins of the other parental virus. this phenomenon, also called phenotypic mixing, may involve capsid proteins, or in the case of enveloped viruses, only membrane glycoproteins (zavada, 1976) . various bacteriophage, plant viruses, and animal viruses have all been observed to produce phenotypically mixed progeny from coinfections (zavada, 1976) . some coinfections result in pseudotyped virions from both parental genomes, while other interactions result in pseudotyped virions of only one type (certo et al., 1998) . for example coinfection of human syncytiotrophoblasts with human cytomegalovirus and human t-cell leukemia-lymphoma virus (htlv-i) results in htlv-1 capsids within cmv envelopes, but not the reverse (toth et al., 1995) . pseudotyped viruses are distinct from helper-dependent virus interactions since neither virus requires components of the other to complete its replication cycle. envelope glycoproteins are the main receptor binding proteins of most enveloped viruses, and therefore pseudotyped virions often have an expanded host range, able to infect the targets of both parental viruses. two herpesviruses have been shown to produce pseudotyped human immunodeficiency virus (hiv) or htlv-1 virions when coinfected with these retroviruses (heng et al., 1994; toth et al., 1995) . in addition to a hybrid virion morphology, these pseudotyped retroviruses possess an expanded host cell range: herpes-pseudotyped hiv is capable of infecting keratinocytes (heng et al., 1994) . this feature of pseudotyped viruses has been used to advantage in the production of recombinant viral vectors, to expand the range of targets they are able to infect (funke et al., 2009; li et al., 2009; wu et al., 2009 ). a third type of direct vvi is viral superinfection exclusion, which occurs when a primary viral infection induces resistance to subsequent infections by similar viruses. superinfection exclusion is known to occur among bacteriophage, retroviruses, hepadnaviruses, arboviruses, and plant viruses (brindley et al., 2008; geib et al., 2003; mcallister and barrett, 1977; nethe et al., 2005; saumet and lecellier, 2006) . mechanisms of exclusion are diverse and have not been determined in all cases, but all mechanisms described so far depend on direct interaction of products of the primary infection with the secondary infecting virus. for example, the cytoplasmic accumulation of borna disease virus (bdv) nucleocapsid components prevents a subsequent infection of a different bdv strain or arbovirus type through interference with the polymerase of the secondary virus, inhibiting early viral multiplication steps (geib et al., 2003) . the sim protein of bacteriophage p1 appears to block injection of nucleic acid by superinfecting phage at the cell membrane (kliem and dreiseikelmann, 1989) , and the equine infectious anemia virus secretes a soluble protein that masks its cell surface receptor, blocking binding of subsequent viruses (brindley et al., 2008) . in plants, interaction of heterologous viral messenger rna molecules which contain sequence homology induces destruction of both messages by the rna silencing mechanism and inhibition of replication (saumet and lecellier, 2006) . although most examples of superinfection exclusion deal primarily with different strains of the same virus, and inhibition of superinfection is not absolute, better understanding of this vvi has the potential to impact future antiviral development (federico et al., 1996) . coinfections with two or more strains of the same virus species in the same host cell can result in progeny virions that are genetic recombinants of the parental viruses. this phenomenon is arguably the vvi with the most serious consequences for human health. influenza virus coinfections, by virtue of the virus's segmented genome, readily produce recombinant progeny (gardner and shortridge, 1979; nelson et al., 2008) . worldwide surveillance networks monitor the appearance of recombinant influenza viruses as the dramatic antigenic shift that results often produces a virus to which few people have immunity, increasing the risk of a pandemic (nuzzo and lam, 2006) . however, due to the relatively short course of influenza infections, coinfections are relatively uncommon (nelson et al., 2008) . human immunodeficiency virus (hiv) in contrast, is a life-long infection and both coinfections (secondary infection prior to seroconversion) and superinfections are well documented. hiv genomic recombination has been shown to facilitate immune escape (streeck et al., 2008) , evolution of replicationdefective hiv variants (iwabu et al., 2008) , and the spread of drug resistance (burke, 1997) , all of which complicate hiv control. life-long herpesvirus infections also result in homologous recombination between strains in vivo, with similar effects on immune control and drug resistance (chou, 1989; haberland et al., 1999; poole et al., 1999) . more recently, recombination between attenuated poliovirus vaccine strains and virulent wild enterovirus strains has led to regeneration of virulent polioviruses and cases of poliolike paralysis in regions targeted for poliovirus eradication (arita et al., 2005; rakoto-andrianarivelo et al., 2008) . although not as carefully monitored as human pathogens, recombination-driven altered virulence of viral pathogens of crop plants has also been reported (ogawa et al., 2008) . whereas genomic recombination involves nucleic acid transfer between viruses with significant sequence homology and similar if not identical genomic organization, embedded viruses are retroviruses that have integrated themselves into the genomes of unrelated, large dna viruses. presumably, integration is random but only those integrations that leave the dna virus capable of productive infection propagate and are detected. two examples, a moth retroviral element embedded into the genome of autographa californica nuclear polyhedrosis virus (friesen and nissen, 1990) , and reticuloendotheliosis virus embedded in the fowlpox genome (hertig et al., 1997) , illustrate that the effects of embedded viruses can be multifaceted. in both cases, the embedded retrovirus gains an alternative transmission and entry pathway to hosts, but in the case of the retrovirus embedded in the baculovirus, retroviral gene expression is also activated (friesen and nissen, 1990) . and for reasons not yet understood, the fowlpox strains carrying the embedded reticuloendotheliosis virus cause immunosuppression in chickens while reticuloendotheliosis virus-free strains do not (wang et al., 2006) . the final type of direct vvi for which we find documentation, involves transactivation of the genes of one virus species by gene products of a heterologous virus. many viruses encode powerful promoters and transactivating proteins in order to appropriate the cellular transcription machinery for maximum viral gene expression. direct binding and transactivation of a heterologous viral promoter is documented in the case of cytomegalovirus transactivating protein ie2-86 which binds to the −120 to 20 region of the hiv-1 long terminal repeat (yurochko et al., 1999) and in the case of human foamy virus bel1 protein which recognizes and binds to the −158 to −118 region of the hiv-1 long terminal repeat (lee et al., 1992) . epstein-barr virus (ebv) and hepatitis c virus (hcv) coinfection results in significantly higher hcv production than hcv infection alone. it is known that the ebv gene product responsible for enhanced hcv replication is the transcriptional activation protein ebna1 (ebv-encoded nuclear antigen-1) (sugawara et al., 1999) and it therefore seems likely that ebna-1 enhances hcv replication by direct transactivation, however, the targeted genes in hcv have not been identified. it is possible, however, that ebna1 activates hcv genes indirectly, as discussed in more detail below. post-transcriptional heterologous transactivation also takes place. herpes simplex virus protein us11 is an rna binding protein that controls post-transcriptional expression of herpes simplex genes. however, during coinfections it also binds and controls splicing of htlv-1 and hiv-1 transcripts normally controlled by the retroviral proteins rex and rev, respectively (diaz et al., 1996) . viral infections can cause many pathogenic changes in the host. often seen during dual infections is acceleration of disease, because of the compounded nature of the two viral cytopathic effects affecting the host in a negative manner. in this section, indirect vvi resulting from alterations in the host environment created by pre-existing or simultaneous coinfections are explored. five subtypes of indirect environmental vvi are currently recognized: indirect transactivation of genes, breakdown of host physical barriers against infection, altered receptor expression, heterologous activation of antiviral pro-drugs, and modification of the interferon-induced antiviral state. while direct binding and activation of viral transactivating proteins to heterologous viral promoters has been documented, more common are reports of viral infections inducing increased expression or activation of cellular transcription factors, which then act on promoters of coinfecting viruses. for example, hepatitis b virus xprotein transactivates promoters containing kappa-b like enhancer elements, both cellular and viral, but not by binding to the elements itself (twu et al., 1989) . human herpes virus 6 (hhv-6) infection of epstein-barr virus (ebv)-infected cells results in transactivation of the ebv zebra gene, but the transactivation appears to be mediated by cellular transcription factors on the ebv promoter (flamand and menezes, 1996) . the human endogenous retrovirus k (herv-k) ltr is transactivated by hsv-1 protein icp0 via increased binding of cellular transcription factor, ap-1 (kwun et al., 2002) . cmv ie1 protein also transactivates an ltr, the hiv ltr, but does so by inducing increased binding of nf-kappa b to the ltr sequence by an unknown mechanism (kim et al., 1996) . a vvi involving another ebv latency protein, lmp2a, illustrates the potential complexity of indirect virus-virus transactivation interactions. ebv infection, and subsequent lmp2a expression in human cells, results in the activation of a superantigen gene encoded by the long integrated and inactivated human endogenous retrovirus k18 (herv-k18) with serious clinical consequences (hsiao et al., 2006) . however, lmp2a appears to cause gene expression from the remnants of this ancient viral infection by binding to an enhancer 13 kb downstream and transactivating a cellular gene encoded on the opposite strand. transcription of the cellular gene most likely displaces repressors on the herv-k18 promoter sequences, resulting in transcription of its env superantigen gene (hsiao et al., 2009) . in summary, indirect heterologous transactivation is probably the most common vvi, with many interactions yet to be discovered. the diversity of mechanisms they represent and our present incomplete understanding of transcriptional control mechanisms increase the challenge of identifying such interactions. most have been investigated because a coinfection was observed to exacerbate a viral disease. there are potentially other cases of indirect heterologous transactivation that reduce pathology, but are more difficult to detect. viral replication and progeny production are often characterized by cytopathic effects. tissue damage that results can compromise physical barriers within the host, allowing secondary infections to gain access to otherwise protected tissues. this type of vvi has been observed in plants, specifically zucchini squash (cucurbita pepo). some cucumber mosaic virus strains infect zucchini squash plants but only cause localized infections. however, in plants coinfected with cucumber mosaic virus and zucchini yellow mosaic virus, the long distance movement of the cucumber mosaic virus is facilitated and systemic infection of both viruses is observed (choi et al., 2002) . this synergistic effect, which may also be mediated by viral movement proteins (melcher, 2000) , is readily observed by the overall deterioration of the plant as well as by molecular analysis (choi et al., 2002) . examples of this type of vvi also exist for animal viruses. humans infected with herpes simplex viruses 1 or 2 (hsv-1 or -2) have a higher susceptibility for acquisition of hiv, and a higher possibility of transmission of hiv to other persons sheffield et al., 2007) . both of these situations are associated with the ability of hsv-2 to cause open skin lesions and to recruit cd4+ t cells to the sites of these lesions (celum, 2004) . the recruitment of these cells makes more potential host cells available for acquisition of hiv in a herpesvirus lesion than are found in a traumatic lesion. in the case of someone already hiv infected, active hsv coinfection increases the probability of hiv transmission because infected cd4+ t cells are recruited to the open hsv lesion, increasing production of infectious virus at the skin surface (celum, 2004) . another example of barrier compromise allowing increased viral spread involves cytokine mediated tissue damage. atencio et al. showed that newborn balb/c and nih swiss mice coinfected with wild-type polyomavirus (a2 strain) and moloney murine leukemia retrovirus (m-mulv) exhibit growth inhibition and kidney inflammation, whereas either virus alone rarely produced these symptoms. coinfection was found to elevate cytokines il-6, ifn-␥, il-1␤ and il-10 early after infection (7 days) much more than single infections, and may be responsible for the kidney inflammation and runting (atencio et al., 1995) . the density of viral receptors on a prospective host cell is a significant factor in determining whether infection is successful (agnello et al., 1999; li et al., 1999) . human immunodeficiency virus, for example, binds to a complex of cd4 protein and either ccr5 or cxcl4 as its receptor, and it therefore almost exclusively infects human cd4+ t cells. coinfections have been shown to alter the cell types infected by hiv by altering expression of cd4 or the co-receptors ccr5 or cxcl4. human herpes virus 6 (hhv6) has multiple effects in this system. it upregulates cd4 expression on t cells that are already cd4+ increasing their susceptibility to the hiv virus, but it also induces expression of cd4 on the surface of cd8+ t cells making them susceptible to hiv infection as well (lusso et al., 1991) . in addition, hhv-6 coinfection boosts the production of the ccr5 ligand, rantes, which binds to ccr5 and inhibits the complex formation between ccr5 and cd4 needed for hiv to infect cells. exogenous rantes alone, can mimic this inhibitory effect of hhv6 on hiv infection, but it is only inhibitory to hiv strains that utilize ccr5 as a co-receptor, not cxcl4-tropic strains (grivel et al., 2001) . human herpes virus 7 (hhv7) infection also alters cell surface receptor expression in a manner protective against hiv. hhv7 is a t-lymphotrophic virus which also utilizes cd4 as a receptor, and competes directly with hiv for binding sites on host cells. in a host first infected by either hiv or hhv7, cd4 expression on t cells is down-regulated, slowing the spread of a subsequent infection by the other virus (lisco et al., 2007; lusso et al., 1994) . a fourth indirect mechanism by which vvi alter infection outcomes by affecting the host environment is the activation of pro-drugs with antiviral activity. many nucleoside analog antiviral drugs, such as acyclovir, gancyclovir, and famcyclovir, specifically target herpesvirus infected cells because they must be phosphorylated by herpesvirus-encoded kinases or phosphorylases before becoming active. once activated, the drugs can be incorporated into nascent herpesvirus genomes by viral polymerases, where they act as chain terminators, preventing replication. recently, it was shown that acyclovir can be activated by one virus and act on another . hiv, lacking a thymidine kinase is usually unaffected by acyclovir. however, in herpesvirus and hiv dual infected cells, acyclovir decreases the replication of hiv as well as the herpesvirus. acyclovir is phosphorylated by herpesvirus kinases and then moves to directly inhibit hiv reverse transcriptase, having an unintended but beneficial effect for the host . a fifth category of virus-induced change in the host environment that may affect coinfecting viruses involves the innate immune mechanism induced in vertebrates by type i interferons known as the antiviral state. the antiviral state consists of increased expression of a combination of enzymes, which if activated, shut down cellular translation (galligan et al., 2006; staeheli, 1990) . the most critical of these enzymes are pkr and 2 -5 oligoadenylate synthetase (2 -5 oas). protein kinase r (pkr) has multiple roles in a cell, but its role in the antiviral state is to phosphorylate eukaryotic translation initiation factor 2 alpha (eif2␣), inactivating it and shutting down protein synthesis (garcia et al., 2007) . the 2 -5 oas synthesizes unique oligonucleotides which activate rnasel, initiating destruction of cellular and viral rna molecules necessary for translation. activation of both enzymes is dependent on the presence of molecules associated with infection, particularly dsrna, and their activation usually results in cell death (staeheli, 1990) . animals with defective type i interferon signaling pathways, pkr, or 2 -5 oas are much more susceptible to viral infections, indicating the effectiveness of the antiviral state (levin and hahn, 1985) . however, most viruses have also evolved antagonists of pkr and or 2 -5 oas (hengel et al., 2005; langland et al., 2006; levy and garcia-sastre, 2001) . from this information it would seem logical to speculate that an antiviral state antagonist from one virus could benefit a coinfecting virus, and this has been shown to be the case in several in vitro systems. murine cytomegalovirus (mcmv) has two proteins that are known to inhibit pkr, m142 and m143. when these proteins are present and active the virus can readily replicate in the host. in the absence of these two proteins, the antiviral state is activated and mcmv replication is inhibited (budt et al., 2009 ). this antiviral state activation can be overcome by introducing the pkr inhibitors encoded by vaccinia virus (e3l) or herpes simplex virus (icp gamma 34.5) (budt et al., 2009) . a herpes simplex mutant lacking icp gamma 34.5, in turn can be rescued in cv-1 cells by coinfection with the polyomavirus sv40, due to the sv40 large t antigen's inhibitory effect on the antiviral state downstream of icp gamma 34.5 (randazzo et al., 1997) , or by inserting human cytomegalovirus genes for pkr antagonists, trs1 and irs1 (cassady, 2005; shah et al., 2007) . one possible natural example of this type of vvi involves the interaction of hepatitis b and hepatitis d viruses. the hepatitis d genomic rna molecule inhibits pkr-mediated inhibition of translation in a cell free translation system, suggesting that it may protect its helper virus, hbv, from the antiviral state (robertson et al., 1996) . although the known examples of this virus-virus interaction are so far only demonstrated in artificial systems, given the multiple in vitro examples it seems likely that this type of vvi also occurs in nature. as the third main category of vvi, we define a subset of indirect virus-virus interactions that occur only in host species with an adaptive immune system. we set these types of interactions apart, because unlike the other indirect vvi, which are dependent on an overlap of the periods of infection of two viruses, immunological interactions can occur between viral infections that are completely separated in time. this is possible because the adaptive immune system of the host organism is permanently changed by its interaction with a virus, even if that infection is completely eliminated from the host, and is changed in a manner very specific to the species and strain of the infecting virus. at present, four types of immunological vvi have been identified. these include altering the activation state of cellular components of the immune system, and induction of autoimmune responses to self-antigens that cross-react with viral antigens. in addition, the humoral immune response to viral pathogens can unexpectedly give rise to antibodydependent enhancement (ade) of subsequent viral infections. and finally, coinfections, as well as sequential infections, also indirectly interact by re-shaping the t cell memory repertoire such that the immune response induced by one infection can impact the outcome of a subsequent viral infection in an interaction termed heterologous immunity (welsh and selin, 2002) . one means by which a virus may sensitize a host for a subsequent infection is by altering the activation state of potential host cells. hiv infection, for example, is associated with human cytomegalovirus (hcmv) infection in part due to hiv's induction of elevated numbers of activated lymphocytes in certain tissues. since activated lymphocytes are permissive for hcmv infection, this hiv-induced activated cellular state results in a two-to threefold enhancement of hcmv replication in these tissues . another example is seen with lactate dehydrogenaseelevating virus (ldv). ldv stimulates polyclonal b lymphocyte activation, resulting in delayed induction of antibodies needed to control other coinfecting viruses. consequently, friend virus (fv) infection, which is normally asymptomatic in mice due to timely production of neutralizing antibodies, will upon coinfection with ldv propagate and cause symptomatic disease (marques et al., 2008) . the prevalence of viral coinfections may also impact the progression of hiv disease in this manner. specifically, it is not unusual for hiv positive patients to be coinfected with gb virus c (gbv-c) and the persistence of certain genotypes of this virus has been shown to lead to slower hiv progression. (schwarze-zander et al., 2006) . this positive effect for the host appears to be mediated by elevated expression of interferon gamma and the immune cell activation that results. higher viral titers of gb virus c are directly correlated with increased serum interferon gamma, which in turn results in increased numbers of circulating mature dendritic cells that may be controlling the hiv infection (lalle et al., 2008) . sequential viral infections have also been associated with generation of autoimmunity in the host. some viruses are able to break immunological tolerance to "self" by expressing a self-like epitope, but are unable to generate the numbers of autoreactive t cells necessary to trigger an autoimmune response. however, a second infection can, by expanding the autoreactive t cell compartment, cause autoimmunity (welsh and fujinami, 2007) . mice transgenic for a lymphocytic choriomeningitis virus (lcmv) nuclear protein remain healthy when infected with lcmv, but a subsequent infection with either poliovirus or vaccinia virus leads to the development of pancreatic inflammation and autoimmune diabetes (christen et al., 2004; evans et al., 1996) . another example of this type of vvi is associated with enteroviruses such as poliovirus. enterovirus infections have been shown to be a risk factor for several autoimmune diseases including insulin-dependent diabetes mellitus (iddm) (dahlquist et al., 1995; grist et al., 1978; hiltunen et al., 1997; hyoty et al., 1995) . multiple epidemiological studies in humans have established that children that manifest iddm have had more exposures to enteroviruses than healthy subjects (andreoletti et al., 1998; clements et al., 1995; d'alessio, 1992) . interestingly, in countries where a live attenuated polio vaccine which is known to confer cross-protection to enteroviruses (juhela et al., 1998) is used to immunize children, the incidence of iddm is lower than in countries where a killed vaccine that does not induce cross-protection is used. this suggests an association between cross-reactivity of immune responses to different viruses and the development of autoimmune diseases (juhela et al., 1999) . in order to infect animal cells, virus particles usually must bind directly to a specific cell surface molecule in a virus-specific manner (flint et al., 2004) . however, several families of virus are known to take advantage of indirectly binding to the cell surface via crosslinking with antiviral antibodies or virus activated complement components which then bind to host cells bearing fc or complement receptors (takada and kawaoka, 2003) . this process in which increased viral replication is produced by exposure to immune sera, is known as antibody-dependent enhancement (ade) of viral infection. it has been observed in vitro for flaviviruses, coronaviruses and retroviruses (cummings et al., 2005) . mechanisms underlying ade are not fully understood, but seem to include increased efficiency of virus binding to host cells, resulting in higher numbers of infected cells. the most well studied case of ade in humans is dengue hemorrhagic fever (dhf). interestingly, the severe form of dengue illness, which is often fatal, is strongly associated with preexisting heterotypic immunity kliks et al., 1988; sangkawibha et al., 1984) . the presence of non-neutralizing antibodies from a previous dengue infection augment viral growth in vitro (kliks et al., 1989) and in vivo (halstead, 1979) . also, individuals suffering from a secondary dengue virus infection have higher viremia than those with primary infections (vaughn et al., 2000) , and the presence of anti-dengue antibodies in mothers has been associated with the occurrence of dengue hemorrhagic fever in newborns (kliks et al., 1988) . the phenomenon of ade is not unique to dengue virus infections. it has been demonstrated to play a role in west nile virus infections in vitro (peiris and porterfield, 1979) and yellow fever virus in vivo (barrett and gould, 1986) . moreover, infections by two other members of the flavivirus family, the yellow fever virus and the japanese encephalitis virus, have also been shown to be enhanced by ade (gould and buckley, 1989) . this process is also thought to be responsible for the enhanced pathogenicity of viral challenges after vaccination with certain formalin inactivated viral vaccines (porter et al., 1972) , including ones for measles (iankov et al., 2006) , respiratory syncytial virus (ponnuraj et al., 2003) , and rabies (prabhakar and nathanson, 1981) . heterologous immunity gives rise to virus-virus interactions when the outcome of the adaptive immune response to a new viral infection is determined in part by immune memory acquired by the host from prior viral infections. development of a primary adaptive immune response to a pathogen results in an immunological memory, which consists of expanded numbers of long-lived, circulating t and b lymphocytes recognizing epitopes of that specific pathogen (welsh et al., 2004) . due to randomized dna rearrangement processes during the generation of the unique specificities of adaptive immune cells, even genetically identical twins have unique t cell repertoires in their naïve state and therefore show some differences in their responsiveness to the same pathogen. notably, these variations seem to have limited significance for the effectiveness of the immune response to a new infection (welsh et al., 2006) . however, if even a small subset of a t cell memory pool is cross-reactive with antigens of a later encountered pathogen, it will outcompete newly activated t cell clones and dominate that response. that the degree of heterogeneity in the immunological responses between genetically identical hosts could be dramatically influenced in different directions by the history of infections with seemingly unrelated viruses was long unappreciated (welsh and fujinami, 2007; welsh et al., 2006) . however, studies in syngeneic mice have confirmed that the unique identity of memory t cells, raised towards one virus but later activated during an infection with an unrelated heterologous virus, dramatically influence the outcome of the second infection (selin et al., 1996) . this heterologous immunity can result in both beneficial and harmful effects (chen et al., 2001) . in mice, for example, immunity to influenza virus protects the host against vaccinia virus challenge, but enhances the virulence of subsequent cytomegalovirus infections (chen et al., 2003) . while difficult to study in outbred populations, syngeneic animal models allow investigation of immunological memory after heterologous infections. when the diverse viruses lcmv, poliovirus, vaccinia virus, murine cytomegalovirus, and vesicular stomatitis virus were sequentially introduced into syngeneic hosts, the immunological memory against one infection was dramatically altered after each successive infection (selin et al., 1996) . thus, the course of each infection is influenced by the t cell memory pool, and with each infection, the t cell memory to previous encountered agents is modified (welsh and selin, 2002) . the effect of heterologous immunity is seen between many different viruses and disease outcome is dependent on both the nature and the specific order in which the sequentially encountered pathogens were encountered (chen et al., 2003) . although relatively unexplored as a field of study, vvi have already been documented to have significant and unexpected effects on viral disease severity, host range, transmissability, immunopathology, and vaccine effectiveness. increased awareness of the potential for virus-virus interactions and a framework for categorizing different types of interactions as described here, would seem to be necessary steps for achieving better understanding of infectious viral diseases in nature. it has long been noted that many viral infections result in mild disease for most infected individuals, moderate disease for some, and fatal disease for a few. the epidemiology of poliomyelitis, influenza, and the recent west nile virus outbreak in the united states are prime examples of this phenomenon. when occurring in otherwise healthy individuals, these differences in susceptibility have largely been assumed to be governed by cryptic immunological defects, either inherited or acquired (kacprzak-bergman and nowakowska, 2005; trammell and toth, 2008) . while this is likely true in some cases, it is also plausible that some variations in susceptibility are determined by virus-virus interactions. evidence for this is strong in the intensively studied case of hiv/aids (lisco et al., 2009 ). investigation of similar interactions for acute viral infections will be challenging, but may allow better identification and protection of the most vulnerable populations during disease outbreaks. it was not possible, of course, to mention every known virus-virus interaction in this article. rather, the goal was to provide a framework into which all vvi can be organized. an online database has been established (www.musc.edu/vvi/) with the aim of compiling a referenced, searchable, comprehensive list of vvi, actively updated by contributions from the scientific community via moderated forum. we anticipate that the number of vvi subtypes may expand, as during the course of this investigation hints of interactions that seem plausible but are not yet documented were found; for example, stabilization of viral rnas by heterologous viral rna binding proteins. in addition, several cases of vvi described above were discovered simultaneously with a new virus, whose existence was not previously suspected. as a wobble in a star's rotation can indicate the presence of an unseen planet, so the perturbation of a viral replication cycle may indicate the presence of an unknown infection, and a virus-virus interaction. curated list of prokaryotic viruses with fully sequenced genomes hepatitis c virus and 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cord-294764-v28wbrqp authors: deval, jerome; jin, zhinan; chuang, ying-chih; kao, c. cheng title: structure(s), function(s), and inhibition of the rna-dependent rna polymerase of noroviruses date: 2017-04-15 journal: virus res doi: 10.1016/j.virusres.2016.12.018 sha: doc_id: 294764 cord_uid: v28wbrqp noroviruses belong to the caliciviridae family of single-stranded positive-sense rna viruses. the genus norovirus includes seven genogroups (designated gi-gvii), of which gi, gii and giv infect humans. human noroviruses are responsible for widespread outbreaks of acute gastroenteritis and represent one of the most common causes of foodborne illness. no vaccine or antiviral treatment options are available for norovirus infection. the rna-dependent rna polymerase (rdrp) of noroviruses is a key enzyme responsible for transcription and replication of the viral genome. here, we review the progress made in understanding the structures and functions of norovirus rdrp and its use as a target for small molecule inhibitors. crystal structures of the rdrp at different stages of substrate interaction have been determined, which shed light on its multi-step catalytic cycle. the in vitro assays and in vivo animal models that have been developed to identify and characterize inhibitors of norovirus rdrp are also summarized, followed by an update on the current antiviral research targeting different regions of norovirus rdrp. in the future, structure-based drug design and rational optimization of known nucleoside and non-nucleoside inhibitors of norovirus rdrp may pave the way towards the next generation of direct-acting antivirals. noroviruses are the primary cause of foodborne illness globally, resulting in sometimes massive outbreaks of gastroenteritis. in this section, we describe in more details the medical burden associated with norovirus, the current status of biomedical efforts to address norovirus infection, and the norovirus genome organization. human noroviruses (hunovs) are category b biodefense agents and the major cause of viral gastroenteritis. less than twenty viral particles can establish a norovirus infection and prior exposure does not lead to protection from a repeat infection. the clinical symptoms of norovirus infection include vomiting, diarrhea, abdominal cramps, and nausea, usually lasting 2-4 days. the centers for disease control and prevention estimates that 900,000 clinic visits among children result from norovirus infection in developed countries and up to 200,000 deaths annually in children less than 5 years of age in developing countries (patel et al., 2009; scallan et al., 2011) . in the united states alone, norovirusassociated foodborne illness is responsible for about 20 million cases yearly, with more than 70,000 hospitalizations and about 800 deaths lopman et al., 2012) . in healthy adults, norovirus causes acute self-limiting disease lasting 1-3 days, but it causes long-lasting infection in elderly, juvenile, and immunocompromised patients. noroviruses are a significant cause of morbidity and mortality in immunocompromised patients such as transplant recipients (boillat blanco et al., 2011; capizzi et al., 2011; roos-weil et al., 2011; saif et al., 2011; schorn et al., 2010; schwartz et al., 2011) . norovirus infections in infants can lead to seizures (chen et al., 2009; medici et al., 2010) and have been linked to necrotizing enterocolitis (turcios-ruiz et al., 2008) . in adults, norovirus infection can exacerbate inflammatory bowel disease (khan et al., 2009) . increased disease severity is linked to the use of statins to regulate cholesterol levels (rondy et al., 2011) . cultured cells treated with statins increases norovirus replication, which is especially a concern given the number of patients using statins (mann et al., 2008) . despite the clear need for medical intervention to prevent and treat norovirus infection, no approved vaccines or small molecule treatment options are currently available. both vaccines and small molecules are currently being evaluated as potential strategies for medical intervention in cases of norovirus infection. although vaccines have been successfully developed to treat diarrheal diseases caused by other rna viruses such as rotaviruses, efforts to develop anti-norovirus vaccines based on live-attenuated virus have been hampered by the inability to grow hunov efficiently in cell culture. therefore, current strategies for developing anti-norovirus vaccines rely on the production of virus-like particles from recombinant and self-assembling protein capsids (for recent review: (tan and jiang, 2014) , (richardson et al., 2013) ). norovirus-like particles can be produced from a variety of expression systems such as baculovirus infection of insect cells, which is among the most advanced and is currently used in the manufacture of clinical trial materials. both orally and nasally administered virus-like particles have been well tolerated and immunogenic in phase 1 clinical trials and even conferred some protection against norovirus gastroenteritis in healthy adults in a human experimental challenge model (atmar et al., 2011; el-kamary et al., 2010; tacket et al., 2003) . a bivalent norovirus-like particle candidate that is safe and immunogenic in adults is currently under evaluation for safety in children and in the elderly and for efficacy in adults (treanor et al., 2014) (clinical trial identifiers nct02153112, nct02661490, and nct02669121). to date, the only small molecule tested in the clinic as a potential anti-norovirus drug is nitazoxanide, which was originally developed and commercialized as an antiprotozoal agent. nitazoxanide has demonstrated some benefit in treating adults suffering from norovirus gastroenteritis (rossignol and el-gohary, 2006; siddiq et al., 2011) . while nitazoxanide's mechanism of action is not well understood, it probably involves multiple host factors rather than directly targeting a norovirus protein. nitazoxanide also inhibits a broad range of other rna and dna viruses including the respiratory syncytial virus, parainfluenza virus, coronavirus, rotavirus, hepatitis b virus, hepatitis c virus, dengue virus, yellow fever virus, japanese encephalitis virus, and the human immunodeficiency virus (rossignol, 2014) . antiviral approaches involving direct-acting antiviral agents remain at early stages of preclinical research and mainly target two viral proteins: the protease and the rna polymerase (kaufman et al., 2014; weerasekara et al., 2016) . the following sections of this review focus on the structure, function, and small-molecule inhibition of norovirus polymerase. noroviruses are a genetically diverse group within the caliciviridae family, which includes six genera: vesivirus, lagovirus, nebovirus, sapovirus, recovirus, and norovirus. the genus norovirus is subdivided into seven genogroups (designated gi-gvii), based on phylogenetic analysis of the capsid gene (vinje, 2015) . genogroups gi, gii and giv infect humans and cause acute gastroenteritis. norovirus strains commonly isolated in cases of acute gastroenteritis belong to gi and gii. within these two groups, cases of gii genotype 4 (gii.4) infection account for the majority of outbreaks of gastroenteritis. noroviruses are non-enveloped viruses with positive-stranded genomes of 7400-7700 nucleotides. the genome typically contains three open reading frames (orfs), named orf1-3 (clarke and lambden, 2000; mcfadden et al., 2011) (fig. 1 ). orf2 and orf3 encode the major and minor structural capsid proteins vp1 and vp2, respectively. ninety dimers of vp1 form the icosahedral capsid of norovirus (donaldson et al., 2010) . vp1 contains two major domains: the shell domain and the protruding domain that binds to receptors on host cell surfaces (prasad et al., 1999; strong et al., 2012) . only a few copies of vp2 reside on the interior surface of the capsid (vongpunsawad et al., 2013) . orf1, located in the first two-thirds of the genome encodes a ∼200 kda polyprotein that is proteolytically processed by the virus-encoded protease to yield 6 non-structural proteins named p48 (ns1/2), ntpase (ns3), p22 (ns4), vpg (ns5), pro (ns6), and pol (ns7) (belliot et al., 2003) (fig. 1) . the function of these non-structural proteins is listed in table 1 . p48 and p22 are involved in viral replication complex formation by recruiting host membrane vesicles. p48 promotes disassembly of the golgi complex and prevents cell surface proteins expression and trafficking to facilitate the recruitment of cellular membranes to its replication complex (ettayebi and hardy, 2003; fernandez-vega et al., 2004) . p22 contains an endoplasmic reticulum export signal that was proposed to promote p22 uptake into the coat protein complex ii vesicles, leading to vesical mislocalization and golgi body disassembly (sharp et al., 2012 (sharp et al., , 2010 . together, p22 and p48 interrupt the host protein secretion pathway and are likely responsible for the rearrangement of the cellular membrane and recruitment of membrane for the replication factory. vpg is covalently linked to the 5 end of the genome and the subgenomic rna. the linkage of vpg to viral rna is thought to occur during viral genome replication whereby vpg is attached as a protein primer to the 5 terminus of the genomic rnas (rohayem et al., 2006b) . protein-primed initiation of rna synthesis is also found in picornaviruses and potyviruses (goodfellow, 2011) . the norovirus rdrp (ns7) is the central enzyme in norovirus rna replication. viral rdrps are important targets for antivirals, as evidenced by the efficacy of drugs that inhibit the hepatitis c virus (hcv) rdrp and contribute to cures of hcv infection. therefore, structural and functional studies of norovirus rdrp are important not only to understand norovirus replication, but also for guiding rational antiviral drug candidate selection and development. multiple high-resolution structures of hunov rdrp have been determined (ng et al., 2004; zamyatkin et al., 2008 zamyatkin et al., , 2009 zamyatkin et al., , 2014 croci et al., 2014a,b; mastrangelo et al., 2012) . these include structures from the gi norwalk virus and the gii.4 hunov that are responsible for the majority of human infections. additional structures for related viruses in the caliciviridae family have also been determined (alam et al., 2012) . the structures are for the apoenzyme, rdrp with divalent metals bound at the active site, rdrp bound to nucleoside triphosphates (ntps), the ternary complex with the template rna and a primer, and several ternary complexes with nucleotide analogs. structures of the hunov rdrp bound to allosteric compounds that inhibit rna synthesis are also known (croci et al., 2014a) . thus, there is extensive knowledge about the structures of the hunov rdrp as they relate to rdrp function. the structure of the norovirus rdrp is highly similar to those of other positive-strand rna viruses (alam et al., 2012; hogbom et al., 2009; ng et al., 2004; zamyatkin et al., 2008) . the rdrp can be described as a partially closed right hand, with fingers, thumb, and palm subdomains ( fig. 2a and b) . the fingers and the thumb subdomains interact to close the hand structure (fig. 2b) , forming a channel where the single-stranded template rna can thread into the polymerase. the template channel merges with the central channel where the 3 end of template rna and the nascent rna exit (fig. 2c) . at the confluence of the template and central channel is the ntp channel where ntps enter. lining the channels are highly conserved residues organized into motifs a through g, which interact with the template, the nascent rna, and the ntps for rna synthesis. the active site lies at the convergence of the template channel and the ntp channel and contains aspartate residues that coordinate divalent metals to promote nucleotide polymerization ( fig. 2) (alam et al., 2012; zamyatkin et al., 2009 ). rna synthesis by the norovirus rdrp active site occurs by a 5-step reaction that is well conserved in rdrps of different viral species (fig. 3) . the five steps are: (1) rdrp bound to the template binds an ntp complementary to the template base to form an open ternary complex; (2) a change in the conformation to form a closed complex; (3) nucleotidyl transfer and translocation of the template; (4) conformational change in the complex; (5) release of the pyrophosphate that allows another ntp to bind to the active site. the steps in rna synthesis involve significant conformational changes in the rdrp and the high quality structures have captured several distinct conformations. the apo-enzyme has the rdrp cterminal tail bound to its active site (ng et al., 2004) . the ternary complex of the norwalk virus rdrp during elongative rna synthesis that was determined by zamyatkin et al. (2008) shows that the binding of the template and primer rna and the substrate ntp displaced the c-terminal tail from the active site and induced a rotation of the central helix in the thumb subdomain of the rdrp to form a binding pocket for the primer rna (zamyatkin et al., 2008) . this effectively represents the closed conformation of the rdrp trapped just prior to nucleotidyl transfer. the active site of the palm subdomain coordinates two divalent metals that mediate catalysis by the rdrp (fig. 2d ). metal a forms an octahedral coordination with asp242 (motif a), asp343, and asp344 (motif c), the 3 oh of the primer, the ␣-phosphate of the ntp, and a water molecule. the water is activated to be a general base to extract a proton from the primer 3 oh to enable it to become a better nucleophile. metal b also forms an octahedral coordination between the carboxylate groups of asp242, asp343, and tyr243 and oxygens of all three phosphates in the ntp. the side chains of the highly conserved arg182 in motif f of the rdrp forms electrostatic interaction with the ␣-, ␤-phosphates of the incoming ntp, likely to stabilize the pyrophosphate as a leaving group. the ribose of the ntp is stabilized by van der waals forces. in addition, the 2 oh forms an h-bond with asn309 and donates an h-bond to ser300 that donates an h-bond to asp247 (fig. 2d) . this network of interactions allows the rdrp to distinguish ribonucleotides from deoxyribonucleotides. following nucleotidyl transfer and the release of the pyrophosphate, the rdrp active site remains in a closed conformation, but helices in the thumb subdomain return to the state similar to that seen in the apo-protein (zamyatkin et al., 2014) . this leads to a loosening of the contacts with the rna duplex, likely to facilitate the translocation of the rna as well as to open the pocket for binding of the next nucleotide. accurate initiation of rna synthesis is critical to prevent the loss of viral genetic information (kao et al., 2001) . the norovirus rdrp specifically recognizes the viral genome for rna synthesis. in murine norovirus (mnv) the positive-or minus-sense rna from the viral sgrna stimulates vpg nucleotidylation, possibly by stabilizing the structure of the viral rdrp (goodfellow, 2011) . the mnv and hunov variants can also preferentially recognize rna segments to direct initiation of the rna towards subgenomic rna . subgenomic rna synthesis by caliciviruses occurs by internal initiation on the minus-strand rna (morales et al., 2004) (fig. 4a) . within the minus-strand rna, a highly conserved stem-loop structure upstream of the start site of the sgrna initiation nucleotide was identified in all caliciviruses (fig. 4b) . in mnv, mutations in this rna structure debilitated mnv sgrna synthesis and suppressor mutations that restored the rna structure were found to restore mnv infectivity (simmonds et al., 2008; yunus et al., 2015) . ns7 from mnv and hunov preferentially recognize their cognate subgenomic promoters and a short stretch of template sequence that contributes to accurate initiation of the subgenomic rnas and efficient rna synthesis. crosslinking of the subgenomic rna hairpin to the mnv and hunov rdrp showed that helix 1 in the thumb subdomain of the mnv rdrp contacts the mnv subgenomic promoter hairpin. notably, this mode of recognition of the promoter for rna synthesis is similar to that of phage rna polymerases, which recognize promoter sequences for accurate initiation of rna synthesis (cheetham et al., 1999; imburgio et al., 2000) . norovirus can initiate rna synthesis through two distinct modes (rohayem et al., 2006b) (fig. 4a) . the replication intermediate minus-strand rna is thought to be initiated by a de novo mechanism (i.e., in the absence of a pre-existing primer). in this case, the first nucleotide serves as a primer to provide the ribose 3 oh for the attachment of subsequent nucleotides. de novo initiation allows rna synthesis to take place opposite to the 3 -terminal nucleotide in the template rna, thus preventing the loss of genetic information. the positive-strand genomic rna and the subgenomic rnas contain the protein vpg covalently attached to their 5 terminus. the ns7 protein or the unprocessed ns6-7 proteins are involved in both de novo-initiated and vpg-primed rna synthesis (belliot et al., 2005) . vpg is essential for translation initiation from the norovirus genomic and subgenomic rnas and appears to also act in the recruitment of ribosomes (daughenbaugh et al., 2003; thorne and goodfellow, 2014) . for rna synthesis, the norovirus vpg contains a tyrosine, likely tyr27 for hunov, whose hydroxyl side chain is involved in rna synthesis, which has recently been confirmed by mass spectrometry (olspert et al., 2016) . in vitro, purified ns7 can add uridylates to purified vpg and then use the vpg-polyu to prime the synthesis of the full-length subgenomic rna (rohayem et al., 2006b) . vpg-primed rna synthesis was not observed with minimal promoter-rna templates, perhaps due to the lack of a sequence such as the cis-replication element that directs the uridylation of the vpg . the cis-acting replication element of noroviruses remain to be identified. de novo nucleotide-initiated norovirus subgenomic rna synthesis suggests that the vpg could be recognized in a way functionally equivalent to an initiation ntp. vpg also increases the activity of the rdrp in a cell-based reporter assay without the production of vpg-primed rna, suggesting that vpg can promote a state of the rdrp that is more competent for rna synthesis ( tion in the nucleotidylation residue reduced the stimulatory effect. the structure of the central region of the mnv vpg determined by nuclear magnetic resonance spectroscopy supports the hypothesis that a large structural rearrangement in the rdrp is required for it to recognize the cognate tyrosine needed for uridylation (leen et al., 2013) . however, details of the molecular interaction between ns7 and vpg are unknown, mainly because a crystal structure of the protein complex is not yet available. activities of the norovirus rdrp are also modulated by the norovirus capsid proteins. vp1 can enhance rna synthesis by the rdrp in a concentration-dependent manner when the proteins are co-expressed in cells. the shell domain of vp1 is responsible for the stimulatory activity and can bind to the rdrp (subba-reddy et al., 2012). overexpression of the mnv vp1 also stimulated the kinetics of mnv replicon replication in cells. the vp1 enhancement of rna synthesis is species specific since the mnv vp1 could not enhance the hunov gii.4 rdrp activity. a regulatory role of the activity of viral structural proteins on viral rna synthesis is becoming an increasingly important theme in rna virology (ni and kao, 2013). a significant increase in the global incidence of norovirus outbreaks since 2002 has been associated with the emergence of the highly transmittable hunov gii.4 lineage (lopman et al., 2004; siebenga et al., 2009) . in this pandemic strain, the rdrp displays reduced replication fidelity, which results in higher mutation rate and rate of viral evolution compared to the nonpandemic and less frequently detected strains (bull et al., 2010) . during the winter season of 2014-2015, a newly emerging gii.17 isolate outcompeted the gii.4 in parts of asia, with risk of spreading globally (lu et al., 2016) . the gii.17 isolate displays evolutionary rates at least 1 order of magnitude higher than those seen with gii.4, further establishing the role of the rdrp in the emergence of new epidemic strains (chan et al., 2015) . the hypothesis that rdrp-driven genetic diversity is vital for viral fitness and pathogenesis is not unique to norovirus, and has been well documented for other virus families such as picornaviruses (vignuzzi et al., 2006; peersen, 2017) . consistent with reports on the poliovirus polymerase, arias et al. reported a high-fidelity mnv polymerase variant associated with the i391l mutation (arias et al., 2016) . when mutated at this position, the corresponding mnv clone displayed delayed replication kinetics in vivo and lower transmission rate compared to the wild-type virus. the impact of a single point mutation in the rdrp on viral replication and transmission further highlights the dynamic interplay between replication fidelity, viral fitness, and the emergence of dominant strains of norovirus. although a number of small molecule anti-norovirus agents have been described, the majority of them remain at early stages of preclinical development. this section reviews these efforts, as well as the assays that have been developed to study such molecules. 3.1. in vitro assays and in vivo models available to characterize norovirus polymerase inhibitors the production of recombinant norovirus ns7 protein is amenable to high yield and purity, which has facilitated the development of norovirus polymerase assays used for inhibitor testing. the most common rdrp assays are based on the incorporation of a radiolabeled nucleotide onto the rna primer strand (fukushi et al., 2004; rohayem et al., 2006a) . rna synthesis is initiated de novo on nonpolyadenylated negative-strand rnas containing a specific promoter sequence. although gel-based assays provide qualitative information about the types of rnas formed enzymatically, their application to inhibitor testing was optimized by adapting the radiometric readout to a 96-well format for higher throughput purposes . the use of fluorescence readout based on the binding of the picogreen dye to double-stranded rna products even further enhanced the compatibility of norovirus rdrp assay with high-throughput screening (mastrangelo et al., 2012) . as described earlier in this review, x-ray crystallography is also a valuable tool to understand the binding interactions at the atomic level between norovirus rdrp and small molecule inhibitors. this has been done both with the apo-protein and with protein in complex with primer/template (ng et al., 2004; zamyatkin et al., 2008) . finally, other biophysical methods have been employed to characterize the binding interaction between norovirus polymerase and small molecules (e.g., the thermal-shift assay measuring changes in melting temperature associated with compound binding (mastrangelo et al., 2012) and the fluorescent polarization assay to measure protein-rna binding ). until recently, all attempts to develop a robust hunov in vitro replication assay have failed because of the lack of permissive cells, including human macrophages and dendritic cells from susceptible humans (duizer et al., 2004; herbst-kralovetz et al., 2013; lay et al., 2010; papafragkou et al., 2014; takanashi et al., 2014) . despite this limitation, the in vitro effect of small molecules on hunov replication has been studied using a stable human hepatoma cell line expressing the part of the norwalk virus rna genome encoding the non-structural proteins and carrying the neomycin resistance gene (chang et al., 2006) . in this norovirus minigenome, or replicon, production of the viral rna leads to the reconstitution of a functional norovirus rna replication complex. the challenge associated with developing an authentic in vitro infection assay for hunov has also led to the exploration of calicivirus surrogates more amenable to cell-based assays. multiple non-human enteric calicivirus systems have been described (for complete review, see rocha-pereira et al., 2014) . among them, mnv is the closest to hunov, and therefore the most broadly used for inhibition studies. mnv replicates well in mouse monocytes and macrophages (wobus et al., 2004; wobus et al., 2006) . in 2014, jones et al., demonstrated successful infection of human b cells with hunov from gii.4, using commensal bacteria as cofactor (jones et al., 2015 (jones et al., , 2014 . the infection of b cells resulted in measurable translation of viral proteins and production of infectious progeny, and the increase in viral genome copies was significant, but overall low. b cells are not the only site of hunov infection. ettayebi et al. recently reported the successful cultivation of human gii.4 norovirus in stem cell-derived human intestinal enteroids isolated from intestinal crypts in human intestinal tissues (ettayebi et al., 2016) . the addition of bile to enteroids was not required for gii.4 virus but enhanced its replication, while other strains only replicated in the presence of bile. taken together, it is likely that the establishment of b cell and enteroid in vitro infection systems will find broad applications in the development of diagnostics, vaccines, and therapeutics. replication of hunov has been described using large animals such as chimpanzees, pigs, and calves (for review, see (rocha-pereira et al., 2014) , but mouse is the most common species for in vivo inhibition studies with small molecules. immunocompetent mice infected with mnv are typically asymptomatic and represent a natural host for mnv infection (karst et al., 2003) . for mnv-1 alone, reactive antibodies have been found in 22% of mouse serum samples from research colonies across the united states and canada (hsu et al., 2005) . other strains of norovirus such as mnv-3, mnv cr6, and mnv o7 have been shown to cause a persistent yet asymptomatic infection in immunocompetent mice (arias et al., 2012; hsu et al., 2006; kahan et al., 2011; shortland et al., 2014) . mnv infection in immunocompromised mice lacking innate immunity factors such as stat1 or interferon receptors can result in persistent or acute virus replication associated with severe diarrhea, weight loss, and mortality (karst et al., 2003; wobus et al., 2004) . in particular, the knockout mouse strain ag129 defective of interferon alpha/beta and gamma receptors has been a widely utilized model to test the in vivo efficacy of norovirus polymerase inhibitors (rocha-pereira et al., 2013) . this particular strain of mice is also used as model for dengue and zika virus infection (aliota et al., 2016; johnson and roehrig, 1999; zmurko et al., 2016) . smallanimal models have also been developed to study hunov infection. balb/c mice deficient in recombination activation gene (rag) 1 or 2 and common gamma chain (␥c) (rag-␥c) are permissive to hunov infection (taube et al., 2013) . replicon cell lines expressing subgenomic components of norwalk virus have enabled fast and simple testing of norovirus replication inhibitors. ribavirin was one of the first small molecules identified to inhibit norovirus in in vitro replicon assays. ribavirin reduced the viral genome and proteins with a weak half-maximal effective concentration (ec 50 ) value of about 40 m (chang and george, 2007; jin et al., 2015) (fig. 5a) . combining interferonalpha with ribavirin showed additive effects on the inhibition of norovirus replication. the mechanism of action was evaluated by adding guanosine to the ribavirin treatment, resulting in moderately reversed antiviral effect. the reversal in the presence of guanosine indicates that ribavirin's effect may be at least partially associated with the depletion of guanosine triphosphate in the cells, suggesting a mechanism other than direct targeting of the polymerase. recent in vitro work from julian et al. using mnv suggested that ribavirin treatment also increases viral quasispecies diversity, which could be contributing to its antiviral effects (julian et al., 2016) . the nucleoside precursor 6-fluoro-3-hydroxy-2pyrazinecarboxamide (t-705, favipiravir) is structurally related to ribavirin (fig. 5a) . t-705 was originally developed against influenza virus (for review: (furuta et al., 2013) ). the molecule has also been shown to inhibit other rna viruses including members of orthomyxo-, noro-, bunya-, arena-, and flaviviridae (furuta et al., 2013) . in vitro, the antiviral potency of t-705 against mnv replication is fairly modest, but more potent than ribavirin (rocha-pereira et al., 2012b) (arias et al., 2014; jin et al., 2015) . time-of-addition assays revealed that t-705 exerts its activity at a timepoint that coincides with the onset of viral rna synthesis, suggesting that t-705 might target the polymerase of norovirus. in cells, t-705 is efficiently converted to a ribofuranosyl 5 -triphosphate form (t-705 rtp) by cellular enzymes (furuta et al., 2009 ). similar to previous work with influenza, t-705 rtp is recognized as substrate during norovirus rna synthesis, which results in enzyme inhibition by delayed and incomplete chain termination (furuta et al., 2005; jin et al., 2013 jin et al., , 2015 . treatment of norovirus-infected cells with t-705 induces viral mutagenesis in vivo through a mechanism similar to that of ribavirin that may be associated with incorporation of the nucleotide analog into viral rna (arias et al., 2014) . the intrinsically low antiviral potency of t-705 prevents experiments aimed to select drug-associated resistance mutations, which would otherwise represent an important orthogonal validation of its proposed mechanism of action. attempts to identify analogs of t-705 with increased selectivity against human polymerases by adding a 2 c-methyl substitution had limited success, with a loss of recognition both by human mitochondrial rna polymerase and norovirus rdrp (fig. 5a) . other promising analogs of t-705 have been tested against influenza virus and its polymerase, but antiviral effect of those analogs against the norovirus counterpart has not been reported (wang et al., 2016) . the nucleoside analog 2 -c-methyl-cytidine (2cm-c) was originally developed against the polymerase of hcv and advanced as valopicitabine/nm283, an orally bioavailable 3 -valine ester prodrug (pierra et al., 2006) (fig. 5b) . although valopicitabine was efficacious in chronically hcv-infected patients, its clinical development was halted because of adverse events. 2cm-c was later found to inhibit the replication of mnv (rocha-pereira et al., 2012a) . in this assay, viral rna and virus-induced plaque formation were inhibited with an ec 50 value of about 2 m. similar to prior work with t-705, time-of-addition assays demonstrated that 2cm-c acts during viral rna synthesis, which is consistent with the viral rna polymerase being the antiviral target. target identification was confirmed in biochemical assays showing that 2cm-c triphosphate directly inhibits norovirus polymerase by terminating rna synthesis . in vitro combination of 2cm-c with ribavirin induced a marked antagonistic antiviral effect by a mechanism that was not clearly understood (rocha-pereira et al., 2012a) . in a separate study, however, no antagonism was observed between 2cm-c and ribavirin (costantini et al., 2012) . 2cm-c also inhibits hunov replication in a norwalk virus replicon assay, with reduced inhibition potency compared with the mnv assay (rocha-pereira et al., 2013) . importantly, the antiviral activity of 2cm-c against hunov was confirmed in the b-cell line bjab infection assay with an ec 50 value of 0.3 m, which is significantly more potent than the value obtained with the hunov replicon . treatment of mnv-infected ag129 mice with 2cm-c prevented virus-induced onset of diarrhea and mortality by reducing viral load in organs (rocha-pereira et al., 2013) , even when treatment started between 12 h and 2 days post-infection . in contrast, 2cm-c did not reduce mortality when treatment was initiated after the onset of symptoms 3 days post-infection. in a norovirus transmission model, prophylactic treatment of sentinel mice with 2cm-c also protected against transmission of mnv infection (rocha-pereira et al., 2015) . 2cm-c also had an antiviral effect against hunov in balb/c rag-␥c-deficient mice . the nucleoside analog 2 -fluroro-2 -c-methyl-cytidine (2 -f-2 -c-mec, or psi-6130) is structurally related to 2cm-c (fig. 5b) . it is the parent nucleoside of the prodrug mericitabine, an hcv inhibitor that acts as a chain terminator and has reached phase 2 clinical development during which it was well tolerated. in cell-based assays, 2 -f-2 -c-mec and 2cm-c have comparable antiviral activ-ity against both mnv and hunov (costantini et al., 2012) . although no biochemical data directly compare the inhibition effect on the target of each ntp, this result suggests that 2 -f-2 -c-mec and 2cm-c are both recognized as a cytidine analog for the norovirus polymerase with similar substrate efficiency. for this reason, it would be interesting to further evaluate the antiviral efficacy of 2 -f-2 -c-mec in a mouse model of norovirus infection. the antiviral activity of 5-nitrocytidine was first described against poliovirus and coxsackievirus b3, demonstrating greater inhibition potency than the control drug ribavirin (harki et al., 2007) . in the same study, 5-nitrocytidine triphosphate (nct) inhibited poliovirus rdrp activity. a high-resolution structure of norovirus polymerase bound to nct was determined by x-ray crystallography (zamyatkin et al., 2008) . in this structure, the rdrp is trapped in a ternary complex that includes a self-complementary primer-template rna and two metal ions, mimicking the conformation of the protein during rna synthesis. the nct molecule occupies the nucleotide binding site and its interactions with the active site residues in the hunov are different from those of natural cytidine triphosphate (i.e., the negatively-charged nitro group of the nct is positioned close to the primer 3 oh to destabilize the transition state of the nucleotidyl transfer reaction) (fig. 6a) . the nitro group also displaces a water molecule from interacting with the ␣-phosphate of the nct and results in increased distance in the coordination between asp344 and metal a. finally, the side chain of arg182 is spatially affected to decrease its interaction with the ␤-phosphate of the nct, likely decreasing the effectiveness of arg182 from stabilizing the pyrophosphate leaving group. although 2 -amino-cytidine triphosphate (act) makes overall similar interactions with the polymerase of norovirus, it also features some significant differences (zamyatkin et al., 2009 ). the nucleobase of act forms watson-crick hydrogen bonds with the template nucleotide and the ribose exhibits primarily a c3endo pucker typically found in rna (fig. 6b) . the ribose and triphosphates of the act are shifted in position to accommodate the 2 -amino group. asp247, which normally forms a hydrogen bond with ser300 to recognize the ribose 2 oh, is out of place. asp242 that binds both divalent metal ions is displaced by 2 å and could impact divalent metal occupancy in the active site. as a result of these relatively subtle changes, the primer 3 oh and the ␣-phosphate in act are not positioned to allow catalysis of nucleotidyl transfer. these interactions could have important implications in the inhibition of norovirus rna replication by nucleotide analogs. although prior studies have shown that other 2 -amino nucleosides can have an antiviral effect on viruses unrelated to caliciviridae (de clercq et al., 1980) , more work is needed to demonstrate that 5-nitro and 2 -amino cytidine have the potential to inhibit norovirus replication. suramin is an antiparasitic molecule originally developed by bayer in 1916. it has a long symmetric structure containing two charged naphthalene-trisulfonic acid heads (fig. 7) . suramin has also been identified in vitro as an antiviral agent potent against human immunodeficiency virus reverse transcriptase and hepatitis b virus (jentsch et al., 1987; offensperger et al., 1993) . more recently, suramin was identified in an in silico docking search for compounds predicted to bind to the active site of norovirus polymerase (mastrangelo et al., 2012) . this modeling exercise was confirmed by measuring the inhibition of the rdrp activity of hunov and mnv polymerase by suramin, resulting in ic 50 values 25 and 70 nm, respectively. nf023, an analog of suramin harboring the same two groups connected by a shorter linker, displayed similar inhibition potency (fig. 7) . interestingly, nf023 is also a known competitive and reversible p2 × 1 receptor antagonist (sneddon et al., 2000; soto et al., 1999) . the analysis of the detailed binding mode of suramin and nf023 to the mnv polymerase domain was made possible by x-ray crystallography. it showed that both molecules occupy the same binding site, between the fingers and the thumb subdomains. the importance of the interaction of the two molecules with tryptophan 42 was demonstrated by site-directed mutagenesis, which resulted in significant loss in inhibition potency. two smaller analogs of suramin, naphthalene di-sulfonate and pyridoxal-5 -phosphate-6-(2 -naphthylazo-6 -nitro-4 ,8 -disulfonate (ppnds), retained some level of biochemical effect and were also able to bind to a second region of the protein located in the thumb subdomain (croci et al., 2014a,b; tarantino et al., 2014) (fig. 7) . one would predict that the high inhibition potency of suramin and its analogs in biochemical assays would translate to a significant antiviral effect in cells infected with mnv. however, suramin has only a modest antiviral effect on norovirus replication, presumably due to low cell permeability . in order to further advance these compounds as potential anti-norovirus drugs, attempts have been made to improve cellular permeability by delivering suramin through liposomes . a separate study showed the in vitro and in vivo antiviral activity of suramin against enterovirus 71, a member of the picornaviridae family (ren et al., 2014) . the same study reported that suramin blocked enterovirus 71 replication by interfering with the viral capsid assembly process. taken together, these results indicate that suramin and its structural analogs are promiscuous binders that can interact with multiple classes of proteins, including but not limited to the norovirus polymerase (morgan et al., 2011; torrente et al., 2014) . to minimize the off-target liability caused by promiscuous binding, it may be possible to increase the selectivity of this class of compounds by further building binding interactions between naphthalene di-sulfonate or ppnds and the second thumb binding pocket in norovirus polymerase. the fluorescence-based picogreen biochemical assay has been used in a high throughput screening campaign to identify small molecule inhibitors of gii.4 hunov rdrp activity (eltahla et al., 2014) , using a model previously established for hcv polymerase (eltahla et al., 2013) . the four most interesting hits were a phenylthiazole carboxamide (nic02) and a pyrazole acetamide (nic04) with ic 50 values around 5 m, and a triazole (nic10) and a pyrazolidinedione (nic12) that were about 2-fold less potent in the polymerase assay (fig. 8) . interestingly, nic12 had already been reported to weakly inhibit poliovirus rdrp (campagnola et al., 2011) , while the three other compounds were not previously known to inhibit viral polymerases. nic02 was the only compound to also show consistent cell-based inhibition in the norwalk replicon and mnv infection assays. in comparison, nic04 was significantly less potent. nic10 and nic12 were inactive in the replicon, and weak (ec 50 ∼35 m) in the mnv assay. mode of action inhibition studies suggested that nic02 and nic04 occupy a different binding pocket of hunov rdrp than nic10 and nic12. however, these findings will need to be confirmed by other methods such as x-ray crystallography and/or identification of resistance-associated mutations. the key role that the norovirus rdrp plays in viral genome replication and the fact that host cells lack an equivalent function make norovirus rdrp an attractive target for development of norovirus-specific antiviral therapies. although many in vitro and in vivo systems are now available to identify and develop inhibitors of norovirus rdrp, current research in this field remains at an early stage of drug discovery, with nitazoxanide being the only small molecule to be evaluated in human clinical trials. the fact that norovirus typically causes a short and self-limiting infection, with a narrow time window for therapeutic intervention has limited the effort and interest in the development of antivirals. however, the consequences of norovirus infection can be severe and prolonged in vulnerable populations such as children, the elderly, and immunocompromised patients. therefore, increased commitment to the development of small molecules treatments alongside the current anti-norovirus vaccine approach is needed. in fact, much is already known about the specific recognition of nucleotides and nucleotide analogs that should guide structure-based inhibitor design. additional locations in the hunov rdrp that bind nonnucleoside inhibitors have also been identified and characterized. finally, since norovirus rdrp shares functional and 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vitro zika virus replication and delays disease progression in a robust mouse infection model we thank x. lin for helpful discussions of the norovirus structure during this work. we also thank peggy korn and anh truong for their editorial comments. key: cord-333180-xevie6tm authors: neumann, simon; el maadidi, souhayla; faletti, laura; haun, florian; labib, shirin; schejtman, andrea; maurer, ulrich; borner, christoph title: how do viruses control mitochondria-mediated apoptosis? date: 2015-11-02 journal: virus res doi: 10.1016/j.virusres.2015.02.026 sha: doc_id: 333180 cord_uid: xevie6tm there is no doubt that viruses require cells to successfully reproduce and effectively infect the next host. the question is what is the fate of the infected cells? all eukaryotic cells can “sense” viral infections and exhibit defence strategies to oppose viral replication and spread. this often leads to the elimination of the infected cells by programmed cell death or apoptosis. this “sacrifice” of infected cells represents the most primordial response of multicellular organisms to viruses. subverting host cell apoptosis, at least for some time, is therefore a crucial strategy of viruses to ensure their replication, the production of essential viral proteins, virus assembly and the spreading to new hosts. for that reason many viruses harbor apoptosis inhibitory genes, which once inside infected cells are expressed to circumvent apoptosis induction during the virus reproduction phase. on the other hand, viruses can take advantage of stimulating apoptosis to (i) facilitate shedding and hence dissemination, (ii) to prevent infected cells from presenting viral antigens to the immune system or (iii) to kill non-infected bystander and immune cells which would limit viral propagation. hence the decision whether an infected host cell undergoes apoptosis or not depends on virus type and pathogenicity, its capacity to oppose antiviral responses of the infected cells and/or to evade any attack from immune cells. viral genomes have therefore been adapted throughout evolution to satisfy the need of a particular virus to induce or inhibit apoptosis during its life cycle. here we review the different strategies used by viruses to interfere with the two major apoptosis as well as with the innate immune signaling pathways in mammalian cells. we will focus on the intrinsic mitochondrial pathway and discuss new ideas about how particular viruses could activately engage mitochondria to induce apoptosis of their host. there is no doubt that viruses require cells to successfully reproduce and effectively infect the next host. the question is what is the fate of the infected cells? all eukaryotic cells can "sense" viral infections and exhibit defence strategies to oppose viral replication and spread. this often leads to the elimination of the infected cells by programmed cell death or apoptosis. this "sacrifice" of infected cells represents the most primordial response of multicellular organisms to viruses. subverting host cell apoptosis, at least for some time, is therefore a crucial strategy of viruses to ensure their replication, the production of essential viral proteins, virus assembly and the spreading to new hosts. for that reason many viruses harbor apoptosis inhibitory genes, which once inside infected cells are expressed to circumvent apoptosis induction during the virus reproduction phase. on the other hand, viruses can take advantage of stimulating apoptosis to (i) facilitate shedding and hence dissemination, (ii) to prevent infected cells from presenting viral antigens to the immune system or (iii) to kill non-infected bystander and immune cells which would limit viral propagation. hence the decision whether an infected host cell undergoes apoptosis or not depends on virus type and pathogenicity, its capacity to oppose antiviral responses of the infected cells and/or to evade any attack from immune cells. viral genomes have therefore been adapted throughout evolution to satisfy the need of a particular virus to induce or inhibit apoptosis during its life cycle. here we review the different strategies used by viruses to interfere with the two major apoptosis as well as with the innate immune signaling pathways in mammalian cells. we will focus on the intrinsic mitochondrial pathway and discuss new ideas about how particular viruses could activately engage mitochondria to induce apoptosis of their host. © 2015 elsevier b.v. all rights reserved. apoptosis is a highly conserved, physiological type of cell death, which is required to eliminate damaged, used-up and misplaced cells within a multicellular organism (kerr et al., 1972; hotchkiss et al., 2009) . in contrast to necrosis or necroptosis, which often leads to pathological, chronic inflammatory immune reactions due to cell lysis, apoptotic cells do not fall apart but are discretely eaten up by macrophages and other non-professional phagocytes in a regulated and non-inflammatory manner (vanlangenakker et al., 2012) . apoptosis is induced by two distinct, yet tightly disruption of the cytoskeleton, inhibition of dna repair, initiation of dna fragmentation and the exposure of so-called eat-me signals, which mediate the uptake and elimination of apoptotic cells by macrophages. the extrinsic, death receptor pathway is indispensable for the execution and limitation of both innate and adaptive immune responses. cells of the innate immune system such as dendritic or natural killer (nk) cells generate a rapid antiviral immune response by directly detecting viral products such as dsrna by toll-like receptor 3 (tlr), by upregulating death ligands such as fasl, trail and tnf␣ and/or by killing infected cells by granule exocytosis (nk cells) (medzhitov, 2001) . later during adaptive immunity, virally infected cells are killed by antigen-specific cytotoxic t cells (ctls) via both the perforin/granzyme and the fasl/fas pathways (chavez-galan et al., 2009 ). subsequently, activated t cells are eliminated by activation-induced cell death involving fasl/fas-mediated killing of the same or an activated neighboring cell after an acute infection (arakaki et al., 2014) . this ensures that highly proliferative, superfluous killer cells are disposed of accordingly, preventing the development of autoimmune or leukemic cells. it is therefore not surprising that viruses have evolved strategies to inhibit the death receptor signaling pathway at several steps although these cells may still be killed by granule exocytosis (benedict et al., 2002; hay and kannouraki, 2002) . as a consequence infected cells are at least partially protected from cytolysis by ctls, nk cells or neighboring cells which express fasl or trail upon activation (ashkenazi and dixit, 1999 ) and tnf␣ may not be able to induce a sufficient inflammatory response. for example, the shope fibroma virus (rabbit poxvirus) produces a tnf receptor ortholog (tnfr2), which neutralizes tnf␣ (smith et al., 1991) (fig. 1) . other tnfr orthologs have been identified in the genomes of lepri-and orthopoxviruses, including cowpox (crmb) (hu et al., 1994) and smallpox (crme) (reading et al., 2002) and in the genome of cmv (ul144 orf) (benedict et al., 1999) . they can either be expressed in the plasma membrane of infected cells or be shed as soluble decoy receptors (for example myxoma virus t2 protein) reading et al., 2002) . these tnf signaling inhibitors clearly contribute to the high virulence of poxviruses because they are mutated in vaccinia viruses, an attenuated form of smallpox. in addition, a trail receptor ortholog (trailr2) was detected in the genome of avian leukocytosis virus (brojatsch et al., 1996) . on the other hand adenoviruses use several proteins encoded in the e3 gene region to promote the internalization and lysosomal degradation of fas, tnfr, trailr1 and r2 (shisler et al., 1997; tollefson et al., 1998; stewart et al., 1995; benedict et al., 2001) . finally, several viruses produce proteins capable of blocking extrinsic death receptor signaling at the level of the disc, i.e. by either inhibiting caspase-8 activation or its proteolytic activity. for example, the viral flip proteins (vflips) are inactive caspase-8 homologs, which are recruited by fadd to form a disc that lacks caspase activity (thome and tschopp, 2001; krueger et al., 2001; subramaniam et al., 2013) . in addition, they can recruit traf2, rip, nik and ikk2, which favor the induction/activation of nfb, a crucial anti-apoptotic transcription factor (kataoka et al., 2000; subramaniam et al., 2013) . vflips are present in the genome of ␥-herpesviruses, including equine herpesvirus 2 (ehv-2), herpesvirus saimiri (hvs), kshv (kaposi), bovine herpesvirus (bhv-4) and moluscum contagiosum virus (mcv) (bertin et al., 1997; thome and tschopp, 2001; benedict et al., 2002) . the vica protein produced by the hcmv viral gene ul36 also associates with caspase-8 and blocks its activation but it has no sequence homology with caspases (skaletskaya et al., 2001) . last but not least, the crma protein from cowpox virus is able to inhibit the proteolytic activity of caspase-8 by binding to and blocking the catalytic center (zhou et al., 1997) . the intrinsic apoptotic pathway is activated by the lack of soluble survival factors or hormones, cell-cell or cell-matrix interactions (deprivation-induced cell death, also called anoikis), the exposure of cells to pathogens such as fungi, bacteria or viruses or the treatment with genotoxic/dna damaging stimuli (irradiation, chemotherapeutic drugs), toxins or pro-oxidants, agents which stress the endoplasmic reticulum, inhibit protein kinases, proteasomal degradation, transcription, translation or perturb the cytoskeleton (fig. 2) (youle and strasser, 2008; hotchkiss et al., 2009; chipuk et al., 2010) . the critical step of this pathway is the permeabilization of the mitochondrial outer membrane (momp), which results in the release of several apoptogenic factors from the intermembrane space of mitochondria. one such protein, cytochrome c, binds to the adapter apaf-1 which then recruits cytosolic pro-caspase-9 into a heptameric complex, called the apoptosome (fig. 2) . in a similar way as caspase-8 aggregates on the disc, monomeric caspase-9 dimerizes on the apoptosomal platform leading to proximity-induced autoprocessing and activation. active caspase-9 then cleaves and activates effector caspase-3 and -7. other proteins released from mitochondria either stimulate yet unknown caspase-independent cell death processes or they enhance caspase-9 and -3 activation (fig. 2) . for example smac/diablo and htr2a/omi bind to xiap, a member of the inhibitors of apoptosis proteins (iaps). xiap is an endogenous caspase-9 and -3 inhibitor, which prevents accidental autoprocessing and activation of these caspases in healthy cells (deveraux and reed, 1999; shi, 2002) . upon sequestration by smac or omi, xiap no longer binds to caspase-9 or -3, therefore allowing the full activation of these caspases in response to apoptotic stimuli, activating the intrinsic mitochondrial pathway (fig. 2) . since momp results in both caspase-dependent and independent cell death signaling it is a crucial life-or-death decision checkpoint ("a point of no return") ( fig. 2) . this checkpoint is controled by the bcl-2 family of proteins (youle and strasser, 2008; chipuk et al., 2010) . the bcl-2 family is subdivided on the basis of structural conservation of so-called bcl-2 homology (bh) domains and comes in three flavors. the pro-apoptotic bh3-only proteins (bim, bid, bad, bik, bmf, hrk, puma, noxa) only share a ca. 20-30 amino acid long alpha-helical bh3 domain with the rest of the bcl-2 family. they act as sentinels/sensors of apoptotic stimuli which activate the intrinsic mitochondrial pathway (happo et al., 2012) . depending on the apoptotic stimulus, particular sets of bh3-only proteins get transcriptionally induced or posttranslationally modified, migrate to and insert into the mom and then activate at this site a second pro-apoptotic subclass of bcl-2 family proteins, the socalled "effectors" bax and bak (happo et al., 2012) . for example, bim is transcriptionally induced by foxo3b in response to growth factor deprivation or phosphorylated by jnk during thymic selection and upon exposure to uv or gliotoxin (happo et al., 2012; geissler et al., 2013) . by contrast, puma and noxa are target genes of p53 after genotoxic stress. bid is proteolytically cleaved to truncated tbid by caspase-8 in response to death receptor ligands such as fasl, trail or tnf␣ defining a second, so called type ii death receptor pathway that crosstalks with the mitochondrial pathway (youle and strasser, 2008; chipuk et al., 2010; happo et al., 2012) . bax and bak directly induce momp. they contain bh1, bh2 and bh3 domains, which form an elongated hydrophobic binding pocket interacting with other members of the bcl-2 family (suzuki et al., 2000; czabotar et al., 2013; brouwer et al., 2014; volkmann et al., 2014; westphal et al., 2014; borner and andrews, 2014) . in healthy cells, bax resides inactive in the cytoplasm (suzuki et al., 2000; schinzel et al., 2004) and constantly shuttles between the cytoplasm and the periphery of the mom without stably inserting in the membrane (retrotranslocation) (wolter et al., 1997; edlich et al., 2011) . bak on the other hand is stably inserted into the mom but held in check by inhibitory proteins such as vdac2 and bcl-2 survival factors (wang et al., 2001; cheng et al., 2003; willis et al., 2005) . three bh3-only proteins bim, tbid and puma are capable of directly activating bax/bak on the mom (letai et al., 2002; cartron et al., 2004; kuwana et al., 2005; gavathiotis et al., 2008; czabotar et al., 2013) . recent structural analysis of the activation process suggests that after their translocation to and insertion into the mom, bim and tbid bind to the hydrophobic pocket in bax/bak via their bh3-domain (czabotar et al., 2013; brouwer et al., 2014) . this changes the conformation of bax and bak in a way that their bh3-regions become exposed for dimeric interaction with the hydrophobic pocket of another bax or bak molecule (dewson et al., 2009; czabotar et al., 2013; brouwer et al., 2014) . for that purpose the bh3-only protein has to dissociate from the hydrophobic binding pocket, which explains why direct interaction between bh3-only proteins and bax/bak is only transient and difficult to detect biochemically. presumably through an additional interaction site in the rear part of the molecule, bax/bak dimers can then assemble into multimers (dewson et al., 2009 ). it is not yet clear if these multimers form a protein pore or perturb the lipid bilayer of the mom to rearrange into a lipid pore that may be generated by a hemifusion intermediate (montessuit et al., 2010; bleicken et al., 2014; borner and andrews, 2014) . newest findings from lipid nanodiscs and fluorescence measurements indicate that bax/bak may form pores of different sizes depending on whether they are monomeric, di-or multimeric (xu et al., 2013; volkmann et al., 2014) . at one point the pores are large enough to allow the passage of cytochrome c or even bigger molecules such as smac and omi to the cytoplasm. the third subgroup of the bcl-2 family is formed by the antiapoptotic proteins bcl-2, bcl-x l , mcl-1, bcl-w and a1 (youle and strasser, 2008; chipuk et al., 2010) . they also contain bh1, bh2 and bh3 domains and some even have an additional bh4 domain at the n-terminus. the 3-dimensional structure of these survival factors looks very similar to that of bax/bak (muchmore et al., 1996; suzuki et al., 2000) . however, in contrast to bax/bak, they are unable to undergo conformational changes to expose their bh3domain, multimerize and form pores, except under unphysiological conditions such as low ph (minn et al., 1997; schendel et al., 1997) . instead their hydrophobic binding pocket interacts with bh3-only proteins in a high affinity manner. on one hand this inhibits the pro-apoptotic action of bh3-only proteins. on the other hand, prebound bim, tbid and puma can be released from the bcl-2 survival factors by other bh3-only proteins activated by apoptotic stimuli such as bik, bad, bmf, hrk and noxa (borner and andrews, 2014) . bim, tbid and puma can then directly activate bax/bak as described above. this explains the effectiveness of bh3-mimetics in anticancer therapy. these are small molecule compounds that bind with high affinity to the hydrophobic pocket of bcl-2 survival factors and thereby release pre-bound bim, tbid and puma for momp activation (oltersdorf et al., 2005; happo et al., 2012) . it turns out that many cancer cells do not only upregulate bcl-2 survival factors but also bim, tbid and puma, a mechanism called "addiction" (certo et al., 2006) . this may explain the high sensitivity of cancer cells to be killed by bh3-mimetics such as abt-267, abt-199 and others (tse et al., 2008; wilson et al., 2010; vandenberg and cory, 2013) . apart from interacting with bh3-only proteins, bcl-2 survival factors also sequester accidently activated bax and bak in healthy cells. since active bax/bak expose their bh3-domain, this region can interact with the hydrophobic pocket of bcl-2 survival factors and hence bax/bak are inhibited willis et al., 2005 willis et al., , 2007 uren et al., 2007) . this may also constitute one of the mechanisms by which mom-inserted bak is kept in check by bcl-2 survival factors. when large amounts of bax/bak are bound by bcl-2 survival factors, bh3-only proteins can displace appreciable numbers of bax/bak molecules from bcl-2 and therefore make more pore-forming proteins available for the activation of momp (youle and strasser, 2008; borner and andrews, 2014) . since bcl-2 survival factors are able to sequester both the "activators" (bh3-only proteins) and the "effectors" (bax/bak) of momp and momp triggers both caspase-dependent and -independent cell death, overexpression of these survival factors is the most efficient way to block apoptosis, even allowing clonogenic survival (which caspase inhibitors are not able to do) (borner, 2003) . given the importance of the intrinsic mitochondrial pathway for apoptosis induced by numerous stimuli, viruses have acquired gene products which mimick the action of bcl-2 survival factors to potently inhibit momp in their host cells. functional homologs of bcl-2 are collectively called vbcl-2s. they are present in various members of the poxviridae (f1l, n1l, m11l, a179l, orfv125), herpesviridae (bhrf1, balf1, vmia, ksbcl-2, mhvbcl-2), adenoviridae (e1b-19k) and birnaviridae (vp5) family of viruses ( fig. 2) (benedict et al., 2002; galluzzi et al., 2008; postigo and ferrer, 2009 ). while some of them like e1b-19k have extensive sequence homology with cellular bcl-2 survival factors in all regions (white, 1998) , others such as fpv039 from fowlpoxvirus only retain sequence homology with the bh1 and bh2 domains while orfv125 of parapoxvirus is homologous in the bh1 and bh3 regions (galluzzi et al., 2008; westphal et al., 2007; banadyga et al., 2007) . other vbcl-2s such as vaccinia virus f1l and n1l, myxoma virus m11l and vmia from cytomegalovirus do not even show any sequence homology with mammalian bcl-2 proteins (galluzzi et al., 2008) . however, the crystal structure of some of these vbcl-2s reveals a close conservation of the bcl-2 family structural conformation. thus, it is not the primary amino acid sequence and/or the conserved bh-domains per se, which determine the function of a bcl-2 survival factor but the helical bundle structure that makes up the hydrophobic pocket where bh3-only proteins and bax/bak are sequestered. other viral proteins inhibit the intrinsic, mitochondrial signaling pathway by modulating bcl-2 family members on the transcriptional level or via post-translational modifications. the tumor suppressor p53 is known to induce the transcription of the bh3only proteins noxa and puma in response to genotoxic stress (oda et al., 2000; nakano and vousden, 2001) and growth factor deprivation (jabbour et al., 2010) contributing to the elimination of stressed and damaged cells (fig. 2) . cells lacking functional p53 survive and proliferate and accumulate gene mutations eventually leading to cancer (harvey et al., 1993) . also, p53-deficient mice are more prone to certain viral infections, indicating that p53 is not only crucial to prevent cancer but also to induce apoptosis of some infected host cells. therefore, viruses have evolved strategies to inactivate p53. the sv40 large t antigen binds to p53 and sequesters it in an inactive complex (lane and crawford, 1979; linzer and levine, 1979) . the human papillomavirus (hpv) e6 protein and the adenovirus e1b-55k protein induce the ubiquitination and proteasomal degradation of p53 (scheffner et al., 1990; werness et al., 1990; steegenga et al., 1998; querido et al., 2001) . also, the x protein of hepatitis b virus (hbx) interacts with p53 and prevents its transcriptional activation of target genes, thereby inhibiting apoptosis (wang, 1995) (fig. 2) . a similar strategy is used by the measles virus v protein, which blocks apoptosis by sequestering the p53 homolog p73 (cruz et al., 2006) . a p53-independent mechanism to inhibit momp is exploited by the human t cell leukemia virus type i (htlv-1) which uses the tax protein to activate the bcl-x l promoter and to repress the bax promoter (tsukahara et al., 1999) . moreover, viruses can effectively block both the intrinsic mitochondrial and the extrinsic death receptor signaling by engaging the transcription factor nfb (sun and cesarman, 2011) . nfb induces transcription of the survival factor bcl-x l , the caspase-8 inhibitor flip or inhibitor of apoptosis proteins (iaps), which often directly or indirectly act as caspase inhibitors (didonato et al., 2012) . for example, herpes simplex virus-1 (hsv-1), through its envelope glycoprotein d uses a tnfr family member, herpes virus entry mediator (hvem) for host surface binding and infection. activation of this receptor triggers a signaling cascade that leads to nfb activation and apoptosis inhibition (medici et al., 2003; sciortino et al., 2008) . similarly, the major b cell-transforming protein in ebv, lmp1, mimics activated cd40 and engages tradd and trafs, which are crucial adaptors used by tnfr to activate nfb. thereby lmp1 prevents b cells from localizing to the follicle and protects cells harboring latent virus from interactions with t cells (uchida et al., 1999) . finally, the nef protein of hiv (wolf et al., 2001) and the u(s)3 protein kinase of hsv-1 (munger and roizman, 2001) were found to mediate phosphorylation of the bh3-only protein bad, thereby preventing it from inducing apoptosis. another possibility for viruses to interfere with apoptosis signaling is to block caspases. as pointed out above cowpox viruses can effectively block initiator caspase-8 of the extrinsic death receptor pathway because this pathway is strictly caspase-dependent (zhou et al., 1997) . the major initiator and effector caspases of the intrinsic mitochondrial pathway, caspase-9 and caspase-3/-7 are held in check by cellular iaps, in particular xiap (fig. 2 ) (deveraux and reed, 1999; shi, 2002) . however, momp also leads to caspase-independent death signaling (fig. 2) . therefore blocking caspase-9 and/or -3 by viruses may not be sufficient to save infected host cells from apoptosis. this explains why an iaportholog strategy is infrequently used in mammalian viruses (in contrast to baculoviruses infecting insect cells) (taylor and barry, 2006) . accordingly, although african swine fever virus and entomopoxviruses encode viral viap such as msepv and amepv these proteins do not contribute to the virulence of these viruses (neilan, 1993; taylor and barry, 2006) (fig. 2) . since viruses have developed so many ways to oppose the intrinsic mitochondrial apoptosis pathway, the question arises, why and when this pathway is activated in infected cells. three scenarios are possible: (i) the infected cell is killed by an external apoptotic signal, (ii) the infected cell "senses" virus entry or assembly and mounts an antiviral stress response which does not only eliminate the virus but also the infected cell or (iii) the virus actively induces apoptosis of its host by using particular viral components expressed in the infected cells (viral proteases, dsrna, dsdna, etc.). in addition to the activation of the fas signaling pathway (described above), ctls and nk cells also kill virally infected target cells via the perforin/granzyme pathway. after successful activation, these cells release perforin and granzyme a and b from their cytotoxic granules at the immunosynapse where they contact the virally infected cell via mhc-i/peptide/tcr and costimulatory molecular interactions (chavez-galan et al., 2009; ewen et al., 2012; thiery and lieberman, 2014) (fig. 2) . perforin oligomerizes into variously sized high molecular structures in the target cell membrane (metkar et al., 2015) . most likely through membrane lipid perturbation, similar to what is proposed for bax and bak on the mom (bleicken et al., 2014) , perforin forms a pore, which allows the entry of the serine proteases granzyme a and b (metkar et al., 2015) (fig. 2) . while granzyme a provokes a still not entirely identified caspase-independent death pathway and is also responsible for inflammatory responses (pardo et al., 2009a (pardo et al., , 2009b joeckel and bird, 2014) , granzyme b induces classical apoptosis in the target cell by two pathways, (i) a direct cleavage and activation of caspase-3 and (ii) the cleavage of bid (similar to what caspase-8 does) and the triggering of bax/bak-mediated momp (pardo et al., 2009a (pardo et al., , 2009b ewen et al., 2012; thiery and lieberman, 2014) . viruses counteract the granzyme b killing pathway by expressing vbcl-2 survival factors. alternatively they produce inhibitors of granzyme b, such as the l4-100k assembly protein from adenovirus (andrade et al., 2001) , crma from cowpox virus (which also inhibits caspase-8) (komiyama et al., 1996) or serp2 encoded by the leporipoxvirus myxoma virus (turner et al., 1999) (fig. 2) . all these inhibitors clearly block granzyme b action in cell free systems. but while the l4-100k protein also potently inhibited granzyme b-mediated cell death (andrade et al., 2001) , this could not be effectively observed with cells overexpressing crma or serp2 or infected with poxviruses (müllbacher et al., 1999; barry et al., 2000; screpanti et al., 2001; pardo et al., 2009a pardo et al., , 2009b . in the following section we focus on rna viruses (fig. 3 ) but similar mechanisms have either been reported or are expected to occur for dna viruses (goubau et al., 2013; unterholzner, 2013) . irrespective of whether virus replication and spread are blocked and viruses are finally eliminated by non-apoptotic or apoptotic mechanism, the infected host cells first have to recognize, i.e. "sense" the respective viruses. this places the molecular mechanisms of sensing at the heart of all anti-viral effects, including the initation of cell death. virus spread is prevented if host cell apoptosis occurs before a virus can form progeny, or if an infected cell is successfully eliminated by ctls. if however the virus can inhibit cell death by expressing own survival factors after infection or if it kills cells after its reproduction, it can successfully propagate and evade the immune system. the best known antiviral "sensing" mechanism of hosts for rna viruses triggers the so called type i interferon response (takeuchi and akira, 2009; ivashkiv and donlin, 2014) . type i interferons such as ifn␣ and ifn␤ are transcriptionally induced after viral infections and play a critical role in mounting innate responses against viruses (fig. 3) . they are secreted and bind to specific interferon receptors on the same as well as on neighboring cells in order to ensure a spreading of the antiviral state to as many cells as possible. activated interferon receptors then trigger a signaling cascade via the janus protein kinase-signal transducer and activators of transcription pathway (jak-stat) that results in the induction of ifn-stimulated genes (isgs) which can interfere with the viral life cycle at different steps, induce host cell apoptosis or both (fig. 3) (ivashkiv and donlin, 2014) . two major components of this ifn-induced signaling system are the rna-dependent protein kinase (pkr) (garcia et al., 2007) and the 2 ,5-oligoadenylate and rnase-l systems (hovanessian, 2007) . activation of rnasel leads to the degradation of viral rna therefore blocking further rna replication and transcription. on the other hand pkr activation inhibits host-cell protein translation via the phosphorylation of elongation initiation-factor 2a (eif2␣) (garcia et al., 2007) (fig. 3) . in addition, active pkr was proposed to induce host cell apoptosis via nfb and irf-1 activation, which then upregulated fasl and trail respectively (tan and katze, 1999; jagus et al., 1999) . although trail is induced by ifns in a variety of paradigms of viral infections and may indeed contribute to apoptosis of host cells, it is unclear if this is really mediated via the pkr/nfb axis (allen and el-deiry, 2012) . also, nfb often acts as a cell death protective rather than apoptosis-inducing transcription factor (didonato et al., 2012) . moreover, we recently reported that pkr was not required for apoptosis induced by the rna virus sfv although it was clearly activated and blocked protein synthesis via eif2␣ phosphorylation (el maadidi et al., 2014) . in order for a host cell to sense a virus it has to recognize it as non-self. this occurs through two principle mechanisms. first, viruses are detected on the host cell surface by so-called pattern recognition receptors (prr) which bind to pathogen associated molecular patterns (pamps) on the virus and other pathogens (pichlmair and reis e sousa, 2007; kumar et al., 2011) . a major class of cell surface transmembrane prr are the toll-like receptors (tlrs) whose pamp-binding domains contain leucine-rich repeats (lrr). tlr3, tlr7, trl9 detect viral nucleic acid ligands such as long dsrna, ssrna or cpg dna, respectively, mostly in the endosomal compartments (subsequent to endocytosis of the virus). after binding, they recruit adaptor proteins such as trif (for tlr3) or myd88 (tlr7/tlr9), which bind specific members of the traf adaptor and activate the irak kinase families. this ultimately leads to the nuclear translocation of nfb and irf3 and irf7 transcription factors through respective phosphorylation and activation of the ikb kinase (ikk) ␣,␤,␥ and ikk/tbk1 complexes (pichlmair and reis e sousa, 2007; bowie and unterholzer, 2008; kawai and akira, 2008; kumar et al., 2011) (fig. 3) . nfb and irf3/7 then cooperate to induce the transcription of ifn␣ and ifn␤. recently, it was reported that activation of the tlr3 pathway by the synthetic dsrna homologs polyic can induce apoptosis by recruiting caspase-8 to tlr3 (weber et al., 2010; estornes et al., 2012) . in an earlier report, trif, an adaptor for tlr3 was shown to induce apoptosis in a fadd/caspase-8-dependent manner (kaiser and offermann, 2005) (fig. 3) . in all cases caspase-8 was activated by proximity-induced dimerization and then cleaved and activated caspase-3 (fig. 3) . this defines a novel, death receptorand mitochondria-independent death signaling pathway involving caspase-8. viruses evolved strategies to inhibit this signaling pathway downstream of tlrs to avoid the transcription of type i interferons. for instance, the ns3-4a protease of hepatitis c virus (hcv) cleaves the adaptor trif (roy and mocarski, 2007) while the ns5a protein decreases the expression of tlr4 (tamura et al., 2011) . vaccinia virus uses its a46 protein to inhibit all tlr-adaptors (myd88, mal, trif, tram), the a52 protein to block traf6 and the protein kinase irak2, the b14 protein to antagonize the ikk␣,␤,␥ complex and the k7 protein to prevent the ikk/tbk1 complex from activating irf3/7 (bowie and unterholzer, 2008) . the latter strategy is also used by hcv ns3, rabies virus phosphoprotein and hantavirus g1. in addition to the recognition of viruses in the endosomal compartment, cells also contain viral sensors in the cytosol (pichlmair and reis e sousa, 2007; kawai and akira, 2008; takeuchi and akira, 2009 ). these are the rig-like helicases (rlh) rig-i, mda5 and lgp-2 (yoneyama et al., 2004; kato et al., 2006) . rig-i and mda5 both contain two caspase recruitment domains (cards), an atpase and a helicase domain (fig. 3) . the helicase domain is used to recognize different kinds of nucleic acids. while mda5 mainly senses long viral dsrna molecules, rig-i is often activated by short dsrna fragments and ssrnas that have a 5 triphosphate (pichlmair and reis e sousa, 2007) . this concept ensures that cellular rnas whose 5 ends are either capped or contain monophosphates, are not recognized by rlhs. activated rig-i and mda5 then use their card domains to interact with the mitochondria-located adaptor protein mavs (also known as ips-1, visa or cardif) (kawai et al., 2005; meylan et al., 2005; seth et al., 2005; xu et al., 2005) (fig. 3) . mavs in turn then triggers ikk␣,␤,␥ as well as ikk/tbk1 phosphorylation that activate nfkb and irf3 and irf7 transcription factors for ifn␣ and ifn␤ induction (takeuchi and akira, 2009 ). similar to tlr3 signaling, sensing through rig-i/mda5/mavs is heavily antagonized by viruses. influenza ns1 binds the rig-i/mavs complex and thereby interferes with its signaling. moreover, paramyxovirus v and polivirus are able to inhibit the function of mda5. finally, hcv ns3-4 abrogates mavs signaling by cleaving it from the mitochondrial membrane. similarly, mavs is degraded by mitochondria-targeted hepatitis a virus 3abc protein (bowie and unterholzer, 2008) . the latter examples show that mavs functions on mitochondria and suggests that this organelle either plays a so far unrecognized role in antiviral type i interferon signaling or that mavs also mediates apoptosis and thereby crosstalks with bax/bak-induced momp. indeed, besch et al. reported that both rig-1 and mda5 could induce apoptosis of human melanoma cells in a type i interferon-independent manner, i.e. at a step before ifn␣/␤ induction (besch et al., 2009) . the molecular mechanisms of this new death signaling pathway had however not been defined at that time. recently, we published new insights into the mechanisms of host cell apoptosis induced by a single stranded, positive-sense rna virus which does not encode for any cell death protective proteins (el maadidi et al., 2014) . semliki forest virus (sfv) is a neurotropic virus that kills mice during the first 21 days of their life, probably due to the induction of apoptosis of immature neurons (strauss and strauss, 1994) . it also causes encephalitis in various animals but is mostly apathogenic for humans (fazakerley, 2002) . we and others however found that sfv induces effective apoptosis of a variety of human and mouse cells in vitro via the intrinsic mitochondrial pathway, i.e. requiring bax/bak and the induction of momp (ubol et al., 1994; grandgirard et al., 1998; murphy et al., 2001; urban et al., 2008) . after infection, sfv produces high amounts of dsrna during the rna replication cycle, and although it is not entirely clear whether dsrna is the trigger for sfv-induced apoptosis and how it could activate bax/bak (via which bh3-only protein?, see below) ( fig. 3) , we reported that sfv-induced apoptosis is clearly rna replication dependent (urban et al., 2008) . moreover, bax/bak double knock-out cells are not entirely protected from sfvinduced apoptosis but exhibit a bax/bak-independent cell death that still requires caspases but does not involve death-receptor signaling. we then found that this novel bax/bak-independent death signaling pathway requires a mda5-mediated activation of mavs on mitochondria (el maadidi et al., 2014) . mavs recruits caspase-8 from the cytosol to mitochondria forming a novel death inducing signaling complex that is capable of processing and activating caspase-3 and inducing apoptosis (fig. 3) . our results indicate that not only the tlr3/trif axis can induce apoptosis in addition to activating nfb, irf3/7 and an antiviral type i interferon response (kaiser and offermann, 2005; weber et al., 2010; estornes et al., 2012) , but the same can be achieved by the mda5/mavs pathway (el maadidi et al., 2014) . both bax/bakand death receptor-independent death signaling pathways use the initiator caspase-8 on novel death platforms (tlr3/trif/caspase-8 or mavs/caspase-8) to activate caspase-3/-7 (fig. 3) . whether this innate immune signaling pathway also accounts for interferon responses and/or apoptosis in response to dna viruses, which use tlr9 (hemmi et al., 2000) and other intracellular sensors such as cgas/sting (sun et al., 2013) or dai (takaoka et al., 2007) remains to be determined. interestingly, it was recently shown that ips-1/mavs may also mediate innate immune signaling in response to dna viruses (zhang et al., 2011; unterholzner, 2013) . thus, this novel mitochondria-associated pathway may be used as a general defence mechanism of host cells against viruses. another possibility how cells can sense and react to virus infection is by mounting an er-stress response (li et al., 2013a (li et al., , 2013b . this is particularly the case for rna viruses such as alphaviruses where the envelope proteins are co-translationally inserted into the er membrane and travel through the exocytotic system to the cell surface for virus budding (li and stollar, 2004; he, 2006; barry et al., 2010; jheng et al., 2014) . in this case the high amount of erlocalized viral proteins induce a classical unfolded protein response (upr). while upr responses are often cell protective by triggering the transcriptional upregulation of chaperones such as bip/grp78 which help in the folding of the massive amount of proteins, a persistent, prolonged upr response, for example via the ire␣/xbp1 pathway can also lead to apoptosis of the infected host cells (lin et al., 2007; sano and reed, 2013; lu et al., 2014) . in this case, the transcription factor chop1 is activated which upregulates the bh3only proteins bim and puma, leading to bax/bak activation and momp via the intrinsic mitochondrial pathway (reimertz et al., 2003; puthalakath et al., 2007; ghosh et al., 2012) . viruses would then counteract host cell death by the expression of bcl-2 orthologs (vbcl-2s). some viruses have developed strategies to actively kill their infected hosts, probably to avoid the presentation of viral antigens to the adaptive immune system. if this happens viruses have replicated and assembled into new virions so that host cell apoptosis does not eliminate the virus as well. both the activation of the extrinsic as well as the intrinsic apoptosis pathways have been observed. targeting the extrinsic pathway, the nef protein of hiv downregulates cd4 and mhc-1 from the cell surface by crosslinking these proteins to the endocytic compartment (piguet et al., 1999) . as a consequence membrane-bound tnf␣ and light are constitutively expressed on the surface of t cells, potentially contributing to the cytotoxic effects of hiv on infected t cells and uninfected bystander lymphocytes (lama and ware, 2000) . moreover, hcmv and reoviruses were shown to actively induce the expression of trail (sedger et al., 1999; vidalain et al., 2000) and hsv and hcmv can enhance the expression of fasl (raftery et al., 1999 (raftery et al., , 2001 . this mechanism is thought to constitute another viral immune evasion tactic to eliminate infiltrating immune cells such as ctls and dcs by counterattack. to activate the intrinsic mitochondrial pathway, the vpr protein from hiv-1 induces swelling of mitochondria and mom permeabilization in lymphoid and transformed cells (stewart et al., 1999; jacotot et al., 2000; muthumani et al., 2002; deniaud et al., 2004) . severe acute respiratory syndrome coronavirus (sars-cov) 7a protein can directly inhibit bcl-xl and other survival factors (tan et al., 2007) . although to date viruses have not been found to express bh3-only orthologs, there is increasing evidence that they somehow activate cellular bh3-only proteins to engage the bax/bak pore machinery on the mom. however, the exact molecular mechanism how this is done has remained enigmatic. the tat protein of hiv-1 was shown to release bim from its inhibitory contraints on the cytoskeleton so that it can directly activate bax/bak (puthalakath et al., 1999; chen et al., 2002) . whether bim indeed is held in check at the cytoskeleton is largely debated. moreover the mechanism of release has not been investigated. ifn␣/␤ production in response to vesicular stomatitis viral infection has been shown to induce expression of noxa (galluzzi et al., 2008) , but the transcription factors involved were not identified. based on the finding that viruses can activate p53 and its homologs such as p73 (kaelin, 1999) , it is possible that noxa, and maybe also puma are transcriptionally induced by these factors in response to certain infections (cruz et al., 2006; liu et al., 2014) . alternatively, virus-specific protein kinases could phosphorylate bh3-only proteins, as it was shown for the u(s)3 protein kinase of hsv-1 on bad (munger and roizman, 2001) . we have recently shown that bim phosphorylation on three sites by jnk1/2 enhances its proapoptotic activity toward bax/bak activation (geissler et al., 2013) . moreover, we have obtained evidence that dsrna produced by sfv does not only trigger a bax/bak-independent death signaling pathway involving the formation of a mitochondrial mavs/caspase-8 complex (see above) (el maadidi et al., 2014) but also uses a so far unknown bh3only protein to activate bax/bak and momp (papaianni et al., submitted) (fig. 3) . how dsrna links to such a bh3-only protein is currently investigated in our lab. finally, it is possible that virus-encoded proteases may cleave bh3-only proteins such as bid. these proteases are required for the viral life cycle because they process large non-structural and structural polypeptide precusors into mature viral proteins (strauss and strauss, 1994) . since they often show degenerate substrate specificity, they could cleave important components of apoptosis signaling and thereby activate them. for example, plv viruses express proteases (2apro and 3cpro), which activate caspase-dependent apoptosis (barco et al., 2000; goldstaub et al., 2000; calandria et al., 2004) . in addition, hiv-encoded protease is known to process and activate caspase-8 both in vitro and in t cells, which then leads to bid cleavage and mitochondria-mediated apoptosis (nie et al., 2002) . indeed bcl-2 overexpression protects cells from the pro-apoptotic effects of the hiv-protease and prevents apoptosis induced by hiv-1 infection of human lymphocytes (strack et al., 1996) . there is increasing evidence that innate immune signaling via tlrs and intracellular prrs do not only trigger an antiviral interferon response and other mechanisms to limit the replication, assembly and spread of viruses, but they also cooperate with the intrinsic bax/bak-mediated mitchondrial pathway to induce apoptosis of the infected host cells. as discussed above, some viruses have clearly evolved strategies to counteract both the intrinsic as well as the innate signaling branch so that they can effectively replicate and reproduce. however, other viruses, such as sfv and other alphaviruses, do not express any cell survival factors and may therefore use these pathways to kill their host cells after their successful reproduction, e.g. to evade detection by the immune system. while we clearly understand how virus-encoded decoy receptors, caspase-8 and other caspase inhibitors as well as bcl-2 orthologs work to inhibit apoptosis of infected cells, we need to further understand how viruses manage to activate bh3-only proteins and/or directly perturb the balance between bcl-2-like survival factors and pore-forming bax/bak proteins on the mom. further challenges will be to identify all the components of the tlr3/trif/caspase-8 and mavs/caspase-8 complexes, which seem to constitute novel discs enhancing bax/bak-mediated momp and to investigate if other viruses such as dna viruses also use innate signaling strategies to kill their hosts. irrespective of whether viruses use apoptotic or anti-apoptotic mechanisms to win their competition with the host, a better understanding will continue to provide considerable 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mechanism involving a cullin-containing complex herpes simplex virus type 1 infection of activated cytotoxic t cells: induction of fratricide as a mechanism of viral immune evasion targeting the function of mature dendritic cells by human cytomegalovirus: a multilayered viral defense strategy vaccinia virus crme encodes a soluble and cell surface tumor necrosis factor receptor that contributes to virus virulence gene expression during er stress-induced apoptosis in neurons: induction of the bh3-only protein bbc3/puma and activation of the mitochondrial apoptosis pathway pathogen subversion of cell-intrinsic innate immunity er stress-induced cell death mechanisms the e6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 channel formation by antiapoptotic protein bcl-2 mechanisms of caspase activation and inhibition during apoptosis conformational control of bax localization and apoptotic activity by pro168 involvement of hvem receptor in activation of nuclear factor kb by hsv-1 glycoprotein d a central role for death receptor-mediated apoptosis in the rejection of tumors by nk cells ifn-gamma mediates a novel antiviral activity through dynamic modulation of trail and trail receptor expression identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 the adenovirus e3-10.4k/14.5k complex mediates loss of cell surface fas (cd95) and resistance to fas-induced apoptosis a cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation t2 open reading frame from the shope fibroma virus encodes a soluble form on the tnf receptor the large e1b protein together with e4orf6 protein target p53 for active degradation in adenovirus infeced cells the adenovirus e3 10.4k and 14.5k proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane lentiviral delivery of hiv-1 vpr protein induces apoptosis in transformed cells apoptosis mediated by hiv protease is preceded by cleavage of bcl-2 the many roles of fas receptor signaling in the immune system the alphaviruses: gene expression, replication and evolution flip: a flop for execution signals nf-b as a target for oncogenic viruses cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway structure of bax: coregulatoin of dimer formation and intracellular localization dai (dlm-1/zbp1) is a cytosolic dna sensor and an activator of innate immune response innate immunity to virus infection hepatitis c virus nonstructural 5a protein inhibits lipopolysaccharide-mediated apoptosis of hepatocytes by decreasing expression of toll-like receptor 4 the emerging role of the interferon-induced pkr protein kinase as an apoptotic effector: a new face of death? induction of apoptosis by the severe acure respiratory syndrome coronavirus 7a protein is dependent on its interaction with the bcl-xl protein near death experiences: poxvirus regulation of apoptotic death perforin: a key pore-forming protein for immune control of viruses and cancer regulation of lymphocyte proliferation and death by flip forced degradation of fas inhibits apoptosis in adenovirus-infected cells abt-263: a potent and orally bioavailable bcl-2 family inhibitor induction of bcl-xl expression by human t-cell leukemia virus type i tax through nfb in apoptosisresistant t-cell transfectants with tax myxoma virus serp2 is a weak inhibitor of granzyme b and interleukin-1beta-converting enzyme in vitro and unlike crma cannot block apoptosis in cowpox virus-infected cells neurovirulent strains of alphavirus induce apoptosis in bcl-2-expressing cells: role of a single amino acid change in the e2 protein mimicry of cd40 signals by epstein-barr virus lmp1 in b lymphocyte responses the interferon response to intracellular dna: why so many receptors? myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contribues to viral virulence apoptosis induced by semliki forest virus is rna replication dependent and mediated via bak mitochondrial permeabilization relies on bh3 ligands engaging multiple pro-survival bcl-2 relatives, not bak abt-199, a new bcl-2-specific bh3 mimetic, has in vivo efficacy against aggressive myc-driven mouse lymphomas without provoking thrombocytopenia many stimuli pull the necrotic trigger an overview measles virus induces functional trail production by human dendritic cells the rheostat in the membrane: bcl-2 family proteins and apoptosis abrogation of p53-induced apoptosis by the hepatitis b virus x gene a role for mitochondrial bak in apoptotic response to anticancer drugs proapoptotic signalling through toll-like receptor-3 involves trif-dependent activation of caspase-8 and is under the control of inhibitor of apoptosis proteins in melanoma cells association of human papillomavirus types 16 and 18 e6 proteins with p53 a novel bcl-2-like inhibitor of apoptosis is encoded by the parapoxvirus orf virus building blocks of the apoptotic pore: how bax and bak are activated and oligomerize during apoptosis regulation of apoptosis by adenovirus e1a and e1b oncogenes proapoptotic bak is sequestered by mcl-1 and bcl-xl, but not bcl-2, until displaced by bh3-only proteins apoptosis initiated when bh3 ligands engage multiple bcl-2 homologs, not bax or bak navitoclax, a targeted high-affinity inhibitor of bcl-2, in lymphoid malignancies: a phase 1 dose-escalation study of safety, pharmacokinetics, pharmacodynamics, and antitumour activity hiv-1 nef associated pak and pi3-kinases stimulate aktindependent bad-phosphorylation to induce anti-apoptotic signals movement of bax from the cytosol to mitochondria during apoptosis visa is an adapter protein required for virus-triggered ifn-beta signaling threedimensional structure of bax-mediated pores in lipid membrane bilayers the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses the bcl-2 protein family: opposing activities that mediate cell death dhx9 pairs with ips-1 to sense double-stranded rna in myeloid dendritic cells target protease specificity of the viral serpin crma. analysis of five caspases our own work mentioned in the review was supported by the deutsche forschungsgemeinschaft (spemann graduate school of biology and medicine, gsc-4, to c.b., u.m and f.h.; research group for2036 to s.n. and c.b.), by the international master programm in biomedical sciences (imbs, to a.s. and c.b.), the virtual liver network (vln) programm of the bmbf (to l.f. and c.b.) and the excellence cluster bioss (centre for biological signaling studies, exc-294, to s.l. and c.b.). key: cord-291086-goidlh08 authors: walker, peter j.; dietzgen, ralf g.; joubert, d. albert; blasdell, kim r. title: rhabdovirus accessory genes date: 2011-09-14 journal: virus res doi: 10.1016/j.virusres.2011.09.004 sha: doc_id: 291086 cord_uid: goidlh08 the rhabdoviridae is one of the most ecologically diverse families of rna viruses with members infecting a wide range of organisms including placental mammals, marsupials, birds, reptiles, fish, insects and plants. the availability of complete nucleotide sequences for an increasing number of rhabdoviruses has revealed that their ecological diversity is reflected in the diversity and complexity of their genomes. the five canonical rhabdovirus structural protein genes (n, p, m, g and l) that are shared by all rhabdoviruses are overprinted, overlapped and interspersed with a multitude of novel and diverse accessory genes. although not essential for replication in cell culture, several of these genes have been shown to have roles associated with pathogenesis and apoptosis in animals, and cell-to-cell movement in plants. others appear to be secreted or have the characteristics of membrane-anchored glycoproteins or viroporins. however, most encode proteins of unknown function that are unrelated to any other known proteins. understanding the roles of these accessory genes and the strategies by which rhabdoviruses use them to engage, divert and re-direct cellular processes will not only present opportunities to develop new anti-viral therapies but may also reveal aspects of cellar function that have broader significance in biology, agriculture and medicine. the rhabdoviridae is arguably the most diverse family of rna viruses. rhabdoviruses have been isolated from a wide range of organisms including placental mammals, marsupials, birds, reptiles, fish, insects and plants (kuzmin et al., 2009) . their ecology includes marine, freshwater and terrestrial environments. transmission can be via insect vectors in which they replicate, by direct horizontal transfer via plant sap (wounds or grafts), aerosol, animal bite or immersion in infected water, or by vertical transovarial passage. the rhabdoviridae is one of only three virus families that include members that infect plants, vertebrates and invertebrates. replication can occur in the cytoplasm or the nucleus of infected cells. infection can be either acute or persistent and rhabdoviruses can cause infections that may be unapparent or result in disease that can vary from being mild to severe or even fatal. over 200 rhabdoviruses have been identified to date and most of them are poorly described (dietzgen et al., 2012) . for many years, knowledge of rhabdovirus molecular biology has been based primarily on extensive studies of vesicular stomatitis virus (vsv) and rabies virus (rabv). these rhabdoviruses, share very similar and relatively simple genome organisations with only five structural protein genes (n, p, m, g and l) that are transcribed sequentially from the (-) rna genome as monocistronic mrnas. although some variations are now evident, such as the long non-coding region in the rabv g gene (designated pseudogene ) (tordo et al., 1986 ) and the small alternative open reading frames (orfs) in the vsv p gene (c and c ) , these viruses have generally been regarded as the rhabdovirus archetypes and as very suitable models for studying virus-host interactions. however, it is becoming evident that the ecological diversity displayed by rhabdoviruses is also apparent in the complexity of their genome organisation and in the increasing array of additional proteins which they encode. although the functions of many of these proteins are yet unknown, preservation of the five canonical structural protein genes (3 -n-p-m-g-l-5 in negative polarity) by all known rhabdoviruses suggests that the additional proteins may not be essential for virus replication in culture and are likely to have functions associated with enhancing replication efficiency, blocking host innate immune defences, modulating cellular signalling pathways, re-directing normal cellular functions, and allowing effective cell-to-cell transmission. this expectation has been supported by limited studies to date on some of these proteins. by analogy with other rna viruses with relatively complex genome organisations (e.g., coronaviruses, lentiviruses, paramyxoviruses), we have therefore adopted the term 'accessory genes' to describe the orfs encoding these novel and potentially informative proteins. rhabdoviruses are non-segmented (-) ssrna viruses that, together with paramyxoviruses, filoviruses and bornaviruses, are classified in the order mononegavirales. the family rhabdoviridae currently comprises six genera containing 46 species, five species unassigned to a genus and more than 150 viruses that have not been formally classified (dietzgen et al., 2012) . several likely species and two proposed new genera (sigmavirus and tibrovirus) are currently under review for classification, and a number of other viruses have been identified as rhabdoviruses by morphology and/or serology but have not been further characterised. the genus lyssavirus includes rabies virus and the rabies-related viruses mokola virus, lagos bat virus, duvenhage virus, european bat lyssavirus 1, european bat lyssavirus 2 and australian bat lyssavirus, irkut virus, khujand virus, aravan virus and west caucasian bat virus as currently designated species. another unclassified lyssavirus, shimoni bat virus, has also recently been identified (kuzmin et al., 2010) . lyssaviruses naturally infect mammals and almost all have been isolated from bats which appear to be the primary natural reservoir (delmas et al., 2008) . transmission is usually by transfer of saliva in a bite puncture wound. they are neurotropic rhabdoviruses and most have been associated with rabies encephalitis in humans or animals (rupprecht et al., 2002) . the genus vesiculovirus comprises an ecologically diverse but genetically similar group of viruses. viruses causing vesicular stomatitis in cattle, pigs and horses in the americas are referred to collectively as serotypes of vesicular stomatitis virus (vsv) and classified as the species vesicular stomatitis indiana virus, vesicular stomatitis new jersey virus and vesicular stomatitis alagoas virus. other recognised mammalian vesiculovirus species include cocal virus, piry virus, carajas virus and maraba virus that are endemic in the americas, isfahan virus from asia and chandipura virus which occurs in both asia and africa. most of these viruses have been isolated from phlebotomine sand flies (lutzomyia, phlebotomus and sergentomyia spp.) or black flies (simulium spp.) which are considered the primary vectors. however, biting midges (culicoides spp.), mosquitoes and other insects have also been implicated as potential vectors and transmission can also occur by direct contact between infected vertebrates (comer and tesh, 1991; letchworth et al., 1999; rodríguez, 2002; stallknecht et al., 2001) . vesiculoviruses can also infect humans and some have been associated with serious disease (rao et al., 2004) . the genus vesiculovirus also currently contains the recognised species spring viraemia of carp virus and several other important pathogens of fish, including pike fry rhabdovirus, trout rhabdovirus, sea trout rhabdovirus, and siniperca chuatsi rhabdovirus, ulcerative disease rhabdovirus, eel virus american and eel virus european x, also appear to be members of the genus (dietzgen et al., 2012; hoffmann et al., 2005) . transmission of fish vesiculoviruses can occur by immersion in infected water and does not appear to involve arthropod vectors (ahne et al., 2002) . the genus novirhabdovirus comprises a second group of fish rhabdoviruses that appear to have a very different evolutionary history from the fish vesiculoviruses. species include infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus which are important pathogens of salmonids, but can also infect a very wide range of other fish species (walker and winton, 2010) . the species hirame rhabdovirus and snakehead rhabdovirus are pathogens of marine fish in temperate and tropical regions of asia, respectively (johnson et al., 1999; kim et al., 2005) . as for the fish vesiculoviruses, transmission is directly through mucosal surfaces or the skin by immersion in infected water and does not appear to involve an arthropod vector. the genus ephemerovirus comprises arthropod-borne rhabdoviruses that have been isolated from mosquitoes and/or biting midges (culicoides spp.) and infect primarily ruminants (walker, 2005) . the type species bovine ephemeral fever virus and the newly recognised species kotonkan virus each cause a disabling febrile disease in cattle and water buffalo (kemp et al., 1973; st. george and standfast, 1988) . other ephemeroviruses, including recognised species berrimah virus and adelaide river virus, and likely species obodhiang virus, kimberley virus, malakal virus and puchong virus, have been isolated only from insects or from healthy cattle and their association with disease is not yet known (walker et al., in press) . plant-adapted rhabdoviruses are separated taxonomically into two genera, cytorhabdovirus and nucleorhabdovirus, based on their site of virus replication and morphogenesis (dietzgen et al., 2012) . cytorhabdoviruses replicate in the cytoplasm of infected cells and virions bud in association with endoplasmic reticulum membranes. the type species of the genus is lettuce necrotic yellows virus and it includes eight additional recognised species. nucleorhabdoviruses replicate in the nuclei of infected plant cells. morphogenesis occurs at the inner nuclear membrane and virions accumulate in perinuclear spaces. the type species is potato yellow dwarf virus and there are nine additional recognised species in this genus. viruses in both genera infect monocot and dicot plants and are transmitted by leafhoppers, planthoppers or aphids in which they also replicate (jackson et al., 2005) . two other genera are currently under consideration for inclusion in the rhabdoviridae. the proposed genus sigmavirus comprises rhabdoviruses infecting only flies (drosophila spp.) with which they have co-evolved. they are transmitted vertically and confer co 2 sensitivity on the host. there are five proposed species including drosophila melanogaster sigmavirus (commonly called sigma virus of drosophila) which is the most extensively studied of the sigmaviruses (longdon et al., 2010 (longdon et al., , 2011 . like the genus ephemerovirus, the proposed genus tibrovirus comprises antigenically related rhabdoviruses that primarily infect cattle and water buffalo, and are transmitted by culicoides species midges. however, tibroviruses and ephemeroviruses have different evolutionary histories as is reflected in their phylogenetic relationships and genome organisations. tibrogargan virus and coastal plains virus, each isolated in australia, are currently proposed as species in the new genus (gubala et al., 2011) . bivens arm virus, isolated from culicoides insignis in the usa, also infects cattle and water buffalo and may be a third species in the proposed genus (gibbs et al., 1989) . complete genome sequences are also available for several other unclassified animal rhabdoviruses that are considered in this review. short sequences in the l and n gene have been used to determine phylogenetic relationships between viruses in the established and proposed genera and many other unclassified animal rhabdoviruses (bourhy et al., 2005; kuzmin et al., 2006) . one monophyletic cluster identified by this approach includes a geographically and ecologically diverse group of viruses termed the hart park group because of the close serological relationship of one of the members, flanders virus, to hart park virus. both of these viruses have been isolated from mosquitoes and infect birds in north america (boyd, 1972) . the hart park phylogenetic group also includes wongabel virus which was isolated from biting midges (culicoides spp.) in australia and appears to infect birds, and ngaingan virus, also isolated from biting midges in australia but whose vertebrate hosts include marsupials (doherty et al., 1973; gubala et al., 2010) . not included in the hart park group but related to each other phylogenetically (but not serologically) are tupaia rhabdovirus isolated from hepatocarcinoma cells of a tree shrew (tupaia belangeri) imported to the usa from thailand (kurz et al., 1986) , and durham virus, which was isolated from the brain of a moribund american coot (fulica americana) in the usa (allison et al., 2011) . oak vale virus, isolated from mosquitoes in australia and infecting feral pigs, and mandarin fish (siniperca chuatsi) rhabdovirus (scrv) are other currently unclassified rhabdoviruses that are considered in this review (cybinski and muller, 1990; zhang et al., 2007) . rhabdoviruses for which complete genome sequences are available are listed in table 1 . the (-) ssrna rhabdovirus genome ranges in size from approximately 11 kb to almost 16 kb. the five structural protein genes are arranged in the same linear order (3 -n-p-m-g-l-5 ) and may be interspersed with one or more additional genes. at each end of the genome, 3 leader and 5 trailer sequences of approximately 50-100 nt contain the promoter sequences that initiate replication of the genome and anti-genome, respectively. the 3 leader and 5 trailer sequences of plant-adapted rhabdoviruses are considerably longer at 84-206 nt and 145-389 nt, respectively (jackson et al., 2005) . in transcription mode, the rna-dependent rna polymerase moves progressively along the genome to transcribe the short leader rna (l) and then to transcribe, cap and polyadenylate mrnas from each gene in decreasing molar abundances (banerjee and barik, 1992) . critical to this process are transcription initiation (ti) and transcription termination/polyadenylation (ttp) sequences flanking each gene (barr et al., 2002) . these sequences are relatively well conserved in each species and are similar in all species within a genus. the termination of transcription and polyadenylation of mrnas are critically dependent on a stretch of seven uridine residues that are usually preceded by a guanosine residue. following polyadenylation, transcription is re-initiated at the next available ti sequence which follows a non-transcribed intergenic region that may be from one to approximately 50 nt in length (banerjee and barik, 1992) . corruption of the [u] 7 stretch in tpp sequences of animal rhabdoviruses leads to read-through, resulting in polycistronic mrnas (barr et al., 1997) . in plant-adapted rhabdoviruses, the incorruptible component of the polyadenylation signal is uumuuuu(u) (jackson et al., 2005) . as discussed below, rhabdovirus genes may contain multiple long open reading frames (orfs), either overlapping or sequentially in the transcriptional units, and these must be expressed by internal initiation of translation. all evidence suggests that the fundamental processes of genome replication and transcription are common to all rhabdoviruses infecting vertebrates, insects and plants. the five major structural proteins of vsv and rabv are amongst the most intensely studied of all viral proteins. although there are reported variations in their ancillary functions, the primary functions and structural characteristics of these proteins are conserved across all rhabdoviruses and have been reviewed in detail on many previous occasions assenberg et al., 2010; coll, 1995; jackson et al., 2005; jayakar et al., 2004; redinbaugh and hogenhout, 2005; roche et al., 2008; rupprecht et al., 2002) . in contrast, the number and locations of accessory genes varies widely both within and between taxa (figs. 1 and 2). in most cases, levels of amino acid sequence identity between cognate accessory proteins are quite low and, with a few exceptions, their specific functions do not appear to be conserved between taxa. as discussed in detail below, some occur as alternative or overlapping orfs within the major structural protein genes; many occur in the regions between the major structural protein genes as new genes containing single or multiple orfs. over 35 rhabdovirus accessory genes have been identified to date and it is likely that many more will emerge as the number of complete genome sequences continues to grow. although the vesiculovirus genome is the smallest and arguably the least complex of all rhabdoviruses, containing only the 5 canonical structural protein genes, additional proteins are expressed from both the p and m genes (fig. 1) . the p genes of most vesiculoviruses encode a small highly basic protein (c) in an alternative orf. in vsv new jersey and vsv indiana, two forms of the c protein (c and c ) are expressed in infected cells by translation initiation at alternative start codons spiropoulou and nichol, 1993) . there is evidence that the c protein is associated with nucleocapsids and increases the activity and fidelity of viral rna polymerase activity in vitro . however, c and c are not essential for replication in cell culture and a vsv infectious clone lacking these proteins has been shown to have identical growth kb 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 16 oth er animal rhabdoviruses table 1 . characteristics to wild-type virus (kretzschmar et al., 1996) . the predicted sizes of the putative c proteins in viruses examined to date range from 41 amino acids (piry virus) to 93 amino acids (cocal virus). interestingly, although spring viraemia of carp virus has the potential to encode highly basic proteins of 83 and 57 amino acids in an alternative orf in the p gene (hoffmann et al., 2002; teng et al., 2007) , there appears to be no alternative orfs in the pike fry rhabdovirus p gene (chen et al., 2009) , suggesting its function may not be conserved across all fish vesiculoviruses. however, these data should be viewed cautiously as a single point mutation kb 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 table 1. during adaptation to cell culture would be sufficient and inhibit expression of these accessory proteins by codon modification. it has also been reported that a small (7 kda) protein is expressed from the vsv p gene from the same orf as the p protein (herman, 1986) . the presence of alternative orfs in the p gene of vesiculoviruses mirrors the situation in paramyxoviruses in which the complex expression of the c and v family of proteins in overlapping open reading frame or by rna editing is very well characterised (nagai and kato, 2004) . the paramyxovirus c and v proteins have various functions associated with infection and pathogenesis including regulation of viral rna synthesis, virion assembly and budding, inhibition of apoptosis, and in binding to components of the jak/stat signalling pathway to antagonize the cellular interferon response (fontana et al., 2008; irie et al., 2010; kurotani et al., 1998) . the vsv m protein is a multi-functional protein that, as well as being a structural component of virions, is involved during infection in inhibition of viral transcription, assembly and budding, inhibition of host gene expression and perturbation of the cell cytoskeleton (black and lyles, 1992; blondel et al., 1990; coulon et al., 1990) . the m protein also blocks the host anti-viral response and promotes apoptosis both indirectly through inhibition of host gene expression and directly by targeting the extrinsic pathway and promoting expression of pro-apoptotic genes (kopecky et al., 2001; pearce and lyles, 2009 ). the vsv m gene is also expressed in infected cells from two alternative initiation codons in the same frame, producing the m2 and m3 proteins (jayakar and whitt, 2002) . little is known of the function of these proteins but they appear to be involved in cell rounding and cytopathology. although alternative initiation codons are available in the m proteins of other vesiculoviruses, it is not known if they are utilised. the lyssavirus genome also conforms to the basic canonical arrangement of the genes encoding the five major structural proteins but lyssaviruses share the common characteristic of a very long 3 non-coding region in the g gene (fig. 1) . the region varies in length from approximately 400 to 700 nt in different lyssaviruses and, in one lineage of laboratory-adapted rabv strains, it is truncated by an additional ttp signal 70 nucleotides downstream of the g protein orf, leading to the suggestion that it may be the remnant of an ancestral gene or pseudogene () (conzelmann et al., 1990; morimoto et al., 1989; tordo et al., 1986) . however, although the long 3 non-coding region is preserved, the additional ttp is absent from wild-type rabies viruses isolated directly from infected animals and from other lyssaviruses (ravkov et al., 1995) . in west caucasian bat virus (wcbv), the region extends 697 nucleotides beyond the g protein orf and, uniquely, contains an orf of 180 nucleotides but there is no evidence of expression of the second orf in wcbv-infected cells (kuzmin et al., 2008) . universal preservation of the region in wild-type lyssaviruses suggests it has functional significance but a rabv infectious clone lacking the region displayed normal growth characteristics in fibroblasts and neuronal cells in culture and no difference in axonal spread or pathogenicity in mice (ceccaldi et al., 1998; schnell et al., 1994) . nevertheless, there is some evidence that, in conjunction with the g protein, the rabv region does contribute to neuroinvasiveness from peripheral sites of infection (faber et al., 2004) . proposed functions of the region include attenuation of l gene expression, stabilisation of the g mrna and interaction of the mrna with host cell proteins (ravkov et al., 1995) . like the vesiculoviruses, lyssaviruses also commonly encode small, highly basic proteins in alternative orfs in the p gene (nadin-davis et al., 2002) . the lyssavirus orfs usually occur in the same region of the p gene as vesiculovirus c orfs but they vary in size in different isolates and do not appear to be selectively retained by a locally constrained mutation frequency (nadin-davis et al., 2002) . it is not known if lyssavirus c proteins are expressed and the variability of their detection in different isolates suggests they may have little functional relevance. however, if their role is to facilitate infection in vivo, sequence data on cell culture-adapted isolates should be regarded cautiously as introduced point mutations in the hypervariable p gene may be selected to interrupt functional c orfs. in addition to the full-length p protein, rabv p gene also encodes four smaller products (p2-p5) in the same reading frame as a result of leaky scanning of the ribosome on the p mrna (chenik et al., 1995) . due to a nuclear localisation signal (nls) in the c-terminal domain and a nuclear export signal (nes) in the n-terminal domain, the various forms of the p protein accumulate differentially in the cell such that the p1 (p) and p2 forms are cytoplasmic whereas p3, p4 and p5 are transported to the nucleus (moseley et al., 2007; pasdeloup et al., 2005) . the p isoforms antagonise cellular interferon responses by several different mechanisms including inhibiting the phosphorylation of cytoplasmic interferon regulatory factor 3 (irf3), binding to nuclear and cytoplasmic stat1 and stat2, and interacting with promyelocytic leukaemia (pml) nuclear bodies (blondel et al., 2002; brzozka et al., 2005 brzozka et al., , 2006 chelbi-alix et al., 2006) . the p3 isoform has also been shown to mediate the sequestration of stat1 away from the nucleus by a microtubule-associated mechanism, preventing nuclear import and activation of the interferon response (moseley et al., 2009 ). the interferon-antagonistic activity of the p protein isoforms are important determinants of the pathogenicity of rabv in mice (ito et al., 2010; rieder et al., 2011) . in the region of the genome occupied by the lyssavirus non-coding region (i.e., between the g and l genes), the novirhabdoviruses contain an additional gene of approximately 500 nucleotides (kurath et al., , 1997 (fig. 1) . the non-virion (nv) orf is flanked by ti and ttp sequences, and encodes a neutral to mildly acidic protein of 110-122 amino acids (13.2-13.6 kda) schutze et al., 1996) . the nv proteins of infectious haematopoietic necrosis virus (ihnv) and viral hemorrhagic septicaemia virus (vhsv) are expressed at low levels in infected cells but do not occur in virions (basurco and benmansour, 1995; schutze et al., 1996) . the level of amino acid sequence conservation amongst nv proteins is quite low (e.g., vhsv and hirame rhabdovirus share only 16.5% identity) but the nv gene is present in all four novirhabdovirus species and there is evidence that they are functionally equivalent (kurath et al., 1997; thoulouze et al., 2004) . the construction of novirhabdovirus infectious clones lacking the nv gene has indicated that it is not essential for virus replication in cell culture but reports vary on the replication efficiency and pathogenicity of the recombinant viruses (biacchesi, 2011) . deletion of the nv gene from an ihnv infectious clone resulted in reduced replication efficiency in epc fish epithelial cells and reduced cumulative mortalities following intraperitoneal injection of rainbow trout (oncorhynchus mykiss) (biacchesi et al., 2000; thoulouze et al., 2004) and similar observations have been reported for an nv-knockout infectious clone of vhsv in yellow perch (perca flavescens) (ammayappan et al., 2010) . however, snakehead rhabdovirus (shrv) infectious clones either lacking the nv gene or in which the nv orf was truncated by point mutations were shown to replicate normally in epc cells, and retained pathogenicity when injected intraperitoneally into zebra fish (danio rerio) (alonso et al., 2004; johnson et al., 2000) . it has been suggested that this apparent difference in the biological response to inhibition of nv expression may reflect the natural host range of the viruses which infect cold water fish species (vhsv and ihnv) or warm water fish species (shrv) and have corresponding temperature ranges for efficient virus replication (biacchesi, 2011) . recent evidence suggests that the vhsv nv protein has an anti-apoptotic function early in infection in vitro (ammayappan and vakharia, 2011) . however, as the m protein of ihnv has pro-apoptotic activity (chiou et al., 2000) and rhabdoviruses generally are known to promote apoptosis, the biological role of the nv protein remains unresolved. novirhabdoviruses also encode small basic proteins in alternative reading frames in the p gene but their functional relevance is unknown. sigmaviruses also contain a single additional gene but it is located between the p and m genes (teninges and bras-herreng, 1987) (fig. 1) . the x gene encodes a polypeptide (pp3) of 298-321 amino acids (landes-devauchelle et al., 1995) . although amino acid sequence identity between the pp3 proteins of sigmaviruses infecting drosophila melanogaster and drosophila obscura is relatively low (∼11%), sequence similarity is relatively high and each is predicted with high probability to have an n-terminal signal peptide. each also has three predicted n-glycosylation sites near the n-terminus and so the mature x protein appears to be a secreted glycoprotein. although original reports suggested that pp3 of drosophila melanogaster sigmavirus may be distantly related to reverse transcriptase of retrotransposons, the relevant amino acid motifs do not appear to be conserved in the drosophila obscura sigmavirus pp3 protein (landes-devauchelle et al., 1995; longdon et al., 2010) . as sigmaviruses have co-evolved with their hosts and are transmitted only vertically in ecological time frames, further studies of the function of pp3 may reveal interesting aspects of virus-insect interactions and innate anti-viral immunity in insects (longdon et al., 2011; tsai et al., 2008) . the genomes of plant rhabdoviruses are 12-14.5 kb in size and have the same basic organisation as their animal-infecting counterparts, 3 -n-p-m-g-l-5 (fig. 2) . the unique feature of plantadapted rhabdoviruses is that they encode a viral movement protein (mp) between p and m genes which facilitates cell-tocell transport (jackson et al., 2005) . there are no characteristic genus-specific differences in genome size or genome organisation between cytorhabdoviruses and nucleorhabdoviruses. however, nucleorhabdoviruses replicate in the nuclei of infected plant cells and most viral proteins are directed to the nucleus, while cytorhabdoviruses replicate in the cytoplasm (as all other rhabdoviruses) and viral proteins are mainly localised to the cell periphery and endomembranes (jackson et al., 2005) . individual members of both genera may encode up to four additional accessory proteins of unknown function interspersed between n-p, p-m and g-l genes (fig. 2) . the number and location of these additional genes transcends the two genera. rice yellow stunt virus (rysv) and rice transitory yellowing virus (rtyv) are strains of the same virus and the nucleotide sequences of their genomes are 98.5% identical (hiraguri et al., 2010) . all other fully sequenced plant rhabdoviruses are only distantly related and have been classified taxonomically as members of distinct virus species. genes located between the p and m genes encode 30-35 kda proteins that appear to be involved in viral intercellular cell-to-cell movement across plasmodesmata, symplastic connections that traverse the plant cell wall of neighbouring cells (benitez-alfonso et al., 2010) . orfs at this position encode the sc4, 4b and y proteins of sonchus yellow net virus (synv), lettuce necrotic yellows virus (lnyv) and potato yellow dwarf virus (pydv), respectively (bandyopadhyay et al., 2010; dietzgen et al., 2006; scholthof et al., 1994) . other plant-adapted rhabdoviruses also contain accessory genes at this location. single accessory genes (designated p3) are located at this position in rice yellow stunt virus (rysv), rice transitory yellowing virus (rtyv), maize mosaic virus (mmv), maize iranian mosaic virus (mimv), taro vein chlorosis virus (tavcv), lettuce yellow mottle virus (lymov) and strawberry crinkle virus (scv) (heim et al., 2007; hiraguri et al., 2010; huang et al., 2005; massah et al., 2008; reed et al., 2005; revill et al., 2005; schoen et al., 2004) . however, maize fine streak virus (mfsv) contains two genes (p3 and p4) and in northern cereal mosaic virus (ncmv) there are four genes (p3, p4, p5 and p6) at this position in the genome (fig. 2) (tanno et al., 2000; tsai et al., 2005) . secondary structure predictions have shown that the synv sc4, lnyv 4b, lymov p3, mmv p3, mfsv p4, rysv p3 and pydv y proteins have some structural similarities in common with those of the '30k' superfamily of plant virus movement proteins (melcher, 2000) . blastp database searches and detailed secondary structure prediction of the lnyv 4b and lymov p3 proteins also revealed close similarities with the movement proteins of viruses in the family flexiviridae which induce movement-associated tubules in infected cells (dietzgen et al., 2006; heim et al., 2007) . lymov p3 has a 58% amino acid sequence identity with lnyv 4b (heim et al., 2007) . the membrane and cell periphery associations of synv sc4 also suggest a role in cell-to-cell movement. direct experimental evidence of cell-to-cell movement function exists only for rysv p3 which is the most characterised protein encoded in the p-m region (huang et al., 2005) . p3 has been shown to trans-complement cell-to-cell movement of a movement-defective heterologous plant virus and binds nonspecifically ssrna in vitro, a common property of mps of numerous plant viruses (benitez-alfonso et al., 2010) . moreover, pull-down assays have shown that p3 interacts with the n protein, providing support for involvement of the nucleocapsid core in cell-to-cell movement (huang et al., 2005) . when expressed as fusion proteins with green fluorescent protein (gfp), pydv y and synv sc4 have been shown to localize to the cell periphery (bandyopadhyay et al., 2010; goodin et al., 2007) whereas mfsv p4 localized to nuclei . the putative mps of synv and pydv self-interact in bimolecular fluorescence complementation (bifc) assays (bandyopadhyay et al., 2010; min et al., 2010) . pydv y and m proteins interacted in planta and the complex accumulated inside the nucleus, while synv sc4 interacted with a soluble truncated form of g protein and complexes were seen on the nuclear envelope and on punctuate loci along the cell periphery. sc4 has also been shown to interact with microtubule-associated host proteins and cytoplasm-tethered transcription activators have been implicated in the intercellular movement of synv (min et al., 2010) . the available information for these four nucleorhabdoviruses indicates potentially different cell-to-cell movement pathways. the functions of the other accessory genes, including the four unique ncmv orfs located between p and m genes, are unknown. three plant rhabdoviruses, ncmv, scv and rysv (and the synonymous rtyv) contain a short orf between the g and l genes (fig. 2) . the ncmv p9, scv p6, and rysv p6 genes are predicted to encode proteins of 52, 68, and 93 amino acids, respectively. they share no identifiable sequence similarities but limited similarity can be identified with other rhabdovirus proteins. the rysv p6 protein shares 24% sequence identity with the n-terminal 110 amino acids of the synv l protein and has limited sequence identity with the nv proteins of novirhabdoviruses (up to 25%) and non-coding regions preceding the l gene in hendra virus (38.3%) and rabv (36.6%). the scv p6 and ncmv p9 proteins are moderately basic (pi ∼ 8.9) but rysv p6 is highly acidic (pi 3.49). the large negative charge of this protein is reminiscent of the acidic n-terminal domain of the rysv l protein, although they lack sequence similarity. of the three small proteins, only rysv p6 has been characterised experimentally (huang et al., 2003) . p6 is phosphorylated in vitro on both serine and threonine residues. it has also been shown to be associated with virions and may have a structural role but it is present only in low amounts during infection. the function of proteins encoded in this genome position is yet unknown. however, distant similarities of rysv p6 with the l proteins of rysv and synv may suggest a close evolutionary relationship with l proteins and potentially an ancillary role in rhabdovirus replication. pydv is unique amongst the sequenced plant-adapted rhabdoviruses in that it has an orf x of unknown function located between the n and p genes. the 11 kda x protein has a high content of proline, aspartic and glutamic acids, with a predicted pi of 4.5. these properties are similar to those of mfsv p3 (10 kda, pi 5.4). however, while mfsv p3-gfp fusions accumulate in punctuate loci in the cytoplasm , pydv x is positive in yeast-based nuclear import assay, indicating its likely nuclear localization (bandyopadhyay et al., 2010) . like the animal rhabdoviruses, some cytorhabdoviruses and nucleorhabdoviruses present alternative orfs in the p gene, but it is unknown if these are expressed in infected plants or vector insects (callaghan and dietzgen, unpublished). ephemerovirus genomes are amongst the largest and most complex of known rhabdoviruses with multiple accessory genes located in a region that extends up to 4.2 kb between the g and l genes (fig. 1) . perhaps the most interesting of these is a second glycoprotein (g ns ) gene that immediately follows the g gene (walker et al., 1992) . the g ns gene occurs in all ephemeroviruses and, until recently, has been regarded as a defining characteristic of the genus. the g ns proteins are 60-90 kda type 1 transmembrane glycoproteins that are highly glycosylated with n-linked glycans (walker et al., 1992; wang and walker, 1993) . they share significant amino sequence identity and structural homology with the g proteins of ephemeroviruses and other rhabdoviruses, and appear to have arisen by recombination or gene duplication (see below). the bovine ephemeral fever virus (befv) g ns protein has been detected in infected cells but is not present in virions (walker et al., 1991) . recombinant g ns expressed from a vaccinia virus vector has been detected in association with amorphous structures at the cell surface but not with budding virus particles (hertig et al., 1996) . despite the structural similarity, the befv g ns protein shares no neutralising epitopes with the g protein (johal et al., 2008) and polyclonal rabbit antibody to the befv g ns protein does not neutralise virus produced in either mammalian or insect cell cultures (hertig et al., 1996) . furthermore, unlike the g protein, befv g ns does not induce spontaneous cell fusion at low ph (johal et al., 2008) , suggesting it functions neither in cell attachment nor fusion in vertebrates or invertebrates. in an attenuated vaccine strain of befv (joubert and walker, unpublished data) and in a highly cell-culture-adapted strain of adelaide river virus (arv), corruptions in the ttp sequence at the end of the g gene allow expression of the g ns protein only as a polycistronic mrna (wang and walker, 1993 ) but this has not been observed in other wild-type or cell culture adapted ephemeroviruses. the function of ephemerovirus g ns proteins is currently unknown. immediately downstream of the g ns gene lies a set of relatively small accessory genes bounded by ti and ttp sequences that vary in number in different ephemeroviruses (mcwilliam et al., 1997; wang et al., 1994) . the ␣ gene occurs in all ephemeroviruses and contains two consecutive orfs (␣1 and ␣2) that are expressed from a single transcript due to the universal absence of intervening ttp and ti sequences. the ␣1 orf encodes proteins of 10.5-14.5 kda, each with a predicted n-terminal ectodomain containing clusters of aromatic residues, a central transmembrane domain and a highly basic c-terminal endodomain (fig. 3) . although sequence identity between the ␣1 protein is relatively low, the structure is highly conserved and strongly suggests they function as viroporins (gonzalez and carrasco, 2003; mcwilliam et al., 1997) . the ␣2 orf encodes proteins that range in size from 10.7 kda to 14.2 kda in different ephemeroviruses and have no identifiable motifs that may suggest a particular function. they share identifiable amino acid sequence homology with significantly higher levels of identity (up to 59%) between the more closely related viruses. conservation of the ␣2 orf in all ephemeroviruses and the preservation of sequence identity suggest that it is of functional significance. in most ephemeroviruses the ␣2 orf initiation codon is in close proximity to the ␣1 termination codon suggesting expression may occur by translation re-initiation as has been described for several other rna viruses (powell, 2010) . in befv, orf ␣2 also contains a 51 amino-acid orf (␣3) in an alternative reading frame but it does not occur in other ephemeroviruses and its functional significance is unknown (mcwilliam et al., 1997) . all ephemeroviruses also contain the ␤ gene which is located immediately downstream of the ␣ gene and is expressed as a monocistronic transcript encoding a 16.9-18.4 kda neutral protein (mcwilliam et al., 1997; wang et al., 1994) . the ephemerovirus ␤ proteins share significant amino acid sequence identity and have been detected both in infected cells and in virions. however, in highly adapted laboratory strains of befv and kimberley virus (kimv) the ␤ proteins are severely truncated by point mutations and are not expressed during infection, indicating that they are not essential for growth in vitro and are not essential components of virions (walker, joubert and blasdell, unpublished data) . they have no identifiable homology with any other known proteins but, although their function remains unknown, they may well have a key role in pathogenesis and/or blocking the host immune response. although absent from the genomes of arv and obodhiang virus (obov), befv, kimv, kotonkan virus (kotv) and berrimah virus (brmv) each also contain a ␥ gene, immediately downstream of the ␤ gene (mcwilliam et al., 1997) . proteins encoded in the ␥ orf are mildly basic and range in size from 11.8 kda to 13.6 kda. they share the highest levels of amino acid sequence identity of any of the ephemerovirus accessory proteins but are not related to any other known proteins and contain no identifiable sequence motifs that suggest a function. the ␥ proteins have been detected in infected cells and in virions. in wild-type viruses, the ␥ genes are transcribed as monocistronic mrnas but in highly cell culture adapted strains, expression of the ␥ protein is severely attenuated by corruption of the ttp sequence at the end of the ␤ gene, allowing expression only as a bicistronic ␤-␥ transcript. selective suppression of ␥ gene expression in culture suggests that its function may be important during infection in vivo. the 15,870 nt kotv genome is the largest yet discovered in any rhabdovirus due to an additional accessory gene immediately downstream of the ␥ gene (walker and blasdell, unpublished data) . the kotv ␦ gene is expressed as a monocistronic mrna encoding a mildly acidic 12.4 kda protein. the ␦ protein has been detected in kotv-infected cells. it is shares significant amino acid sequence identity with the pleckstrin homology (ph) domain of coactivatorassociated arginine methyl transferase (carm1) which is involved in chromatin re-modelling and transcriptional activation of nf-b dependent gene expression (covic et al., 2005; troffer-charlier et al., 2007) . kotv ␦ may mimic this domain, inhibiting activation of the anti-viral innate immune response (joubert and walker, unpublished data) . like most other rhabdoviruses, several ephemeroviruses also encode small highly basic proteins in alternative orfs in the p gene but their functional significance is unclear. unlike the ephemeroviruses, the complex genomes of the hart park group of rhabdoviruses contain multiple accessory genes inserted at various locations (fig. 1) . wongabel virus (wonv) contains five additional orfs (u1-u5) interspersed at various locations across the 13,196 nt genome (gubala et al., 2008) . the u1, u2 and u3 orfs are located between the p and m genes and are each bounded by ti and ttp sequences. orf u1 and orf u2 encode acidic proteins of similar size and net charge (21.2 kda/pi 5.2 and 21.9 kda/pi 4.5, respectively). they have no remarkable characteristics but do share a significant level of amino acid sequence homology (∼16% identity; 60% similarity) and are more closely related to each other than to any other known protein. the wonv u3 gene follows the u2 gene and also encodes a relatively small acidic protein (16.5 kda/pi 4.6) that is unrelated to any other known proteins but shares sequence homology with the wonv u1 protein (∼22% identity; 57% similarity), including the common sequence [ksxydfvwpxxxlxxg] in the central region of each protein (fig. 4a) . preliminary data suggest that the u3 protein binds to the iap (inhibition of apoptosis) protein apollon and a component of the chromatin remodelling complex and may be involved in promoting apoptosis (joubert and walker, unpublished data). wonv orf u4 is located within the n gene as a second orf in the 142 nt region that follows the n protein termination codon. it encodes a putative small acidic protein (5.8 kda/pi 4.0) of 49 amino acids that could only be translated by internal initiation on a bicistronic (n-u4) mrna. wonv orf u5 overlaps the g protein orf and thus also must be translated by internal initiation on a bicistronic (g-u5) mrna. translation from the first initiation codon would result in a 14.9 kda protein with that is similar in size and structure to the ephemerovirus viroporin-like ␣1 proteins (fig. 3) . several low molecular weight proteins (range 20-25 kda) have been detected in infected cells using wonv hyperimmune antiserum but they are larger than the predicted u1-u5 proteins and may well be degradation products of larger viral proteins (gubala et al., 2008) . the flanders virus (flav) genome organisation appears to closely resemble that of wonv. the available sequence data (gen-bank ah012179) is dated and appears to be corrupted either by sequencing ambiguities or as a result of cell culture adaptation of the virus. however, through alignment with the wonv genome, it is reasonable to infer that flav also contains three accessory genes between the p and m genes and a 13.8 kda viroporin-like protein encoded in a second orf in the g gene (fig. 3) . the flav u1, u2 and u3 orfs each appear to encode proteins of 18-19 kda and, as for the corresponding wonv proteins, they share an unusual level of sequence similarity suggesting a common origin and/or functional association. this is most evident between flav u1 and u2 (also referred to as the 19k protein) that share ∼20% amino acid sequence identity and ∼58% similarity (fig. 4b ). there is no second consecutive orf in the flav n gene corresponding to wonv u4 but there is a small alternative orf within the n gene encoding a polypeptide of 66 amino acids (7.9 kda). in addition to the n, p, m, g and l proteins, three virus-induced proteins have been reported in flav infected cells and in virions (boyd and whitaker-dowling, table 1. 1988). however, only one of these proteins (19 kda) corresponds to the predicted size of any flav accessory gene product. the 15,764 nt ngaingan virus (ngav) genome is one of the largest and most complex of known rhabdoviruses (gubala et al., 2010) . as in wonv and flav, the ngav genome contains three orfs between the p and m genes (u1, u2 and u3) encoding neutral to mildly acidic proteins of 15.1-17.1 kda that share identifiable sequence similarity. however, unlike wonv and flav, orf u1 and orf u2 are encoded in overlapping reading frames within the same gene and orf3 is encoded in a separate gene bounded by ti and ttp sequences. downstream of the u3 gene, the ngav genome organisation is very complex and varies significantly from wonv and flav. between the m and g genes the ngav u4 gene encodes a 9.7 kda protein with a tyrosine-rich c-terminal domain but otherwise has no remarkable features. downstream of the g gene, however, is a region that resembles the ephemerovirus genomes including a second glycoprotein gene (g ns ), followed by three additional orfs. the g ns gene encodes a type 1 transmembrane glycoprotein that is most closely related to the ephemerovirus g ns proteins and shares sequence homology and structural characteristics with the ngav g protein and those of all animal rhabdoviruses (fig. 5) . the two accessory genes immediately downstream of the ngav g ns gene also resemble the ephemerovirus ␣ and ␤ genes in format, size and location. the first contains two overlapping orfs (u5 and u6) that are similar in size to the ephemerovirus ␣1 and ␣2 orfs. however, the 12.6 kda u5 protein does not have the characteristic structure of the viroporin-like ␣1 proteins and, unlike the featureless ephemerovirus ␣2 proteins, the u6 protein has the structure of a small (10-12 kda) membrane-anchored class 1 glycoprotein with a signal peptide domain, a transmembrane domain and an nterminal ectodomain containing a single n-glycosylation site. the ngav u7 gene contains a single orf encoding an 18.2 kda protein that is similar in size to ephemerovirus ␤ proteins but shares no identifiable sequence homology and has no remarkable features. lyssavirus g a minimum evolution phylogenetic tree constructed from a clustal x multiple sequence alignment of animal rhabdovirus g protein and gns protein sequences. the tree was constructed using the mega program. viruses assigned to established genera and proposed genera are shaded. abbreviations of virus names are as listed in table 1 . in addition to these independent accessory genes ngav encodes two small basic proteins (p 1 and p 2) in alternative reading frames within the p gene and a 10.8 kda protein in an alternative reading frame in the m gene. if all these potential orfs in translated, ngav could potentially express a total of 16 functional proteins. whole genome sequences are available for two viruses that have recently been proposed as species in a new genus tibrovirus. the 13.2-13.3 kb tibrogargan virus (tibv) and coastal plains virus (cpv) genomes share a similar organisation with 3-4 accessory protein genes (gubala et al., 2011) (fig. 1) . in each virus, orfs u1 and u2 are located between the m and g genes. each is bounded by ti and ttp sequences and encodes a protein of 17-20 kda. the u1 proteins are acidic and the u2 proteins are basic, and there is extensive amino acid sequence homology between the corresponding proteins. an n-terminal signal peptide is predicted with high probability in the cpv u1 protein, suggesting it may be secreted but it is not predicted in the tibv u1 protein despite the high level of overall sequence homology and so its significance is uncertain. features of the u2 proteins are unremarkable. the tibv and cpv genomes also each contain orf u3 encoded in an independent gene located between the g and l genes. the u3 proteins have the structural characteristics of viroporin-like proteins that occur in ephemeroviruses as the ␣1 proteins and in wonv and ngav as the u5 proteins. in each case, the viroporin orfs are located between the g and l genes, either directly following the g gene or following the g ns gene in those viruses in which it occurs. in tibv, u4 is an alternative orf that overlaps the g protein orf and could potentially encode a very small basic protein (6.6 kda/pi 9.6). however, u4 does not occur in cpv and its significance, if any, is unclear. as in many other rhabdoviruses, the cpv p gene also contains an alternative orf encoding a small basic proteins but none is evident in the tibv p gene. several other unclassified rhabdoviruses isolated from insects, mammals, birds and fish share a similar genome organisation (fig. 1) . tupaia rhabdovirus (tupv), durham virus (durv) and oak vale virus (oakv) each encode small hydrophobic (sh) proteins in orfs flanked by ti and ttp sequences in the region between the m and g genes (allison et al., 2011; springfeld et al., 2005) . however, although superficially similar, the putative proteins expressed from these genes have different characteristics. the tupv orf encodes a small protein with a predicted signal peptide and a central transmembrane domain that would generate an 8.6 kda non-glycosylated membrane-anchored protein with short ectoand endo-domains. in durv, the sh protein is predicted to have similar membrane topology with predicted signal peptidase cleavage generating a 6.9 kda non-glycosylated membrane-anchored protein. these proteins resemble the small hydrophobic protein encoded in the ngav u5 gene (see above). the oakv sh protein is predicted to have an n-terminal signal peptide but no membrane anchor to generate a 4.2 kda non-glycosylated secreted product (genbank gq294474). the mandarin fish (siniperca chuatsi) rhabdovirus (scrv) genome does not contain an independent sh gene but does encode a small hydrophobic protein as an alternative reading frame in the upstream m gene (tao et al., 2008) . the predicted structure is similar to that of the oakv sh protein with a high probability signal peptidase cleavage site generated a secreted product of only 2.9 kda. although it has been reported that the putative scrv sh protein could not be detected in infected fish cells, this may not be unexpected if it is indeed secreted. the x genes of sigmaviruses also encode a protein that is predicted to be secreted (see above) but it is very much larger than these small peptides. several of these unclassified rhabdoviruses also encode small basic proteins in alternative orfs in the p gene. although characterised by remarkable diversity and complexity, a global view of rhabdovirus genomes does reveal some patterns in the nature and location of accessory genes (fig. 6) . in various rhabdoviruses, accessory genes occur in each of the junctions between the structural protein genes and as alternative or overlapping orfs within all structural protein genes except the l gene. alternative or overlapping orfs occur least commonly in the n gene (and of course the l gene), possibly reflecting the structural and functional constraints on amino acid sequence variations, and in the g gene that requires sufficient genetic flexibility to evolve under the pressure of neutralising antibody. alternative orfs occur most commonly in the p gene in which the preservation of function is far less dependent on conservation of the amino acid sequence, allowing more scope for the evolution and stabilisation of new orfs in alternative frames (nadin-davis et al., 2002; spiropoulou and nichol, 1993) . the n-p, p-m, m-g, and g-l gene junctions have all been utilised as sites for accessory genes, although in rhabdoviruses examined to date, the p-m and g-l junctions have been utilised more commonly. the primary evolutionary constraint on the occupation of these sites may be the resulting attenuation of transcription in the downstream genes. for example, rare use of the n-p gene junction may be due to the importance of the balance of expression of the n and p proteins in modulating replication and transcription (banerjee and barik, 1992) . alternative orfs in the p gene occur very commonly and usually encode small basic proteins. although their function has not been examined in any detail, by analogy with paramyxoviruses, they may have multiple roles in replication, virion assembly and in modulating host responses to infection (fontana et al., 2008; irie et al., 2010; kurotani et al., 1998) . the p-m gene junction has been utilised as a site of accessory genes in many rhabdoviruses including genes encoding movement proteins in cytorhabdoviruses and nucleorhabdoviruses and the apparently secreted and glycosylated pp3 protein of sigmaviruses. the p-m junction is also the location of the set of three related 15-22 kda proteins in the hart park group viruses that may be involved in the promotion of apoptosis. the m-g gene junction is occupied less commonly but genes encoding small proteins that are either secreted or membrane-anchored do occur here, including the tibv u1 protein, and the small hydrophobic (sh) proteins of the unclassified viruses tupv, durv and oakv (allison et al., 2011; springfeld et al., 2005) . interestingly, in the mandarin fish rhabdovirus (scrv), and sh protein is encoded just upstream as an alternative orf in the m gene (tao et al., 2008) . the g-l gene junction is the most commonly occupied site of accessory genes in animal rhabdoviruses. genes encoding the non-structural glycoprotein (g ns ) invariably occur immediately downstream of the g gene and share a common genetic lineage (gubala et al., 2010; walker et al., 1992; wang and walker, 1993) . genes encoding viroporin-like proteins also usually occur in this region, immediately following g ns gene in ephemeroviruses (mcwilliam et al., 1997; wang et al., 1994) and following the g gene in flav and tibroviruses which lack g ns (gubala et al., 2011) . interestingly, the wonv viroporin-like protein is encoded immediately upstream of the g-l junction in an orf that overlaps the end of the g gene. the viroporin-like proteins all have a similar structure with a central hydrophobic domain and a highly basic c-terminal tail, and so may have evolved from a common ancestor (fig. 3) . many other accessory genes are located in the g-l intergenic region including the novirhabdovirus nv gene, and genes coding proteins of unknown function in ephemeroviruses, ngav and some plantadapted rhabdoviruses. the g-l intergenic region is also the site of the lyssavirus region (tordo et al., 1986) . sequence insertion at this site may be most favoured as it would primarily affect expression of the l protein which is required only in catalytic amounts. the ecological diversity of rhabdoviruses suggests they have an ancient evolutionary history, perhaps originating as insect viruses that have adapted to replication in plants or animals (hogenhout et al., 2003) . there is recent evidence that endogenous viral elements (eves) derived from rhabdoviruses occur commonly in insect genomes with which they have co-evolved over many millions of years (katzourakis and gifford, 2010) . rhabdovirus genomic diversity will also therefore have evolved over geological time frames, possibly involving rare events that may appear highly improbable in a contemporary ecological context. we have argued previously that the evolution of rhabdovirus accessory genes may have occurred by a copy-choice mechanism involving polymerasejumping (wang and walker, 1993) . according to this model, upstream relocation of the polymerase during replication would generate a sequence duplication in a process mirroring the downstream relocation that has been proposed for the generation of defective-interfering (di) genomes (lazzarini et al., 1981; perrault, 1981) . there seems no prima face reason to suggest that upstream relocation and downstream relocation should not occur with similar frequency but purifying selection would enrich for rapidly replicating di genomes and quickly remove larger genomes unless the duplication event has provided a specific selective advantage. although survival of such larger genomes may be exceedingly rare, preservation of the region in lyssaviruses does indicate that the selective advantage of maintaining long stretches of apparently non-functional sequence may sometimes be cryptic (kuzmin et al., 2008; ravkov et al., 1995) and sequence similarities in adjacent genes of some rhabdoviruses do suggest that duplication may have contributed to the evolution of accessory genes. such sequence similarities are evident in the g ns genes in ephemeroviruses and ngav (gubala et al., 2010; walker et al., 1992; wang and walker, 1993) , the related u1, u2 and u3 genes in the p-m region of hart park group viruses (see above), and the p6 gene in rice yellow stunt virus (huang et al., 2003) . other possible mechanisms for the acquisition of accessory genes include homologous genetic recombination and lateral gene transfer (lgt). life-long persistent infections occur commonly in insects and mixed infections with different viruses have been reported (chen et al., 2004; mourya et al., 2001; thavara et al., 2006) . although genetic recombination is believed to occur rarely in (-) ssrna viruses (holmes, 2009) , possible recombination events have been detected and may have contributed to their long term evolution (chare et al., 2003; han et al., 2008; lukashev, 2005; qin et al., 2008; sibold et al., 1999) . unlike gene duplication, in which the new gene must be preserved while evolving a new function, recombination could offer immediate selective advantage in larger more complex genomes by increasing tissue tropism, host range or replication efficiency or more effective blocking of host defensive mechanisms. homologous genetic recombination must therefore be considered an equally plausible explanation for the generation of genomic structural diversity in rhabdoviruses. lgt also offers a mechanism by which accessory genes could be obtained from the host or an unrelated virus to provide immediate selective advantage (holmes, 2009) . there is evidence that the coronavirus haemagglutinin-esterase glycoprotein was acquired from influenza c virus (luytjes et al., 1988) and that the envelope glycoprotein of the tick-borne orthomyxovirus thogoto may have been derived from a baculovirus (morse et al., 1992) . several other examples of possible lgt involving transfer of host genes to viruses have been reported but these events appear to be very rare and none has yet been reported to our knowledge in rhabdoviruses or other unsegmented (-) ssrna viruses (holmes, 2009) . it can also be argued that purifying selection should drive evolution towards smaller and perhaps more efficient genomes rather than large complex genomes. indeed, the generation of di particles, which occurs with high frequency during infection in vitro, would appear to provide the mechanism for rapid elimination of redundant sequences (lazzarini et al., 1981; perrault, 1981) and phylogenetic analyses do suggest that accessory genes may have been lost during the evolution of some lineages. as shown in fig. 5 , phylogenetic analysis of available rhabdovirus g protein sequences places ngav with wonv and flav to form the hart park group and analyses of the l and n protein sequences supports these relationships (bourhy et al., 2005; gubala et al., 2010) . however, the ngav g ns protein clusters with the ephemerovirus g ns proteins (fig. 5) , suggesting that it was obtained by a common ancestral virus and subsequently lost from wonv and flav. alternatively, ngav may have acquired g ns independently in a later event involving recombination with a virus in the ephemerovirus lineage. all arguments considered, perhaps the most congruent hypothesis is that the evolution of rhabdovirus genomic diversity is a dynamic process in which accessory genes have been gained and lost during the course of continual adaptation and purifying selection. despite the importance of rhabdoviruses as pathogens of humans, livestock, fish and plants, it is evident from this review that very little is known of the functions of the proteins encoded in their many accessory genes. in some respects, this is very surprising as five major rhabdovirus structural proteins have been the subject of intensive investigation and have provided some of the most important models for understanding fundamental processes during viral infection. on the basis of available evidence, and by analogy with other viruses that are rich in accessory functions (e.g., hiv, sars coronavirus), we can anticipate that many of the rhabdovirus accessory proteins may be important determinants of virulence and pathogenesis and function to increase the efficiency of viral replication, block host innate and/or adaptive immune responses, regulate apoptosis or induce cytopathology. understanding the strategies employed by rhabdoviruses to engage, divert and re-direct cellular processes will not only present opportunities to develop new anti-viral therapies but may also reveal aspects that have broader significance in biology, agriculture and medicine. one of the most attractive targets for future study is the role of rhabdovirus accessory genes during infection in insects and in transmission to mammals or plants. despite their importance as vectors of disease, and over 100 years of immunology, the antiviral defences of insects remain poorly understood (huszar and imler, 2008) . rnai has been identified as an important anti-viral mechanism and there is evidence of involvement of the toll and imd pathways that function primarily in innate immune responses to bacteria and parasites (kemp and imler, 2009 ). in drosophila melanogaster, the innate immune response to sigmavirus infection has been shown to involve upregulated peptidoglycan reporter and antimicrobial peptide gene expression (tsai et al., 2008) . however, innate anti-viral sensor and effector mechanisms in insects are largely unexplored and may be critical determinants of their susceptibility to infection and ability to act as efficient vectors. as most rhabdoviruses naturally infect insects, their accessory genes may well be involved in engaging defensive responses and so provide useful 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of ruminants, with large and complex genomes proteins of bovine ephemeral fever virus the genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes emerging viral diseases of fish and shrimp complex genome organization in the gns-l intergenic region of adelaide river rhabdovirus adelaide river rhabdovirus expresses consecutive glycoprotein genes as polycistronic mrnas: new evidence of gene duplication as an evolutionary process isolation and characterization of a rhabdovirus from co-infection of two viruses in mandarin fish key: cord-304424-048xo7jn authors: greninger, alexander l. title: a decade of rna virus metagenomics is (not) enough date: 2018-01-15 journal: virus res doi: 10.1016/j.virusres.2017.10.014 sha: doc_id: 304424 cord_uid: 048xo7jn it is hard to overemphasize the role that metagenomics has had on our recent understanding of rna virus diversity. metagenomics in the 21st century has brought with it an explosion in the number of rna virus species, genera, and families far exceeding that following the discovery of the microscope in the 18th century for eukaryotic life or culture media in the 19th century for bacteriology or the 20th century for virology. when the definition of success in organism discovery is measured by sequence diversity and evolutionary distance, rna viruses win. this review explores the history of rna virus metagenomics, reasons for the successes so far in rna virus metagenomics, and methodological concerns. in addition, the review briefly covers clinical metagenomics and environmental metagenomics and highlights some of the critical accomplishments that have defined the fast pace of rna virus discoveries in recent years. slightly more than a decade in, the field is exhausted from its discoveries but knows that there is yet even more out there to be found. in 1986, using direct cloning of 16s rdna genes norman pace presciently noted that the method "seems to have no upper limit as to the complexity of the populations" (pace et al., 1986) . thirty years later, pace's maxim only seems ever more true. deeper and deeper sequencing technologies have enabled the discovery of thousands of viral species. metagenomic viral discovery can be applied to almost any sample type and novel viruses emerge. growth in virome and metagenomics papers is outpaced only by that of the viruses discovered ( fig. 1 ). this review is meant to cover the field of modern-day rna virus metagenomics, where rna is perhaps best defined as "not dna", mostly due to the use of dna-specific nucleases in library preparations. many excellent reviews on viral metagenomics already exist, but no specific review focuses on rna virus metagenomics (bexfield and kellam, 2011; delwart, 2007; edwards and rohwer, 2005; mokili et al., 2012; rosario and breitbart, 2011; tang and chiu, 2010) . since there is absolutely no way to cover all the samples sequenced or genome organizations found even if one was restricted to a single order or family, the author apologizes in advance if he failed to cite your contribution to the field. where do we begin with rna virus metagenomics? construction and sequencing of cdna libraries share most of the features of rna viral metagenomic protocols and have existed since the late 1970s (sim et al., 1979; efstratiadis et al., 1977) . one could argue about the metaor the −genomic nature of the cdna sequencing example, but the method is nearly unchanged since its introduction. a single organism contains multitudes and now one of the more common ways of discovering new viruses is from searching said cdna sequencing data (feng et al., 2008; shi et al., 2016; krishnamurthy et al., 2016) . clearly, the depth of sampling has changed in recent years but sequencing even two clones could be considered metagenomics. rna virus metagenomics began with a dna virus and a contaminant. in a prescient 2001 manuscript, allander et al. added dnase to spin-filtered serum prior to extraction to enrich for viral particles (allander et al., 2001) . nucleic acids were extracted and separately processed for dna and rna libraries and then randomly amplified, enabling the discovery of two bovine parvoviruses from the commercial bovine serum that was used to dilute their viral spike-in controls. surprisingly, the novel dna viruses were present in the rna half of the experimentthe side that was specifically reverse transcribed and amplified in order to test the protocol's ability to detect gb virus c (allander et al., 2001) . this experiment is worth focusing on as it foreshadowed several themes in the world of rna virus metagenomics. the protocol used in the paper is surprisingly similar to that used today (conceição-neto et al., 2015) . host or environmental background dna dwarfs most nucleic acids and sequencing of rna species circumvents the problem so well that it may be the best way to find high concentration dna viruses that are being actively transcribed. even today concerns over whether metagenomics is sufficiently sensitive bedevil its use as a gold standard (schlaberg et al., 2017; wylie et al., 2012) . finally, in the world of metagenomics, you do not know what is in your sample until you sequence, and the sensitivity of your assay is perhaps best measured by your ability to recover ubiquitous reagent contaminants (bhatt et al., 2013; naccache et al., 2013) . environmental rna virus metagenomics arose somewhat later. as recently as 2003, degenerate pcr targeting the rna-dependent rna polymerase from picorna-like viruses had shown the potential for viral discovery in marine virus communities with the detection of four new rna virus families (culley et al., 2003) . large scale, high-profile efforts such as sailboat-based shotgun sequencing of the sargasso sea came two years later and used 0.1-3.0 μm filters that predominantly enriched for dna from microbial communities (venter et al., 2004) . three years later, robert edwards and forest rohwer were still calling for the development of methods that would allow for sequencing of rna viruses (edwards and rohwer, 2005) . allander's original method that detected the bovine parvovirus contaminants gave rise to clinical metagenomics in 2005 by discovering a novel human parvovirus (human bocavirus) and detecting a novel human coronavirus (coronavirus hku1) by screening pooled human nasopharyngeal aspirates (allander et al., 2005; woo et al., 2005) . reads to influenza a virus, human orthopneumovirus (rsv), metapneumovirus, human adenovirus, and ttv-like virus were also recovered from the 864 reads. that same year 36,789 colony sequences from dnase-treated rna extracted from viral concentrates of human feces revealed a preponderance of pepper mild mottle virus and other plant viruses, but no new human viruses (zhang et al., 2005) . the first non-human environmental-focused rna viral metagenomic survey used dnase-treated extracted nucleic acids from nuclease-treated viral concentrates from coastal communities in british columbia (culley et al., 2006) . the resultant pure rna was reverse transcribed and double-stranded cdna was synthesized, blunt-end ligated, and cloned to reveal nothing, for there was not enough rna present even in 40-60l of seawater, highlighting the importance of amplification in most any rna virus metagenomic preparation. after amplification, two novel picorna-like viruses and one novel +ssrna virus that remains taxonomically unassigned were discovered at high concentrations among the 355 reads. it is incredible to note that only one year later, researchers were reporting rna viral metagenomic sorcery of honeybees with hundreds of thousands of reads (cox-foster et al., 2007; koonin, 2007) . nextgeneration sequencing and rapid bioinformatics gains allowed researchers unbelievable opportunities ( fig. 1) (delwart, 2007) . due to the short read length of solexa sequencing at the time, initial viral metagenomic efforts required 454 sequencing, which allowed for hundreds of thousands of reads a few hundred bases long (rothberg and leamon, 2008) . the result was an array of human viral discoveries, including merkel cell polyomavirus, human bocavirus 2, salivirus, lujo arenavirus, dandenong arenavirus, sfts bunyavirus, and a variety of new human astroviruses including mlb1 and va1 through va5 (briese et al., 2009; feng et al., 2008; finkbeiner et al., 2008a finkbeiner et al., , 2008b holtz et al., 2009; kapoor et al., 2009a,b; koonin, 2007; li et al., 2009; meyer et al., 2015; palacios et al., 2008; yu et al., 2011) . groups often found the same viruses within days of each other and discrepant names for the same virus were common holtz et al., 2008 holtz et al., , 2009 kapoor et al., 2008; li et al., 2009) . pyrosequencing yielded impressive gains in environmental rna virus sequencing with profiling of a freshwater lake yielding tens of novel ssrna and dsrna viruses (djikeng et al., 2009 ). the rna virus metagenomic age was officially underway. 3. why has rna virus metagenomics been so successful? while early studies of metagenomics lamented the lack of rna viruses discovered compared to dna viruses, rna virus metagenomics publications by year for "rna virus metagenomics" (a) and "virome" (b) over the past decade using the "results by year" graph from pubmed. number of viral species (c), genera (d), and families (e) assigned by the ictv over the past four decades ("international committee on taxonomy of viruses (ictv), " n.d.). changepoint analysis with the "at most one change" statistical test was significant at year 1996 for genera and family data and 2015 for species data ("changepoint, " n.d.). a.l. greninger virus research 244 (2018) 218-229 appeals to the viral discoverer for a number of reasons. the first is that success in viral metagenomics is often measured by the genetic distance of a newly discovered virus from other known viruses. while finding a new virus in the environment is not necessarily a problem in either dna or rna, the low fidelity of rna polymerases and the sequence space they are capable of sampling, along with the possibility of recombination, lend themselves to new species and genera that are the trophies of metagenomic viral discovery. these properties also favor the metagenomic library preparation process. sufficient homology to land pcr primers for amplicon-tiling library preparation approaches may not exist, and the number of oligos needed to cover the sequence space of a given rna virus such as hepatitis c virus can make hybrid capture approaches cost-prohibitive. de novo assembly methods that allow for the rapid reconstruction of near complete genomes have also dramatically improved over the past decade and can handle widely disparate coverage for each contig (grabherr et al., 2011; bankevich et al., 2012) . all of these properties often make metagenomics the first method of choice when trying to recover sequence from even a known rna virus (greninger et al., 2017a; ogimi et al., 2017) . another reason for the provision of rna virus metagenomics is the ease and increased depth and sensitivity associated with removing background dna. indeed, one of the difficulties of rna virus metagenomics is that rna virus genome sizes are generally measured in the kilobases, but the size and quantity host dna background measures in the gigabases. if not partitioning sequence selectively across rna and dna, a single contaminating host cell can be the equivalent of half a million virions. removal of host dna is often required for one to recover complete rna virus genomes. the library preparation process simply requires a 30-min treatment with dnase i or benzonase to remove all background dna leaving the rna viruses behind. in addition, rna viral capsids are measured in nanometers and can be selectively enriched using 0.22 um or 0.45 um filtration. even if one is interested in dna viruses, rna metagenomics allows the recovery of mrnas that are associated with actively replicating dna viruses, which may be helpful information when so many dna viruses can be latent or integrated into host genomes (perlejewski et al., 2015) . recovery of these rna viruses is also made easier by the fact that, once host dna is removed, they can be present at extraordinarily high concentrations in total rna or constitute a surprisingly large proportion of transcripts in an mrna library, up to almost 90% of non-ribosomal rnas in some invertebrates (li et al., n.d.; shi et al., 2016) . based on total masses of nucleic acid in virus like particles, it has been estimated that half of marine viruses are rna viruses due to the larger burst size of eukaryotic rna viruses (steward et al., 2013) . a particularly instructive example was the discovery of the lake sinai virus in honeybees (runckel et al., 2011) . it was originally discovered and assembled metagenomically at such high titer that when researchers went to perform northern blots, they could see the viral rna staring back at them after rna electrophoresis. the author has also previously taken part in studies where the method of screening for known segmented rna viruses such as rotavirus was to run gel electrophoresis on extracted stool rna without amplification and look for banding (greninger et al., 2010; yu et al., 2012) . mining of publicly available transcriptome data has contributed greatly to the discovery of novel rna viruses (basler et al., 2005; schomacker et al., 2004) . indeed, genome-transcriptome studies of eurkaryotes sometimes miss the viruses present in their sequence when they map their rna-seq reads to their assembled dna-seq genome (babb et al., 2017; clarke et al., 2015) . rna viruses are lurking in the unaligned portion of the reads and often can be readily assembled and aligned to ncbi to find many new viruses in a given host (bekal et al., 2011) . most recently, six novel rna viruses were discovered in publicly available orb weaver spider transcriptome data (debat, 2017) . such sequences can be downloaded from the short read archive, assembled, and deposited into ncbi's third party annotation database ("ncbi third party annotation, " n.d.). research parasites of the world can now identify with their obligate intracellular parasitic brethren (longo and drazen, 2016) . rapid increases in sequencing depth achieved with next-generation sequencing have been matched by the increasing ease of library preparation. original viral sequencing protocols involved the preparation of multiple micrograms of nucleic acid and about a week of work (thurber et al., 2009; willerth et al., 2010) . protocols were slowly refined to reduce library preparation to a matter of days (greninger et al., 2010) . the development of transposon-based library creation and amplification have reduced rna metagenomic library preparation such that most labs perform the process in 12 days, and when coupled to nanopore sequencing the entire sample-to-sequence process can be as little as 6 h (greninger et al., , 2017b . a typical rna virus metagenomic protocol entails filtration, nuclease treatment, double-stranded cdna synthesis, followed by nextera xt tagmentation and pcr amplification (alexander l. hall et al., 2014) . the benefit of the protocol is the minimal hands-on time, low cost given the dilutability of the transposase, ease of dual-indexing, and direct compatibility with illumina sequencers. the protocol can be performed in as little as 4-6 h and is amenable to automation (greninger et al., 2017b (greninger et al., , 2017c . variants include the use of additional pcr cycles or amplification prior to tagmentation. however, in the author's experience these are not necessary given the amplification step present in the tagmentation library preparation protocol and the possibility of extra pcr cycles creating coverage bias (conceição-neto et al., 2015) . steps to increase the amount of nucleic acid used as input are helpful for the retrieval of longer reads and reducing pcr jackpotting, although often practitioners move forward with the rna present. while no doubt the best way to enrich for viral like particles, and perhaps to help define a sequence as likely viral in origin, the use of ultracentrifugation is not necessarily required for viral metagenomics. rather ultracentrifuge is kept for defined "virome" work, especially where there is a high bacterial background such as feces (kohl et al., 2015; temmam et al., 2015; thurber et al., 2009) . indeed a direct headto-head comparisons of ultracentrifugation and filtration for viral particle enrichment found the main benefit of ultracentrifugation to be the removal of host dna background, which is less of a concern for rna viral metagenomics if extracted nucleic acids are dnase treated (kleiner et al., 2015) . as evidenced from the early history, discovery of new etiological agents of human disease drove the adoption of viral metagenomics (lysholm et al., 2012; tang and chiu, 2010) . clinical metagenomics, or direct shotgun sequencing from clinical samples with minimal sample processing, for diagnostic testing has largely been driven by the diversity of rna viruses that preclude the routine use of single-plex or even multiplex pcr for broad clinical detection. indeed, current rapid diagnostics followed by 16s or its pcr and sanger sequencing cover most of the actionable and more exotic organisms present in clinical specimens. while metagenomics delivered on the promise of finding novel human viruses, viral discovery in humans has increasingly become a tragic story of patients interacting with the wrong squirrel or tick on the wrong day and most samples sequenced are frankly negative (hoffmann et al., 2015; mcmullan et al., 2012) . in part because of the declining number of viral discoveries in humans, clinical metagenomics has taken on the mantle of detecting all known pathogens instead (naccache et al., 2014; wood and salzberg, 2014) . metagenomics has been especially successful at finding known rna viruses in unexpected sample types (doan et al., 2016; naccache et al., 2015) . provided there is sufficient coverage, it can also provide single nucleotide resolution to define strain relatedness for outbreak epidemiology and, where available, antiviral resistance (barzon et al., 2011; capobianchi et al., 2013; greninger et al., 2017b greninger et al., , 2017c . furthermore, the vast majority of clinical samples sent for clinical metagenomic sequencing are entirely negative, yielding a greater potential role for "ruling out" an infectious cause through metagenomic testing prior to treating for other diseases such as autoimmune disorders with immunosuppressant medication. the excitement over using clinical metagenomics for one definitive test for clinical microbiology is tempered by questions around its sensitivity, actionable-ness of the sequence recovered (including crosscontamination), and cost. since clinical labs are highly unlikely to perform ultracentrifugation, metagenomics likely has the best sensitivity in acellular environments such as cerebrospinal fluid rather than respiratory or stool specimens (schlaberg et al., 2017) . human cells become an interfering substance and at concentrations much lower than those that interfere with other clinical chemistry measurements (ranjitkar et al., 2017) . detection of viruses at low concentration such as zika virus make shotgun metagenomics problematic for a rule-out result in diagnostic virology (bingham et al., 2016; landry and st george, 2017; naccache et al., 2016) . as with other culture-independent testing, culture will likely be required to provide antimicrobial sensitivity. while early detection can reduce further diagnostic testing, most rna viruses in humans have no treatment and are essentially unactionable. the ease and ubiquity of rna virus metagenomics is perhaps best illustrated by the increasingly diverse samples that are sequenced. when sequence divergence is the metric of success, metagenomicists boldly go where no human has gone before. for the purposes of raw discovery, the use of extraordinary mixed samples allow for one-stop shopping and diversify discovery risk, albeit with the drawback that host species cannot be confidently defined. and the continuing need to fund and sell discovery from the basis of security has focused metagenomic efforts on species with high zoonotic pandemic potential (racaniello, 2016; temmam et al., 2014) . here, bat guano routinely ranks high on the rna virus metagenomic list as it checks all three requirements listed above. bats aggregate a wide array of arthropods, plants, and other animals and produce copious amounts of guano and have been associated with multiple zoonoses. cameroonian fruit bats, chinese bats, myanmar bats, hungarian bats, french bats, american bats, big brown bats, tricolored bats, and little brown bats have all been profiled showing a vast array of rna viruses from eukaryotic hosts (dacheux et al., 2014; donaldson et al., 2010; ge et al., 2012; he et al., 2013 he et al., , 2017 kemenesi et al., 2014; li et al., 2010; yinda et al., 2017) . bats have been used to put an upper limitin the hundreds of thousands − of the total number of eukaryotic viruses that remain to be discovered (anthony et al., 2013) . other insect and arthropod aggregators such as spiders and birds have also yielded many new rna virus species (debat, 2017; ducatez and guérin, 2015; shean et al., 2017; zhou et al., 2015) . the greatest paradigm shifter in recent viral metagenomics work has been the sheer number and diversity of novel rna viruses present in arthropods and invertebrates. yong-zhen zhang and team at the chinese cdc have been systematically profiling the transcriptomes of invertebrates to upend our understanding of animal virology. a haul of 112 novel negative-stranded rna viruses from 70 arthropods surpassed the known diversity of mononegavirales (li et al., n.d.) . these included the first circular negative stranded rna viruses ever found, which were part of a larger family of viruses (chuviridae) that may yet become an order given that it is phylogenetically ancient to existing segmented and unsegmented viruses and was arranged in a number of topologies. and yet those were just the negative-stranded viruses. when the team increased the number of hosts profiled to 220, they found 1445 novel rna viruses (shi et al., 2016) . the genomes found illustrated widespread recombination among rna viruses, viruses with multiple copies of structural genes, loss of structural genes, as well as transfer of genes between virus and host. indeed, these discoveries so dwarf all previous rna virus discoveries, especially in the picorna-like superfamily, that the paper itself would be worth reprinting here in full. three additional novel families of positive-stranded viruses and two novel families of negative stranded viruses were described. rather than detailing what was found, the surprises are the families in which novel rna dependent rna polymerase (rdrp) sequences were not found such as the arenaviridae, picornaviridae, hepeviridae, paramyxoviridae, arteriviridae, and secoviridae. of course, whole undiscovered taxa basal to these taxa were also recovered. perhaps most impressively, zhang's team finished complete genomes with rapid amplification of cdna ends (race) for each of these viral genomes, a feat that not many metagenomic viral discovers undertake when finding more than a few novel viruses. no doubt evolutionary rna virologists will have just begun to comb through this data trove before the next ten thousand whole genomes of novel rna viruses is deposited from peat moss. most importantly for veterinarians, rna virus metagenomics has revealed a number of candidate etiological agents for a variety of animal ailments including avian proventricular dilatation disease, mink shaking syndrome, snake inclusion body disease, python respiratory illness, dairy cow disease, and tilapia die-offs (kistler et al., 2008; gancz et al., 2009; blomström et al., 2010; stenglein et al., 2014 stenglein et al., , 2012 uccellini et al., 2014; hoffmann et al., 2012; bacharach et al., 2016) . animal feces has proven fruitful hunting for metagenomicists, with canine, feline, porcine, equine, goose, duck, and shrew fecal rna viromes turning up a plethora of mostly novel picorna-like viruses, albeit with an unclear relationship to disease and perhaps some more closely linked to the invertebrates indicated above (fawaz et al., 2016; greninger and jerome, 2016; li et al., 2011; moreno et al., 2017; nagai et al., 2015; ng et al., 2014b; phan et al., 2011; reuter et al., 2012; sano et al., 2016; sasaki et al., 2015; zhang et al., 2014) . the recovery of ancient northwest territories cripavirus from 700-year old frozen caribou feces illustrated an incredible stability of encapsidated rna (ng et al., 2014a) . screens for fish viruses are still in their infancy and yet the phylogenetic diversity of fish and perhaps their intense exposure to marine rna viruses may prove them to be the chordate arthropods of viral discovery. novel members of the orthomyxoviridae, picornaviridae, and reoviridae have recently been described in fish (bacharach et al., 2016; reuter et al., 2013 reuter et al., , 2015 . our understanding of reptile and amphibian viral diversity is also still in its early days. the discovery of a non-old world, non-new world arenavirus in boid snakes with an envelope protein most closely related to filoviruses proved a tantalizing hint toward new variations on old taxonomies (stenglein et al., 2012) . for the basic scientists, a number of novel rna viruses have been discovered in model organisms such as c. elegans or d. melanogaster (félix et al., 2011; webster et al., 2015) . thousands of new viruses have been found in plants through metagenomics, with most awaiting further characterization or even finished assembly (roossinck et al., 2010) . plant virus metagenomics is probably one of the more fertile areas of growth for viral discovery in the coming years given their incredible biodiversity (roossinck, 2012) . rna viral metagenomics has made a number of contributions to our understanding of viral diversity at the far reaches of life. sequencing of a hot, acidic lake and wastewater revealed a novel ssdna virus with an rna virus-like capsid protein, suggesting past recombination between dna and rna viruses (diemer and stedman, 2012; "rdhv-like virus sf1 replication-like protein and capsid protein genes, complete cds," 2015). while there are still no definitive rna viruses of archaea, viral rna metagenomics isolated a 5.6 kb contig that contained an capsidlike protein and an rna-dependent rna polymerase that differed from the rdrps of viruses from eukaryotes and bacteria (bolduc et al., 2012) . the same can be said of the ciliates, who were previously noted among the eukaryota for having no known rna viruses (koonin et al., 2008) . metagenomic sequencing of wastewater after a rainstorm revealed two rna contigs that were highly suggestive of ciliate rna viruses in a metagenomic background of tetrahymena and several viruses best translated in the ciliate genetic code were found in the invertebrate surveys detailed above (greninger and derisi, 2015a; shi et al., 2016) . given the incredible growth in bacteriophage diversity from metagenomics, rna phages were a surprisingly late arrival to the metagenomic discovery party. as recently as 2016, genome sequences from rna phages were surprisingly few and far between with only 11 ssrna and 5 dsrna genomes compared to over 1000 dna bacteriophage genomes from 494 species (greninger and derisi, 2015b; krishnamurthy et al., 2016) . mining of metagenomic datasets yielded an additional 122 partial genomes from novel rna phages covering > 100 novel species including the first known rna phage number of a gram-positive bacteria (krishnamurthy et al., 2016) . these sequences included multiple unique genomic arrangements as well as orfs that had no similarity to known proteins. invertebrate virus sequencing efforts detailed above also found a similar number of levi-like viruses (shi et al., 2016) . given the many uses of the ms2 phage coat protein for understanding rna-protein interactions and as a tool for bioengineering rna affinity purification, the expansion of the rna phageome will likely create a number interesting tools for molecular biology that can build on ms2. the ridiculous number of virus discoveries in the past several years has put an incredible strain on the whole system of viral taxonomy (fig. 1c-e) . the inclusion of uncharacterized metagenomic viral sequence in taxonomy has long been of some debate at the international committee on taxonomy of viruses (ictv), the adjudicator of novel families, genera, and species of the viral world. only this past year did the ictv codify its existing policy through issue of a consensus statement on the inclusion of metagenomic data in its consideration of taxonomical placement of viruses . the torrid pace of discoveries has forced the hand of viral discoverers to come up with euphonious names for viruses for which there is almost no biological understanding ("international committee on taxonomy of viruses (ictv), " n.d.). convention previously dictated something like location, host, and number, even if the world health organization now recommends against ruining the tourist economy along the ebola river or in coxsackie, new york (fukuda et al., 2015) . since sequence similarities govern our understanding of genomic function, there is a temptation to name based on homology and let past discoveries anchor the novel (e.g., picobirnavirus or dicipivirus/cadicivirus/picodicistrovirus) (woo et al., 2012) . default nomenclature is approaching that of drug manufacturers or therapeutic antibody naming, with alternating consonant-vowel pairs that might have some basis in latin or relation to the location but obscures the actual place (e.g. avisivirus, aquamavirus, hunnivirus, harkavirus) reuter et al., 2012) . recently, five ancient chinese states formed the basis for naming novel family lineages (shi et al., 2016) . detection of ancient recombination in rna virus sequences is revealing genomic abominations that only a liger could love. what do you get when you cross a picornavirus and a calicivirus? probably a non-functional, nonreplicating piece of rna, but picalivirus a-d are still a thing (greninger and derisi, 2015c; ng et al., 2012) . tombusviridae and nodaviridae? tombunodavirus (grasse and spring, 2017; greninger and derisi, 2015d) . the inventiveness of nomenclature for novel virus discovery in a space of anarchy combats a steady march of rules and reason by the ictv . not even clinically relevant viruses such as human parainfluenza viruses or respiratory syncytial virusesnow human respirovirus, rubulavirus, orthopneumoviruscan resist binomial nomenclature and taxonomical reassignment (adams et al., 2017) . however, if discoverers get out far enough of the rationalization, their original names can take advantage of the movement to have an outsized influence on viral nomenclature runckel et al., 2011; woo et al., 2012) . in a field with seemingly no end to frontiers, it is curious to define even more. no doubt the 'virtuous cycle' of metagenomic sequencing will only increase in breadth and depth in the coming years with a concomitant growth of new bioinformatic algorithms leading to discovery of even more new organisms and further decrease in so-called unalignable viral "dark matter" (fig. 2) krishnamurthy and wang, 2017) . turning the crank on rna virus discovery metagenomics is now the provenance of undergraduate theses shean et al., 2017; zaaijer et al., 2016) . other than sampling more broadly and maintaining the exponential growth in genbank, what are orthogonal challenges for future rna virus metagenomic studies? fig. 2 . the conjoined circles of metagenomic success. the increased output of modern-day sequencers has led to increased metagenomic sequencing of samples, both environmental and clinical. this in turn has led to an explosion in the ncbi genbank and wgs databases. new discoveries beget the discovery of more divergent new viruses and organisms as they are now alignable to new references in the database. rather than searching through gapped alignments, the increased coverage of the genbank reference database allows for more exact k-mer searching, which allows for faster, more sensitive alignments of reads. this in turn makes metagenomic sequencing more useful, especially in the clinic, which in turn begets more sequencing. a.l. greninger virus research 244 (2018) 218-229 8.1. easier, faster, better, broader protocols to start in the wet lab, better methods of host depletion and recovery of full viral genomes are absolutely required. too many analogies to "finding a needle in a haystack" in the literature necessitate the use of cellulases (allen et al., 2009; kowalchuk et al., 2007; lax and gilbert, 2015; lecuit and eloit, 2014; naccache et al., 2014; soueidan et al., 2015) . even if rna viral transcripts constitute > 50% of an arthropod, they rarely do so in mammalian tissue (feng et al., 2008; shi et al., 2016) . in both clinical metagenomics and complex mixtures there are always low concentration viral sequences, either due to time of sampling, viral biology, or the long tail of ecological abundance, which deeper sequencing alone may not solve. synthetic biology and programmable nucleases may allow new options for depletion beyond the ribosome although current efficiencies must be improved (gu et al., 2016; matranga et al., 2014) . even though sample preparation methods have become considerably easier in the past decade, they still take several hours of hands-on time and have not routinely been ported to automated liquid handlers. with plunging sequencing costs, library preparation costs for rna virus metagenomics have taken a front seat. the time and cost of library preparation hamper adoption of metagenomics in the clinical virology lab. similar to the expanding suite of cas programmable sequence-guided nucleases, an rna-directed transposase for adapter tagging followed by one-step, dual-indexed amplification would speed up library preparation as the bulk of time in current preparations is spent on double-stranded cdna synthesis (gertz et al., 2012) . such an enzyme might be discovered through the approaches highlighted here, or perhaps through directed evolution of existing dna transposases (adey et al., 2010) . another solution might be a one-pot mix of enzymes that performed library preparation in a similar fashion as gibson cloning. even in a host-free environment, current amplification methods do not recover full viral genomes from end-to-end. instead, the common paradigm is random priming with amplification followed by a follow-up step of race to recover ends (li et al., n.d.; shi et al., 2016) . this second step is highly laborious, especially for segmented rna viruses, and difficult to execute on viral sequences at low concentration or in complex mixtures. furthermore, current transposase-based library methods can produce inversions or sequencing artifacts at the end of genome segments or in areas of rna secondary structure. as "unbiased" or "agnostic" as metagenomics can be, we still rely on sequencing by synthesis and a four base read-out. the ultimate immunoevasion strategy may be virally-encoded nucleotides (bryson et al., 2015; murphy et al., 2013; weigele et al., 2017) . host and virallyencoded rna editing already bedevils calling of accurate whole genome sequences in both positive-and negative-strand rna viruses (park et al., 2015; pelet et al., 1991; piontkivska et al., 2017; vidal et al., 1990) . adenosine-based modifications of viral genomes have shown effects on viral and host biology, but detecting them requires additional modifications to most sequencing protocols (gokhale and horner, 2017; gonzales-van horn and sarnow, 2017; kennedy et al., 2016 kennedy et al., , 2017 . our understanding of rna base modifications in viral genomes is still in its infancy. here, nanopore-based approaches to sequencing may hold the key to new discoveries as they can directly detect modified nucleotides, although current approaches are as dependent on the biology of pore proteins as we are on polymerases (ayub et al., 2013; carlsen et al., 2014) . the ultimate answer may be direct mass spectrometry-based detection of viral nucleotides (gooskens et al., 2014; cobo, 2013) . improvements in sensitivity of nanopore sequencing in terms of sequencing depth and the host depletion strategies highlighted above are required before nanopore-based metagenomics becomes routine or meaningful . other than the retroviruses, rna viruses do not readily provide host linkage information in metagenomics like dna viruses and phages often do. linking rna virus and host has been imputed based on transcript levels or dinucleotide usage, although the latter has recently been shown to more correlated with viral family than host species (kapoor et al., 2010; giallonardo et al., 2017; shi et al., 2016) . even when sequencing discrete organisms such as a singular honeybee, the co-existence of eukaryotic parasites means that imputation of viral host cannot be exact (runckel et al., 2011) . host promiscuity seems to be a common theme among newly-described rna viruses (nunes et al., 2017) . highly-indexed libraries, chemical linkage of rna species, and physical partitioning through single cell transcriptomics currently provide the best potential solution to the host imputation problem in complex mixtures (burton et al., 2014; chow et al., 2015; turner et al., 2009) . to date, most metagenomics has adopted the 19th century british naturalist approach of cataloging inventory and diversity. this focus of the field has been somewhat punishing for those involved, with burnout of both scientists and reviewers from not fully understanding the why of it all (canuti and van der hoek, 2014) . this author does not necessarily have a better plan, but perhaps phenotype might be a reasonable start. mass spectrometry was available to previous generations of biochemists, but they did not necessarily stop to catalog the contents of every fraction, focusing instead on function. this is not to understate the critical impact of the technical training that comes from viral metagenomics, nor to minimize the power of the method. but once we know that viruses are diverse, some concern for function is most likely in order. although they are now no doubt contributing, viral metagenomicists have previously borrowed liberally from the biochemical functions assigned to strings of sequence without confirmation. genomics has been the wires that allowed biochemistry to scale (167, 168). a number of options for functional characterization of these viruses are available but they unfortunately require work. additional culture models are needed to handle all the new discoveries and genetic engineering methods for more easily establishing cell lines from exotic species are worth pursuing (ettayebi et al., 2016; finkbeiner et al., 2012; stenglein et al., 2012; bell-sakyi and attoui, 2016; janowski et al., 2017) . focusing discovery on genetically tractable organisms has allowed for relatively rapid functional studies of the new discovered c. elegans orsay virus jiang et al., 2017) . a number of culture-independent methods exist now to characterize the novel viruses and their genes. biochemical assays for rdrp, proteases, helicases, capsids, and internal ribosome entry sites all exist and synthetic biology allows for easy cloning from a database to test these new proteins found through metagenomics (ladd effio et al., 2016; o'donoghue et al., 2012; peersen, 2017) . profiling known functions across the new viral species, such as rna binding strength and sequence specificity of novel rna phage proteins related to ms2 phage or viral protease specificity and kinetics, might be a place to start (o'donoghue et al., 2012) . affinity purification-mass spectrometry is a powerful method that allows discovery of viral-host protein-protein interactions in the absence of culture (jäger et al., 2011; greninger et al., 2012; medina et al., 2017; greene et al., 2016) . for vertebrate viruses, serological assays have long been used to confirm roles in disease (o'sullivan et al., 1997; bao et al., 2011; coller et al., 2016) . the ubiquity of these viruses also leads to questions of how we publish in the absence of experiments and what a sequence is worth. the number of novel viruses discovered needed for a high-profile paper has increased by logarithms (krishnamurthy et al., 2016; shi et al., 2016) . in the absence of particular phenotypic data or wet-lab viral characterization, many authors have turned to genome announcements, biorxiv, or simply uploading to genbank with extra metadata, figuring that sequencing is the most likely future method of both detection and discovery (debat, 2017; greninger and derisi, 2015e,f; karamendin et al., 2016; sharman et al., 2016; sparks et al., 2013) . given the glut of new viruses, expansion of sequencing, and the time it takes to publish, scientists may be more likely to align to your novel virus rather than read about it in a journal and decide to screen for it. this method has allowed for rapid sharing of data and de facto global screens of novel rna virus prevalence, tropism, and evolution that then lead to something resembling a story. perhaps the most difficult challenge to spring from sequence gazing at all the novel rna viruses is our understanding of the origins of rna viruses. at what point in host evolution does an rna viral pathogen arrive? and what exactly did the host look like then? rna virus metagenomics still does not provide the answer. results from initial rna virus genomics work suggested picorna-like viruses predated the radiation of the five supergroups of eukaryotic organisms (koonin et al., 2008) . since then, hints of genetic exchange between hosts and viruses, prominent in dna virus and bacteriophage genomics, are showing up in rna viruses (hughes and stanway, 2000; sasaki and taniguchi, 2008; shi et al., 2016; staring et al., 2017) . accounting for each of the domains commonly present in rna viruses is a challenge for any origin story, though greater knowledge of domain organization and swapping between different rna viruses may help firm up these dates (koonin et al., 2015) . biochemical characterization of the incredibly diverse rna viruses discovered through ongoing metagenomic screens will also test theories of the origin of rna viruses and better date the origin of different rna virus clades relative to different epochs of eukaryotic evolution. recent genetic and proteomic screens of eukaryotic rna viruses have indicated key reliance on host lipid modifying enzymes that regulate vesicular transport sasaki et al., 2012; greninger, 2015; carette et al., 2011; marceau et al., 2016; borawski et al., 2009; berger et al., 2011; arita et al., 2013; nagy and pogany, 2012; xu and nagy, 2016; salloum et al., 2013) . membrane repurposing is a critical determinant of rna virus replication which should be added to the "hallmark genes" of rna viruses, though they currently escape sequence alignment profiling (koonin and dolja, 2014) . these results have led to a hypothesis in which part of the rna virus origins involve selfish vesicular transport that likely expanded with the rise of intracellular eukaryotic transport (greninger, 2015; koonin et al., 2006; kuehn and kesty, 2005) . the list of rna viruses that have acquired a cellular envelope continues to grow feng et al., 2013; mcknight et al., 2017) . new concepts in bacterial vesicular transport might be consistent the close evolutionary link between the eukaryotic rna virus polymerases and bacterial retroelements (jan, 2017; kuehn and kesty, 2005) . to put it scientifically, it is nuts that all this and much more have happened in only approximately a decade. if they were not already, rna viruses have become the star child of evolutionary biologists, whether for day-by-day or eon-by-eon examples of evolution (koonin, 2007; koonin et al., 2008 koonin et al., , 2015 shi et al., 2016; xue et al., 2017) . with our world expanded and our place in it shrunk, there is still much to do regarding viral sequencing for our own quotidian human rna viruses (tang et al., 2017) . there is but one way to ensure that we find no more viruses. go sequence something. changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses rapid, low-input, low-bias construction of shotgun fragment libraries by 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the first sicinivirus isolate from chickens in mainland china by using viral metagenomics the author would like to thank keith jerome samia naccache, sue greninger, and ryan shean for helpful comments. key: cord-351766-ik89889i authors: juybari, kobra bahrampour; pourhanifeh, mohammad hossein; hosseinzadeh, azam; hemati, karim; mehrzadi, saeed title: melatonin potentials against viral infections including covid-19: current evidence and new findings date: 2020-08-05 journal: virus res doi: 10.1016/j.virusres.2020.198108 sha: doc_id: 351766 cord_uid: ik89889i viral infections are dangerous diseases for human health worldwide, which lead to significant morbidity and mortality each year. because of their importance and the lack of effective therapeutic approaches, further attempts should be made to discover appropriate alternative or complementary treatments. melatonin, a multifunctional neurohormone mainly synthesized and secreted by the pineal gland, plays some roles in the treatment of viral infections. regarding a deadly outbreak of covid-19 across the world, we decided to discuss melatonin functions against various viral infections including covid-19. therefore, in this review, we summarize current evidence on melatonin therapy for viral infections with focus on possible underlying mechanisms of melatonin actions. melatonin, the main hormone secreted by the pineal gland, plays crucial roles in pharmacological and pathological conditions in both animals and humans . numerous investigations have reported wide spectrum physiological and pharmacological functions for melatonin (bahrami et al., 2018; dehdashtian et al., 2020; mehrzadi et al., 2016) . melatonin has various properties such as antioxidant, antiinflammatory, anti-excitatory, sleep initiation, and immunoregulation (daryani et al., 2018; hosseinzadeh et al., 2018a; hosseinzadeh et al., 2019; juybari et al., 2019) . melatonin protects mitochondria against free radicals, modulates mitochondrial permeability transition pore, effects on mitochondrial electron flux, and influences energy metabolism . melatonin is effective as a therapy for sleep disturbances, cardio-vascular diseases, ocular diseases and other pathologies. furthermore, as a complementary therapeutic agent, melatonin has shown beneficial effects in neonatal care, in vitro fertilization haemodialysis and anesthesia (sanchez-barcelo et al., 2010) . viral infections are serious life-threatening and problematic human diseases which contribute to mortality and morbidity in individuals with primary immunodeficiency disorders across the world. moreover, finding effective treatments for these diseases has been noticed. in addition to possess diverse biological and therapeutic benefits, melatonin also has antiviral properties (silvestri and rossi, 2013) . it is well-known that melatonin as an anti-oxidative and anti-inflammatory agent counters acute lung injury (ali)/acute respiratory distress syndrome (ards) induced by viral and bacterial infections. melatonin can be beneficial in critically ill patients via reducing vessel permeability, inducing sedation, decreasing agitation and increasing sleep quality. these beneficial properties of melatonin may highlight this hypothesis that melatonin may exert further clinical outcomes for covid-19 patients . this review aimed to summarize available data on melatonin therapeutic j o u r n a l p r e -p r o o f effects on viral infections with focus on coronaviruses, especially coronavirus disease 2019 . as mentioned earlier, melatonin is primarily secreted from the pineal gland during the dark period of a circadian cycle (dubocovich, 1988) . circadian rhythm disruption interferes with nocturnal melatonin signals leading to the impairment of several physiologic cell actions and homeostatic metabolic rhythms causing acceleration of malignancy (stevens et al., 2014) . melatonin interacts with numerous cellular proteins such as signaling molecules, transporters, channels, and enzymes liu et al., 2019) . in addition to antiinflammation, anti-oxidation, biological rhythms resynchronization, and sleep induction, melatonin has multiple biological impacts, including apoptosis induction and immunomodulation (carlberg, 2000; pourhanifeh et al., 2020) . crucial effects of melatonin such as oncostatic properties are mediated through receptorindependent and receptor-dependent mechanisms (srinivasan et al., 2008) . the mt1 receptor is thought to be implicated in melatonin suppressive effects in mammalian brains to modulate brain functions; this type of receptor is primarily distributed in the retina, skin, liver, hypothalamus suprachiasmatic nuclei, and pars-tuberalis of the pituitary gland (carbajo-pescador et al., 2011; reiter, 1991) . the mt2 receptor is involved in phase-shifting circadian activity rhythms; this receptor is mainly located in the retina, vessels of extremities, and osteoblasts. receptor-independent mechanisms of melatonin are associated with the prevention of tumor metabolism, circadian disruption, and suppression of migration and angiogenesis (hill et al., 2015; srinivasan et al., 2008) . melatonin easily penetrates into cells and exerts diverse potential impacts through interacting with intracellular and cell surface receptors, or direct scavenging free radicals (hosseinzadeh et al., 2018b) ; these actions of j o u r n a l p r e -p r o o f (henderson et al., 2005; sigurs et al., 2010; zomer-kooijker et al., 2014) . the rsv infection results in incomplete immunity, which contributes to the recurrent infection throughout life. the rsv infection outcome determinants are not well-recognized, but both host and viral factors play a part (johansson, 2016) . due to the failure of persistent immune response induction against rsv antigen, development of an efficient vaccine for rsv infection has not been successful (ruckwardt et al., 2019) . inflammation (nuriev and johansson, 2019) and oxidative stress (wang et al., 2018) have been recognized as two principle events implicated in rsv pathogenesis. the rsv infection causes inflammation of the airways and epithelial cell injuries resulting in severe breathing problems. airway inflammation stimulates cytokine production and augmented mucous release in immune-compromised patients and children (rudd et al., 2005) . in patients with rsv infection, enormous levels of inflammatory cells infiltrate into the perivascular space of lung. therefore, in order to treat different rsv-mediated diseases, inflammation prevention possesses critical therapeutic significance (rudd et al., 2005) . oxidative stress leads to the modification and disruption of cellular molecules during immune-inflammatory response to viral infections (bakunina et al., 2015) . respiratory syncytial virus has been indicated to mediate the activation of v-rel reticuloendotheliosis viral oncogene homolog a (rela) through inducing reactive oxygen species (ros) generation (jamaluddin et al., 2009) . in airway epithelial cells infected by rsv, antioxidants could inhibit the enhancement of interferon (ifn) regulatory factor (irf)-3 signals and over-production ros (liu et al., 2004) . recently, some studies have been conducted to demonstrate anti-inflammatory and antioxidant functions of melatonin against rsv infection. huang et al. evaluated the suppressive effect of melatonin on rsv infection through modulating toll-like receptor (tlr)-3 signaling, in vitro. the downstream pathway from tlr-3 results in the activation of nuclear factor-κb (nf-kb), irf-3, and j o u r n a l p r e -p r o o f subsequent expression of various inflammatory mediators. they showed that melatonin timeand dose-dependently attenuates tlr-3-induced gene expression in macrophages infected by rsv; repression of nf-kb activity by melatonin seems to be the strategic event leading to reduction of the expression of inflammatory genes. however, melatonin did not affect tlr-3 and myeloid differentiation factor 88 (myd88) expressions. these findings show the immunoregulatory roles of melatonin (huang et al., 2008) . in another study, huang and colleagues performed a research to assess melatonin potentials against rsv infection. melatonin administration considerably decreased levels of malondialdehyde (mda) and nitric oxide (no), and increased glutathione (gsh) and superoxide dismutase (sod) activities in mice intranasally inoculated with rsv. furthermore, melatonin suppressed pro-inflammatory cytokine production in serum of rsvinfected animals, demonstrating ameliorative effects of melatonin on rsv-mediated lung injuries through blocking oxidative stress and pro-inflammatory cytokine production (huang et al., 2010) . obviously, further investigations are required to explore exact underlying mechanisms of melatonin actions and prove its therapeutic potential for rsv infection treatment. venezuelan equine encephalitis/encephalomyelitis (vee) virus, a member of the togaviridae family of viruses, causes flu-like symptoms such as pharyngitis, nausea, fatigue, fever, and myalgia in humans. among 14% of subjects, severe neurological complications can happen following encephalitis, such as coma, blurred vision, confusion, and seizures. progression to encephalitis probably leads to chronic neurological deficits and dying of approximately 1% of patients (de la monte et al., 1985; gardner et al., 2008; weaver et al., 2004) . recent studies have demonstrated that vee virus replicates in the brain, resulting in the inflammation and subsequent damage of blood-brain barrier leading to the enhanced permeability. this event contributes to neuroinvasion and subsequently causes long-lasting neurological sequelae (cain et al., 2017) . furthermore, microglia responds to the infection through releasing pro-inflammatory factors (keck et al., 2018) . evaluation of vee virus-infected mice brain indicated a complex immune response to vee virus infection; genes involved in the various immune responses, apoptosis, and inflammation over-expressed in the brain of mice infected by vee virus (sharma et al., 2008) . during vee viral infection, the initiation of unfolded protein response (upr) pathway and subsequent activation of early growth response protein 1 (egr1) have important roles in virus-mediated apoptosis outcome (baer et al., 2016) . alteration in immune responses and/or oxidative stress could be involved in vee viral infection. in this regard, the immunomodulatory and pro-oxidant activities of no have been demonstrated (burrack and morrison, 2014) . melatonin, as a potent antioxidant agent, may show antiviral function against vee virus through inhibition of oxidative stress . melatonin also diminishes increased brain expression of apoptosis marker proteins and formation of mda and nitrite in vee virus-infected mice both in vivo and in vitro. moreover, melatonin enhances survival rate (montiel et al., 2015) and decreases nitrite and lipid peroxidation products levels in the brain of affected animals (valero et al., 2007) . valero et al., showed that melatonin markedly reduces the nitric oxide concentrations in infected splenocytes. these findings show that splenocytes infected with the vee virus generate important amounts of nitric oxide. melatonin is suggested to protect the vee virus-infected mice through reducing nitric oxide concentration in the tissue (valero et al., 2005) . j o u r n a l p r e -p r o o f acute liver failure (alf) is a serious situation characterized by extensive liver necrosis. clinical presentations include a severe disruption of liver functions with development of jaundice, impairment of coagulation and progression of encephalopathy within 8 weeks of first signs and symptoms onset, at least in subjects without pre-existing hepatic diseases. infection with liver-tropic viruses, autoimmune diseases, metabolic disorders (including wilson's disease), and paracetamol toxicity are the main alf causes, which is also known as fulminant viral hepatitis (bernal and wendon, 2013; ganger et al., 2018; stravitz and lee, 2019) . of note, disease outcome is poor and its survival rate is fewer than 20% in the absence of liver transplantation; however, after liver transplantation, survival rates may reach 80% (bernal et al., 2015; lemon et al., 2017) . according to recent reports, viral hepatitis will be eliminated until 2030. in this regard, the number of infected individuals and associated mortality should be reduced by 90% and 65%, respectively (mendlowitz et al., 2020) . rabbit hemorrhagic disease (rhd) is a greatly lethal viral infection in rabbits characterized by severe necrotizing hepatitis and disseminated intravascular coagulation in the liver, kidney and spleen (beller et al., 1969) . very little is known about the pathogenesis of this disease. furthermore, few studies are available realted to melatonin effects on viral hepatitis. hepatoprotective roles of melatonin in fulminant hepatic failure partially induced by the activation of nuclear factor erythroid 2-related factor 2 (nrf2) pathways leading to the inhibition of oxidative stress and elevation of antioxidant enzymes activities (crespo et al., 2010) . suppressive effects of melatonin on apoptotic liver damage is related to the inhibition of endoplasmic reticulum (er) stress by modulating the three arms of upr signaling (tuñón et al., 2013) . in rhd virus (rhdv) infection, the sphingosine kinase 1 (sphk1)/sphingosine 1-phosphate (s1p) system activates viral replication resulting in the induction of inflammatory pathways. melatonin attenuates elevated s1pr1 receptor expression, s1p j o u r n a l p r e -p r o o f production, interleukin-6 (il-6), tumor necrosis factor alpha (tnf-α), and tlr-4 expression in rabbits with rhdv infection (crespo et al., 2016) . melatonin prevents rhdv-induced hepatic regenerative/proliferative response by antiinflammatory function and stimulation of regenerative mechanisms (laliena et al., 2012) . melatonin dose-dependently inhibits liver apoptosis-induced by rhdv infection. this antiapoptotic effect is associated with the elevation of b-cell lymphoma-extra large (bcl-xl) and b-cell lymphoma 2 (bcl-2) expressions and reduction in the cytosolic release of cytochrome c, expression of bcl2 associated x (bax) and activation of caspase-9 (tuñón et al., 2011) . rabbit hemorrhagic disease virus also induces autophagic responses which is remarkably repressed by melatonin administration (san-miguel et al., 2014) . therefore, more research should be performed to prove safety and effectiveness of melatonin for humans myocarditis, inflammation of cardiac muscle tissue, is caused by infiltration of immunocompetent cells following cardiac injuries. infectious causes include a wide range of fungi, protozoa, bacteria or viruses, but it is most commonly caused by inflammatory events directed against viral pathogens. up to now, a shift is seen from enteroviruses and adenoviruses such as coxsackievirus b3 (cvb3) to human herpes virus 6 and parvovirus b19, as the most commonly recognized cardiotropic viruses in endomyocardial biopsies (kuhl et al., 2005; verdonschot et al., 2016) . among young people, viral myocarditis is known as a leading cause of sudden death. this viral disease contributes to cardiac dysfunction and can progress to dilated cardiomyopathy (dcm). 5-year survival rate of dcm patients is only 55% under current heart failure therapy, showing the requirement for more appropriate approaches (van linthout et al., 2014) . nowadays, no efficient medications have been discovered to intervene with myocarditis progression and routine therapeutics strategies are unsatisfactory. some mechanisms have been addressed for viral myocarditis pathogenesis. the exact role of autophagy in cardiac tissue is not fully understood. as postmitotic cells, cardiomyocytes utilize basal autophagy levels for general organelle homeostasis and cellular maintenance (de meyer and martinet, 2009; gottlieb et al., 2009) . notably, autophagy is further detected in failing cardiomyopathic hearts (miyata et al., 2006; shimomura et al., 2001) and ischemic cardiomyocytes (yan et al., 2005) ; autophagy suppression has been reported to augment cardiac hypertrophy development (pfeifer et al., 1987) . regarding the fact that autophagy also clears intracellular pathogens (levine, 2005; richetta and faure, 2013) , diverse microorganisms use strategies to escape or to counteract this cellular process for their own survival and replication advantage. the replication of cvb3 relies on intracellular membrane rearrangement into double-membrane vesicles (yoon et al., 2008) . coxsackievirus b3 uses the autophagy to gain replication advantages on autophagosomes surface (wong et al., 2008) . after ischemic insult, apoptosis (orzalli and kagan, 2017) is activated in the myocardium; apoptosis is another kind of programmed cell death playing roles in viral infection prevention. promoted viral replication subsequently enhances cvb3-mediated myocardial apoptosis (wang et al., 2017b) . apoptosis mechanisms are complex, ander stress has recently been introduced as a novel transduction signaling implicated in apoptosis (xin et al., 2011) . the endoplasmic reticulum is a crucial intracellular organelle which supports several activities such as integration into the membrane, translocation across the membrane and protein synthesis (anelli and sitia, 2008) . the suppression of unfolded protein accumulation, oxidative stress, and protein glycosylation in the er lumen may impair normal er functions and stimulate unfolded protein responses called er stress (ron and walter, 2007) . coxsackievirus b3 initiates apoptosis in cardiomyocytes through induction of er stress by activation of protein kinase r-like er kinase (perk) pathway (wang et al., 2017a) . in j o u r n a l p r e -p r o o f addition to mentioned cellular events involved in the pathophysiology of viral myocarditis, mitochondrial damage is another important mechanism leading to myocardial injury and cardiac dysfunction (lin et al., 2017) . few recent investigations have indicated beneficial potentials of melatonin in the treatment of viral myocarditis. ouyang et al. conducted a study to evaluate the protective role of melatonin in the setting of viral myocarditis with a focus on mst1-hippo pathway, er stress, and mitochondrial dysfunction. they showed that melatonin ameliorates cardiac function and represses virusinduced cardiomyocyte apoptosis. melatonin also repressed er stress and maintained mitochondrial dysfunction. furthermore, mst1 was upregulated by virus infection, which reduced by melatonin (ouyang et al., 2019) . sang and co-workers performed an in vivo study to investigate protective effects of melatonin on viral myocarditis and explore possible mechanisms. melatonin treatment significantly ameliorated the myocardial injuries through improving myocarditis via repressing inflammation. furthermore, melatonin regulated the rate of autophagy and inhibited apoptosis in mouse hearts with cvb3-induced myocarditis. therefore, melatonin should be further considered as a new therapeutic agent for viral myocarditis (sang et al., 2018) . current studies on melatonin treatment for mentioned viral infections are summarized in table 1 . in addition to mentioned viral infectious diseases, melatonin has been shown to play therapeutic roles in infection induced by ebola virus. ebola virus disease, a rare but deadly illness, occurs following the transmission of ebola virus from wild animals to humans . the ebola virus increases blood coagulation, weakens the immune system, and mediates noticeable inflammatory responses leading to the oxidative stress-induced organ and cellular damages. especially, the endothelial injury of blood vessels causes hemorrhage shock, a deadly complication of ebola infection (murray, 2015; nicastri et al., 2019) . melatonin is a potent free radical scavenger (reiter et al., 2016a) , has anti-inflammatory properties (hardeland, 2018) , triggers the immune system , and affects thrombin formation and platelet physiology (geisbert et al., 2003) ; with these effects, melatonin can combat with ebola. the vasculopathy is obviously a substantial event and contributes to hemorrhagic shock syndrome leading to death in subjects with ebola infection (lyon et al., 2014) . junaid et al. (junaid et al., 2020) used melatonin against ebola-induced hemorrhagic shock and observed that melatonin decreased vascular permeability. this improvement in vasculopathy makes melatonin a therapeutic candidate to limit ebola infection. furthermore, melatonin probably induces the expression of heme oxygenase 1 (ho-1), which reduces the replication of ebola virus (hill-batorski et al., 2013) ; this demonstrates possible direct antiviral effects of melatonin (boga et al., 2012) . the utility of melatonin has been suggested in human populations infected with ebola virus (tan et al., 2014) melatonin also has beneficial properties in controlling animal models of west nile virus (ben-nathan et al., 1995) and semliki forest virus (ben-nathan et al., 1995) . furthermore, melatonin has slight proviral impacts upon dengue virus type-2 infection in hek293t/17 cells, but no impact was observed in hepg2 cell (paemanee et al., 2018) . altogether, antiviral effects of melatonin should be further proved, especially in human studies. due to the vast distribution of covid-19 around the world, and because of increasing number of affected individuals as well as enhancing number of deaths, the therapeutic effect of melatonin in fighting against this life-threatening viral illness will be discussed in the next parts in details. coronavirus disease 2019 (covid-19, named by the world health organization (who) on feb 11, 2020) is cused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a j o u r n a l p r e -p r o o f novel class of coronavirus. covid-19 was first identified in late december 2019 in wuhan, hubei province, china (huang et al., 2020a) . this coronavirus was detected in a cluster of patients with pneumonia of an unknown etiology, which epidemiologically linked to a seafood and wet animal wholesale market in wuhan, hubei province, china (lu et al.) . covid-19 is spreading rapidly to other geographical locations after the outbreak in china. on march 2020, who declared a covid-19 pandemic to emphasize the gravity of the situation and urge all countries to take actions for detecting infection and limiting or preventing transmission (remuzzi and remuzzi, 2020) . the first case of infection was reported in the united states on january 19, 2020 (holshue et al., 2020) . the clinical manifestations of covid-19 include fever, headache, sore throat, dry cough, chest pain, dyspnea, myalgia, fatigue, and diarrhea. laboratory test may indicate normal or decreased total number of white blood cells, and chest ct imaging shows pneumonia (ahmed, 2020; salehi et al., 2020) . most covid-19 patients, about 80%, have mild symptoms and recover within one to two weeks; symptoms will be mostly confined to the upper and conducting airways. about 20% of infected patients will develop severe pneumonia with dyspnea requiring hospitalization. a small proportion of these patients are critically ill with very severe lung dysfunction, requiring mechanical ventilation (thomas-rüddel et al., 2020) . the overall case-fatality rate for covid-19 has been estimated to be around 2%; however, the chances of dying from covid-19 vary markedly with age (wu and mcgoogan, 2020) . in terms of physiopathological point of view, sars-cov-2 infection shares many similarities with sars-cov infection. sars-cov-2 virus like sars-cov causes excessive host inflammatory responses, leading to profound pulmonary injuries. hence, the severity of disease related to both viral infection and host response (tay et al., 2020) . the crown-like spikes (s protein) on their surface of both viruses sars-cov and sars-cov-2 intermediates the entry of virus into the target cells. the s protein has two subunits including s1 and s2. the s1 subunit comprises a receptor-binding domain (rbd) and an aminoterminal domain (wong et al., 2004; xiao et al., 2003) . the rbd binds to ace2 (angiotensin-converting enzyme 2), as its host cell target receptor starting the infection process. in fact, the interaction between rbd and ace2 initiates endocytosis of the sars-cov-2/ace2 complex into the target cells, leading to the exposure of virion to endosomal proteases (simmons et al., 2005) . virus-induced internalization of pulmonary ace2 and loss of its function may be considered as an important cause in the pathology of sars-cov-2 associated acute respiratory distress syndrome. ace2 has been known to be involved in the modulation of renin-angiotensin system (ras). the major role of ace2 is the conversion of angiotensin ii (ag ii) to ag 1-7, which is a vasodilator peptide and exhibits protective properties in the cardiovascular organ. ace2 internalization by sars-cov-2 may result in a dysfunction of ras and amplification of pulmonary tissue destruction initially inflamed by sars-cov-2. therefore, reduced expression of ace2 and ras dysfunction following sars-cov-2 infection may influence fluid/electrolyte balance and blood pressure. on the other hand, the diminution of ace2 may exacerbate airway inflammation and vascular leakage, contributing to chronic loss of pulmonary function, and enhanced tissue fibrosis (south et al., 2020) . in addition to the presence of ace2 in the pulmonary system, ace2 isextremely expressed in other organs such as cardiovascular tissues, neuronal cells, tubular epithelium of kidneys, etc. in patients with cardiovascular disease (cvd), the diminution of ace2 induced by sasrs-cov-2 would be expected to worsen cvd (yousif et al., 2012) . furthermore, in consistent with previous reports about the neurovirulent effect of sars-cov, the recent studies on sars-cov-2 has been shown that the entry of ace2/virus complex into neuronal cells could lead to infected cell death mao et al., 2020) . brain tissue involvement may even interfere with the proper function of autonomic nervous system in the regulation of blood pressure and potential respiration (wrapp et al., 2020) . in the same way, the loss of this enzyme in kidney tissue may interfere with tubular sodium transport, promoting blood volume and pressure along with acute and chronic kidney damages (e silva and teixeira, 2016; williams and scholey, 2018) . the mechanistic basis for the innate resistance of bats to counteract viral disease is poorly understood and stayed on the level of hypothesis. the role of melatonin in bat's anti-viral immunity is not thoroughly known. however, it is thought that melatonin may play a crucial role in the sars-cov-2 virus (shneider et al., 2020) . in accordance with heideman et al and tresguerres et al studies, endogenous melatonin concentrations in bats range from 60 to 500 pg/ml during the night and 20-90 pg/ml during the day depending on the species. melatonin production level in humans is significantly lower than in bats, particularly in the elderly ones (heideman et al., 1996; tresguerres et al., 2006) . given that the elderly people were excessively affected by sars-cov-2 than people under the age of 20, besides other factors, it could be hypothesized that high levels of melatonin exert protective properties in bats against the severity of sars-cov-2. the p21-activated kinases (paks), a family of serine/threonine kinases, have been known as downstream effector proteins for the small gtpases in mammalian cells. these kinases are classified into two major categories; group i includes (p21 (rac1) activated kinase (pak) 1, pak2 and pak3, and group 2 includes pak4, pak5, and pak6. in the last decade, paks have acquired great attention in medicine due to their contribution to a diversity of cellular functions (kumar and vadlamudi, 2002) . among them, pak1 is considered as a pathogenic enzyme and its unusual activation could be responsible for a broad range of pathologic j o u r n a l p r e -p r o o f conditions such as aging, inflammation, malaria, cancers immunopathology, viral infections, etc (maruta, 2014) . in a recent study conducted by oh et al. (2016) "chloroquine" (cq) (an antimalarial drug used as an experimental medication in covid-19 treatment protocol) was found to increase the expression of p21 that was downregulated by pak1 in th1 cells (oh et al., 2016) . furthermore, lu et al. (2020) beside oxidative cell injury induced by sars-cov-2, serum inflammatory markers such as d-dimers, c-reactive protein, and ferritin, neutrophil count in a complete blood count (cbc), and inflammatory cytokines, and chemokines increase in severe covid-19 patients (merad and martin, 2020a; ruan et al., 2020) . in the most severe patients, the systemic cytokine profiles have a lot in common with those found in cytokine storm syndrome (gong et al., 2020; yang et al., 2020) . these cytokine profiles include macrophage activation syndrome in association with the enhanced formation of cytokines such as tnf-α, il-6 and, il-7. in addition to these cytokines, some chemokine ligands including cxc-chemokine ligand 10 (cxcl10), cc-chemokine ligand 2 (ccl2), and cc-chemokine ligand 3 (ccl3) as well as the soluble interleukin-2 receptor have also been detected in serum profile of covid-19 patients (mehta et al., 2020b; merad and martin, 2020a; schulert and grom, 2015) . according to the above-aforementioned explanations, it can be hypothesized that the dysregulation of the mononuclear phagocyte (mnp) system contributes to acute inflammation associated with covid-19 (merad and martin, 2020a) . it is though that sars-cov-2 causes severe acute respiratory via triggering pyroptosis, a highly inflammatory type of programmed cell death that is often observed following cytopathic viruses. as the molecular signaling pathways of destructive effects of sars-cov-2 have not yet been completely identified, data on the inflammatory mechanisms of sars-cov have been used in this review. the viral protein encoded by orf8b directly binds to a pyrin domain-containing receptor 3 and nucleotide-binding domain leucine-rich repeat (nlr) protein such as nlrp3 inflammasome (shi et al., 2019) ; this results in the stimulation of the inflammasome adaptor protein apoptosis-associated speck-like protein containing card (asc) and caspases 4, 5 and 11. this process causes the cell membrane disruption with a j o u r n a l p r e -p r o o f discharge of inflammatory products to the extracellular space (shi et al., 2017) . therefore, inhibition of pyroptotic cell death induced by nlrp3 in the lungs could be considered an essential clinical need (shneider et al., 2020) . in response to sars-cov-2 infection and the subsequent destruction of bronchial epithelial and alveolar cells, the local immune system is stimulated. monocytes and macrophages are recruited to affected tissues, which results in the release of cytokines and initiation of t and b cell-mediated immune responses. although the infection will be commonly eliminated by triggering the immune system, severe pulmonary injury and even systemic pathological abnormalities can be observed due to a dysfunctional immune response (tay et al., 2020) . a cascade of local pro-inflammatory cytokines and chemokines such as monocyte chemoattractant protein 1 (mcp1), il-6, infγ, and interferon gamma-induced protein 10 (ip-10) or cxcl10 enter into the bloodstream of afflicted patients (huang et al., 2020b; . indeed, the emergence of such reactions is hallmark of t helper 1 (th1) cellpolarized response (huang et al., 2005) . similar responses could be observed in sars-cov and middle east respiratory syndrome (mers)-cov infections. moreover, the production of mentioned chemokines and cytokines attracts immune cells especially monocytes and t lymphocytes from the bloodstream into the sites of inflammation . therefore, the recruitment of immune cells from the bloodstream to lungs of patients with covid-19 infection may express decreased lymphocyte-neutrophil ratio and lymphopenia in approximately 80% of these patients (qin et al., 2020; tay et al., 2020) . it seems that cytokines, hyper-inflammatory state, and lymphopenia play crucial roles in covid-19 pathogenesis; thus it can be concluded that immunoregulatory agents are probably able to reverse the situation and treat the infection (saghazadeh and rezaei, 2020) . pineal melatonin, as an immunoregulatory agent (mańka and majewska, 2016) , is important j o u r n a l p r e -p r o o f to the dampening or resetting of immune cells at night; these effects seem to be mediated by driving the shift from glycolytic metabolism to oxidative phosphorylation. this 'resetting' of metabolic function is important and is proposed to underpin the immune senescence that is evident in the elderly. a growing body of data shows that the management of melatonergic pyruvate dehydrogenase complex provides a link between glycolysis and the krebs cycle through the conversion of pyruvate to acetyl-coenzyme a (acetyl-coa), thereby elevating oxidative phosphorylation and atp generation. suppression of pineal melatonin could fail the circadian "resetting" of mitochondrial metabolism that is very important in the management of immune cells (anderson and reiter, 2020) . following respiratory viral infection, viruses are phagocytosed by dendritic cells and antigens are presented to t cells. afterward, cytotoxic cd8+ t cells trigger apoptotic signaling in infected cells through production and release of pro-inflammatory cytokines (rogers and williams, 2018) . the virus and apoptosis processes are capable of the activation and amplification of immune responses. overproduction of cytokines, the unmanageable destruction of respiratory epithelial cells and extreme immune cell recruitment into infected sites cause a vicious circle in acute lung injury (ali)/acute respiratory distress syndrome (ards) (yang et al., 2018) . in the clinical profile of patients with sars-cov-2, a significant reduction is observed in the count of lymphocytes, cd8+ t cells and neutrophils in peripheral blood . the efficacy of melatonin in the regulation of the immune system has been shown in both in vivo and in vitro studies. melatonin plays an important role in proliferation and maturation stages of natural killer cells, b and t lymphocytes, monocytes and granulocytes in bone marrow as well as other tissues (miller et al., 2006) . furthermore, melatonin treatment could ameliorate antigen presentation through upregulating major histocompatibility complex class (mhc) class i and class ii antigens, complement receptor 3 (cr3), and cluster of differentiation 4 (cd4) antigens on macrophages/microglia in postnatal rat brain. in c3h/hen mice, an inbred strain with a robust melatonin rhythm, it has been demonstrated that mice exert strong regulatory effects on the count of individual types of lymphocytes as the length of the day changes. notably, reduction of cytotoxic t (tc) lymphocytes, activation of b and t lymphocytes and elevation of natural killer cell counts are illustrated in c3h/hen inbred mice, at night. (kaur and ling, 1999) . it has been shown that the activation of mt1 and mt2 receptors plays a prominent role in a variety of melatonin physiological and pharmacological functions. however, some reports indicate a potential role for melatonin, which is mediated by alpha7 nicotinic acetylcholine receptor (α7nachr) activation. this receptor is located in brain, spleen, and lymphocytes of lymph nodes. the α7nachr-induced melatonin effects can be regulated by circadian melatonin (markus et al., 2010) . the interaction of melatonin with α7nachr modulates mitochondrial function, mitophagy, autophagy and activation of immune responses. it has also been reported that melatonin produced by immune-competent cells could improve macrophage efficacy and modulation via α7nachrs (anderson and maes, 2016) . furthermore, macrophage-synthesized melatonin develops phagocytosis through autocrine action by switching m1-like macrophages to m2like phenotypes. these outcomes along with other studies accomplished in this area propose a possible therapeutic potential for melatonin in viral infections (muxel et al., 2012) . conversely, there are some experimental studies indicating that melatonin may have j o u r n a l p r e -p r o o f inhibitory effects on immunity responses. santello et al., (2008) reported that melatonin treatment significantly suppresses the production t helper 2 (th2) lymphocyte in rats infected with trypanosoma cruzi (santello et al., 2008) . moreover, crespo et al., (2020) showed that activation of innate immunity was considerably repressed by melatonin (crespo et al., 2020) . melatonin also is able to down-regulate macrophages in trypanosoma cruziinfected wistar rats (brazão et al., 2011) . therefore, according to contradictory studies, more detailed experiments are required to track all molecular mechanisms of melatonin and its regulatory pathways in the immune system (pohanka, 2013) . both inflammation and oxidative stress are implicated in covid-19 pathogenesis (iddir et al., 2020; merad and martin, 2020b) . melatonin acts as a free radical scavenger and antioxidant. the metabolites of melatonin including cyclic-3-hydroxymelatonin (c3ohm), n1-acetyl-n2-formyl-5-methoxykynuramine (afmk), and n1-acetyl-5-methoxykynuramine (amk) are generated via direct scavenging free radicals. these metabolites have also been shown to possess protective properties (reiter et al., 2016b) . apart from its direct action, melatonin and its metabolites function as anti-excitatory agents via reducing prooxidant enzyme activity, inducing glutathione synthesis, and elevating antioxidant enzymes. furthermore, melatonin plays an essential role in improving mitochondrial function with reference to elevation of mitochondrial complex i and complex iv activities and suppression of electron leakage (juybari et al., 2019) . a number of studies indicate that melatonin has the ability to ameliorate inflammatory conditions through regulating various pathways both in vivo and in vitro (tyagi et al., 2010; wu et al., 2011; xu et al., 2007) . related to anti-inflammatory properties, yu et al., (2017) have shown that melatonin reduces lps-stimulated expression of positive acute-phase proteins (apps), pro-inflammatory cytokines, and chemokines including il-1β, il-6, tnf-α, j o u r n a l p r e -p r o o f ccl2, ccl5, c-reactive protein, serum amyloid a, haptoglobin, ceruloplasmin, granulocytemonocyte colony-stimulating factor (gm-csf), and α-1 antitrypsin. besides this, melatonin treatment could enhance the expression of the negative app fibrinogen and the antiinflammatory cytokine il-1rα (yu et al., 2017) . moreover, melatonin exerts an inhibitory effect on the nlrp3 inflammasome. in a recent study performed by zhang et al., (2016) , melatonin has been found to be a potent inhibitor for the nlrp3 inflammasome in an lpsinduced ali mouse model. this beneficial effect of melatonin improves the pulmonary damage and reduces the influx of neutrophils and macrophages into the lungs (zhang et al., 2016) . the first reports on melatonin and the cardiovascular system have been presented 40 years ago, suggesting that pinealectomy could provoke hypertension in animal studies (holmes and sugden, 1976; karppanen et al., 1975; meneuvonen and karppanen, 1971) . researchers documented that melatonin functions as a vasoconstrictor or vasodilator through mt1 and mt2 receptors in vascular smooth muscle, respectively. in general, melatonin administration causes a reduction in blood pressure due to its sympatholytic effect (campos et al., 2013; slominski et al., 2012) . in a study conducted by simko et al., (2013) , a close relationship has been reported between a significant decrease in the serum melatonin level and cardiovascular problems particularly myocardial fibrosis and hypertension progression (simko et al., 2013) . in this regard, the mechanisms by which melatonin attenuates target organ injuries are highly complicated. based on available evidence, angiotensin and melatonin could have an opposing effect on the cardiovascular system. the opposing effect of melatonin on ag ii is mediated through its anti-inflammatory, antioxidant and antihypertensive properties (dominguez-rodriguez, 2012) . moreover, limited data demonstrate that melatonin affects neurohumoral functions principally the renin-angiotensin-aldosterone system (raas) (simko et al., 2018) . in addition, bader et al., (2001) and campos et al., (2004) reported that both angiotensin and melatonin are generated in the brain. angiotensin synthesizes locally in central nervous system (cns) in nuclei involved in fluid-electrolyte balance and cardiovascular modulation and cooperates with other systems including vasopressinergic and sympathetic nervous systems (bader et al., 2001; campos et al., 2004) . besides cardiovascular protection, melatonin is expected to improve metabolic defects associated with diabetes and insulin resistance via inhibition of the ras system. it seems that melatonin treatment in combination with a ras blocker could exert more efficient in this condition (campos et al., 2013) . as mentioned, severe covid-19 infections can lead to a high generation of inflammatory factors and organ destruction through an overreaction of the immune system. inflammatory factors cause a cytokine storm consisting of an uncontrolled production of immune cells and cytokines (mehta et al., 2020a) . in recent years, stem cell therapy has appeared as a potential treatment in the management of incurable diseases. although considerable improvement has been provided in the use of stem cell-based in the therapeutic field, some important restrictions such as ethical concerns, immunogenic challenges, limited cell source have not been unraveled yet (golchin et al., 2019) . overall, regarding the destructive effects of sars-cov-2 fallowing stimulation of immune system, mesenchymal stem cell (msc) intravenous injection may suppress the cytokine storm induced by the immune system and enhance respiratory regeneration because of their reparative properties (golchin et al., 2020) . the regenerative response could ameliorate injured alveolar epithelial cells, lung dysfunction, pulmonary fibrosis, and consequent pneumonia induced by covid-19 (leng et al., 2020) . however, one of the important limitations of this method is providing clinical-grade human mscs source and then the speed of extraction and sample preparation for clinical application. reliable and secure stem cell resources or banks can play an important role in such an j o u r n a l p r e -p r o o f emergency condition. thereby, these findings seem to highlight that mscs-based therapy alone or in combination with currently supportive treatment may probably be a promising candidate for clinical studies of covid-19 patients (golchin et al., 2020) . in a recent clinical study performed by chen et al., (2020) the application of mscs has been proposed as a beneficial method to protect against inflammation associated with virusinduced cytokine storm in patients with ards caused by influenza a (h7n9) . furthermore, there is a case report from china on a 65-year-old female patient with sever covid19 pneumonia indicating that treatment with umbilical cord mscs for 21 days could considerably ameliorate the symptoms of the disease. in this patient neutrophil and lymphocyte serum levels showed alterations; an elevation of 87% in neutrophil level and a reduction of 9.8% in lymphocyte level. despite treatment of the patient with lopinavir/ritonavir, oseltamivir, ifn-α, moxifloxacin, immunoglobulin, and methylprednisolone, noninvasive mechanical ventilation was used to improve ventilation and respiratory muscle fatigue. after the second injection of mscs, the symptoms of pneumonia were clearly attenuated and the blood count of neutrophil and lymphocyte adjusted to normal levels. most importantly, the number of cd8+ t cells, cd4+ t cells, and cd3+ t cells were noticeably elevated (liang et al., 2020) . in another study conducted by leng et al., from january 23 to february 16, msc transplantation was also applied for 7 patients with covid19 pneumonia hospitalized in beijing youan hospital. inflammation, clinical symptoms, and variations in immune function were evaluated for 14 days after msc treatment. the outcome of this study illustrated that msc transplantation was capable of recovering the patients within two days after transplantation. in addition, mscs were able to stimulate cytokine-secreting immune cells including cxcr3+ cd4+ t cells, enhance serum lymphocyte and il-10 levels, and reduce natural killer (nk) cxcr3+ cells and tnf-α levels on day 6 in mscs treated group j o u r n a l p r e -p r o o f in comparison with the conventionally treated group. therefore, results from two mentioned studies recommended that treatment based on mscs in combination with immune modulators may provide a better condition for treating patients with acute covid-19 pneumonia (leng et al., 2020) . in addition to a variety of biological functions, melatonin regulates the fate of mscs during different pathological and physiological circumstances. in vivo data support that melatonin exerts antioxidant activity with or without specific receptors. there are two g proteincoupled receptors (gpcrs, mt1 and mt2) contributing to the regulation of mscs functions in mammalian. based on the type and physiological status of stem cells, the expression levels of melatonin receptors would be different (hu and li, 2019) . for instance, in order to enhance survival rate, cell proliferation, and degree of differentiation of placenta-derived mscs in vitro, melatonin operates through activation of mt1 receptor, but not mt2 receptor (kaneko et al., 2011) . melatonin treatment reduces the expression level of bax, improves the expression levels of manganese superoxide dismutase (mnsod), and copper-zinc-superoxide dismutase (cuznsod). furthermore, treatment with melatonin decreases ros generation in a dose-dependent manner, leading to both protecting mscs against injuries and improving their biological activities (liu et al., 2013) . in a study conducted by chen et al., (2014) , therapeutic effects of melatonin-mscs on sepsisinduced ali were evaluated. in this research, melatonin in association with mscs could reduce acute lung ischemia-reperfusion injury through ameliorating oxidative stress, inflammation, apoptosis, and fibrosis (chen et al., 2014) . the immunomodulatory effects of melatonin on co-cultured human peripheral blood mononuclear cells and mscs have been analyzed. melatonin is reported to reduce inflammatory responses through the management of pro-inflammatory cytokines such as interleukin 1 beta (il-1β), tnf-α, and il-6. j o u r n a l p r e -p r o o f furthermore, melatonin treatment could efficiently attenuate t cell proliferation in association with the down-regulation of il-6 and il-10 expression (heo et al., 2019) . the immunomodulatory role of melatonin has been illustrated both in preclinical and clinical studies. the production and secretion of melatonin are believed to be correlated with daily and seasonal alterations in the immune system (srinivasan et al., 2005) . since melatonin is also produced by human lymphocytes, it suggests the role of melatonin in the regulation of human immune responses (carrillo-vico et al., 2004) . melatonin promotes both cellmediated and humoral immunity. it motivates the synthesis of progenitor cells for macrophages and granulocytes, nk cells and t-helper cells specifically cd4+ cells. in addition, melatonin supplementation induces the production of family cytokines with pleiotropic functions including il-2, il-6 and, il-12 and reduces cd8+ cells generation (garcia-maurino et al., 1997; srinivasan et al., 2005) . although it has never been released a vaccine for human coronavirus, many companies are now working hard to produce safe and effective vaccines against sars-cov-2. however, even if such a vaccine would be established, vaccine efficacy is probably considered to be inferior for the elderly and other high-risk population groups compared to people who are healthy and young (shneider et al., 2020) . the immune responses to vaccines have been shown to be limited in the aforementioned groups because of a weakened immune system (chen et al., 2009) . therefore, using immunomodulatory agents such as melatonin as an effective adjuvant besides vaccination may boost the vaccine's effectiveness in patients with both compromised and healthy immune systems (srinivasan et al., 2005) . as abovementioned, melatonin is capable of enhancing the count of natural killer and cd4+ cells and amplifying the production of cytokines needed for effective vaccine response (carrillo-vico et al., 2006) . furthermore, sleep deprivation weakens immune response to viral infection j o u r n a l p r e -p r o o f (ibarra-coronado et al., 2015) , and melatonin has been proved to be a critical factor in improving sleep quality (habtemariam et al., 2017) . taken together, covid-19 has contaminated more than 2.4 million people and global deaths exceed tens of thousands of individuals around the world. although numerous conventional medications such as remdesivir, hydroxychloroquine/chloroquine, favipiravir, atazanavir/lopinavir, etc. have been suggested to moderate severe covid-19 patients, none of these medications have shown a promise effect in this condition. the race to design or new medication for covid-19 is proceeding. however, their progress and testing will take time for months to years. thus, to manage this crisis, there is an urgent medical necessity for finding promising agents to deal with covid-19 disease. various evidence indicate that melatonin may play an important role in the treatment of covid-19 when it is given prophylactically or therapeutically alone or in combination with other drugs . this study has provided a comprehensive overview of numerous beneficial properties of melatonin in different viral complications even viral respiratory disorders associated with oxidative stress, inflammation, and immune dysfunction. literature evidence supports that the management of oxidative stress and inflammatory responses, as well as the regulation of immune responses may be critical to target respiratory virus infections such as sars-cov-2. due to a positive correlation between immune dysfunction and disease severity in patients with covid-19, it is necessary to consider this condition for preparing the optimal vaccine. the safety of melatonin profile has been broadly examined in different preclinical and clinical studies on wide-range doses. because of the lack of an available vaccine or effective antiviral treatment for covid-19, the use of melatonin as an adjuvant might be worth consideration. although the direct protective action of melatonin against covid-19 is j o u r n a l p r e -p r o o f unknown, its 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inflammasome increased risk of wheeze and decreased lung function after respiratory syncytial virus infection this research did not receive any specific grant from funding agencies in the public, commercial, or not for-profit sectors.j o u r n a l p r e -p r o o f key: cord-344084-z4t2wkgk authors: ellwanger, joel henrique; kulmann-leal, bruna; kaminski, valéria de lima; rodrigues, andressa gonçalves; de souza bragatte, marcelo alves; chies, josé artur bogo title: beyond hiv infection: neglected and varied impacts of ccr5 and ccr5δ32 on viral diseases date: 2020-05-30 journal: virus res doi: 10.1016/j.virusres.2020.198040 sha: doc_id: 344084 cord_uid: z4t2wkgk the interactions between chemokine receptors and their ligands may affect susceptibility to infectious diseases as well as their clinical manifestations. these interactions mediate both the traffic of inflammatory cells and virus-associated immune responses. in the context of viral infections, the human c-c chemokine receptor type 5 (ccr5) receives great attention from the scientific community due to its role as an hiv-1 co-receptor. the genetic variant ccr5δ32 (32 base-pair deletion in ccr5 gene) impairs ccr5 expression on the cell surface and is associated with protection against hiv infection in homozygous individuals. also, the genetic variant ccr5δ32 modifies the ccr5-mediated inflammatory responses in various conditions, such as inflammatory and infectious diseases. ccr5 antagonists mimic, at least in part, the natural effects of the ccr5δ32 in humans, which explains the growing interest in the potential benefits of using ccr5 modulators for the treatment of different diseases. nevertheless, beyond hiv infection, understanding the effects of the ccr5δ32 variant in multiple viral infections is essential to shed light on the potential effects of the ccr5 modulators from a broader perspective. in this context, this review discusses the involvement of ccr5 and the effects of the ccr5δ32 in human infections caused by the following pathogens: west nile virus, influenza virus, human papillomavirus, hepatitis b virus, hepatitis c virus, poliovirus, dengue virus, human cytomegalovirus, crimean-congo hemorrhagic fever virus, enterovirus, japanese encephalitis virus, and hantavirus. subsequently, this review addresses the impacts of ccr5 gene editing and ccr5 modulation on health and viral diseases. also, this article connects recent findings regarding extracellular vesicles (e.g., exosomes), viruses, and ccr5. neglected and emerging topics in “ccr5 research” are briefly described, with focus on rocio virus, zika virus, epstein-barr virus, and rhinovirus. finally, the potential influence of ccr5 on the immune responses to coronaviruses is discussed. inflammatory cells play a crucial role in protecting the host from viral infections. leukocyte migration is a fundamental step of the inflammatory response to viruses, a process regulated by the interaction between chemokines and their receptors. therefore, dysregulations in the chemokine-mediated inflammatory process may contribute to viral pathogenesis (glass et al., 2003) . the c-c chemokine receptor type 5 (ccr5) interacts primarily with the chemokines ccl3 (mip-1α), ccl4 (mip-1β), and ccl5 j o u r n a l p r e -p r o o f (rantes) , which act as ccr5 agonists by stimulating cell migration and mediating inflammatory responses. on the other hand, the chemokine mcp-3/ccl7 is the main ccr5 antagonist ligand (blanpain et al., 1999; zlotnik and yoshie, 2000; glass et al., 2003; alkhatib, 2009 ). in addition to regulating the migration of non-specific leukocytes during inflammatory responses, ccr5 controls the action of specific cell types, including natural killer (nk) cells (khan et al., 2006; weiss et al., 2011) and regulatory t (treg) cells (wysocki et al., 2005; tan et al., 2009; dobaczewski et al., 2010) . ccr5 is also expressed by tissue-resident memory t cells. these ccr5 + cells support barrier immunity (davis et al., 2019) . the ccr5 is a g-protein-coupled receptor (gpcr), containing seven transmembrane α-helices, three extracellular loops, and three intracellular loops (tan et al., 2013) . figure 1 shows the structure of ccr5, highlighting its transmembrane domains and extra and intracellular loops. the specificity of interaction between ccr5 and chemokines is mediated by the second extracellular loop (samson et al., 1997) . helices 2 and 3 have a fundamental role in chemokine-induced ccr5 activation (govaerts et al., 2003) . the steps required from ligand binding culminating in cell migration encompass a series of intracellular interactions, including the g-protein heterotrimer and downstream effectors (lacalle et al., 2017) . after stimulation by chemokines or natural reactive antibodies and subsequent triggering of chemotaxis, ccr5 is phosphorylated and internalized in the cytoplasm (signoret et al., 2000; venuti et al., 2015; venuti et al., 2016; lacalle et al., 2017; venuti et al., 2017; venuti et al., 2018) . the number of available receptors on the cell surface is related to the rate of internalization and recycling of ccr5, which affects the activation of ccr5 and consequent signaling of specific pathways that culminate in chemotaxis processes (mueller et al., 2002) . of note, intracellular pools of ccr5 can be detected in the cells. these pools are probably formed by internalized or immature/precursor forms of ccr5 molecules (mirzabekov et al., 1999; kohlmeier et al., 2008; achour et al., 2009; guglielmi et al., 2011; shirvani et al., 2011) that can be rapidly expressed on the cell surface in response to viral stimuli and inflammatory responses (kohlmeier et al., 2008) . in other words, ccr5 molecules are recycled by cells. specifically, ccr5 recycling can be mediated by degradation followed by de novo synthesis (in response to stimulation by natural antibodies) or occur in the classic short-term system without de novo synthesis (in response to stimulation by ccl5, for example) (venuti et al., 2015; venuti et al., 2016) . the traffic of ccr5 between the plasma membrane and the intracellular medium is mediated by different molecules, including clathrins, β-arrestin 2, and extracellular signal-regulated kinase (erk) 1 (venuti et al., 2015; venuti et al., 2016; venuti et al., 2018) . also, intracellular cd4 regulates the expression of ccr5 on the cell surface (achour et al., 2009 ). the human ccr5 protein (352 residues) is encoded by the ccr5 gene [chromosome 3 (3p.21.31)], which is very polymorphic (blanpain et al., 2000; hoover, 2018) . among polymorphisms of the ccr5, the ccr5δ32 (rs333) has been intensively studied in different human populations. the frequency of the ccr5δ32 is quite variable. in general, the δ32 allele frequency is high in european-derived populations (for example, 16% in norway and 11% in germany) and low or absent in african and asian populations j o u r n a l p r e -p r o o f (solloch et al., 2017) . however, although the δ32 allele is more frequent in european populations, there are exceptions due to migratory events. for example, the frequency of the δ32 allele is high in south africa (13%) and chile (12%) (solloch et al., 2017) . also, the frequency of the δ32 allele can be quite variable within the same country. in brazil, the frequency of the allele in the general population is around 4-5% (silva-carvalho et al., 2016; solloch et al., 2017) . in the southern region of the country, the frequency can reach up to 9% due to the past migration of european populations to this region (boldt et al., 2009; pena et al., 2011; schauren et al., 2013) . figure 2 summarizes basic aspects of ccr5 and shows the frequency of the δ32 allele in various countries. the ccr5δ32 is the most studied genetic variant of the ccr5 gene because of its strong protective effect against hiv infection (considering susceptibility to ccr5-tropic strains). hiv entry into cd4 + t cells is mediated by the interaction of the virus with cd4 and with a co-receptor, usually ccr5. the ccr5δ32 variant is a 32 base-pair deletion in the ccr5 coding region, which causes a frameshift, resulting in a truncated protein that is not directed to the cell surface. ccr5δ32 in heterozygosis promotes a decrease in the expression of functional ccr5 on the cell surface compared to ccr5 wild-type cells. therefore, individuals with heterozygous genotype for ccr5δ32, if infected with hiv, have a small protection against disease progression due to the reduced expression of ccr5 on the surface of cd4 + t cells (reduced hiv-ccr5 interaction). in ccr5δ32 homozygous cells, no ccr5 is expressed in the plasmatic membrane. therefore, homozygous individuals for this polymorphism (δ32/δ32) show virtually total protection against hiv type 1 infection, since no ccr5 expression is verified on cell surface (no hiv-ccr5 interaction at cell surface is possible) (deng et al., 1996; dragic et al., 1996; huang et al., 1996; samson et al., 1996; wu et al., 1997; proudfoot, 2002; venkatesan et al., 2002; picton et al., 2012) . figure 3 illustrates the phenotypic effects of ccr5δ32 in human cells. the main results involving the triad "ccr5, hiv, and ccr5δ32" were published in 1996 in nature, cell and science papers by different groups (parmentier, 2015) . since then, the research involving ccr5 has explored the role of the ccr5 protein and ccr5δ32 polymorphism in different diseases, as well as the therapeutic potentials of ccr5 blockade. currently, the physical interaction of ccr5 with hiv is known in detail (shaik et al., 2019) ryst, 2015; latinovic et al., 2019) . also, a recent study has shown that molecules that inhibit ccr5 trafficking to the plasma membrane also have a therapeutic potential against hiv infection (boncompain et al., 2019) . research involving ccr5 has also brought other important advances in combating hiv infection. of note, there are already two cases of sustained remission of hiv infection following stem-cell transplantation using ccr5δ32 homozygous donor, the "berlin patient" (hütter et al., 2009) and the "london patient" (gupta et al., 2019; gupta et al., 2020) . articles that evaluated the involvement of ccr5 or ccr5δ32 in the infections caused by the mentioned viruses were analyzed. subsequently, the google scholar (https://scholar.google.com.br/) was consulted to detect relevant papers that were not indexed in pubmed, using the terms "ccr5" in association with the name of each of the viruses covered in the review. the authors of this review tried to include the largest number of original articles that addressed the topic covered in this work, intending to write a broad and complete article. however, some papers were not included because it was not possible to obtain clear conclusions from the studies. the reference lists of the consulted articles were also used to complement the selection of articles for this review. as previously mentioned in the introduction, a discussion addressing the involvement of ccr5 in tbev infection was not included in this work. articles addressing the involvement of ccr5 and ccr5δ32 in hiv infection were included only to present to the reader some basic and historical aspects related to such topics, cited mainly in the introduction section and figures, but not included as a major section. considering the importance of the coronavirus disease 19 (covid-19) pandemic, a section addressing the potential influence of ccr5 on the immune responses to coronaviruses was included in the article. review articles were also selected in pubmed and google scholar for writing the sections and paragraphs that address the basic aspects of viruses, exosomes, and diseases (e.g., epidemiological, molecular, clinical aspects). exceptionally, review articles with outstanding discussions regarding the role of ccr5 in viral infections were also cited in this work. regarding tables, it is important to note that the data available in the "population" columns are limited and often represent only general characteristics of the evaluated population. in many studies, a population can be composed of individuals from various ethnic groups. this limitation must be taken into account when evaluating the results of the studies cited in the tables. finally, also in "population" columns, "information not available" was used when this information was not clearly described in the cited article. west nile virus (wnv) is a neurotropic positive-sense single-stranded rna flavivirus endemic in various parts of the world. wnv transmission to humans occurs through the bite of infected mosquitoes, especially species of the culex genus (suthar et al., 2013) . although different animals can participate in the wnv transmission cycle, birds are the classic amplifier hosts. humans, horses and other mammals are deadend hosts (kramer et al., 2008; cdc, 2018) . among humans, blood transfusion, organ transplantation, and breast milk can also transmit the virus. vertical transmission may also occur. however, compared to transmission by mosquito bites, these routes of transmission are rare (kramer et al., 2008) . about 25% of wnv-infected individuals develop west nile fever, a clinical condition with variable symptoms and severity. in less than 1% of the infected individuals, wnv invades the central nervous system (cns), causing neurological manifestations (neuroinvasive disease), including meningitis, encephalitis, and acute flaccid paralysis. of note, west nile neuroinvasive disease shows a 10% to 20% fatality rate. severe illness is associated with older age and other factors, including genetic traces (petersen et al., 2013; sejvar, 2016) . wnv infection is considered the leading cause of arboviral encephalitis in the world (ciota, 2017) . the treatment of wnv infection is supportive (petersen et al., 2013) . it is known that the ccr5 protein interferes in the clinical course of wnv infection (glass et al., 2005; diamond, 2009; michlmayr and lim, 2014) , but the effects of ccr5δ32 on the susceptibility to this infection and disease progression are different. according to lim et al. (2010) and danial-farran et al. (2015) , ccr5δ32 has no important effect on susceptibility to wnv infection. in accordance, loeb et al. (2011) found no association between ccr5δ32 and wnv infection. conversely, there is robust populationbased data showing a strong association between the ccr5δ32 homozygous genotype and increased risk of developing symptomatic wnv infection lim et al., 2008; lim et al., 2010) . also, bigham et al. (2011) showed that the ccr5δ32 variant was associated with symptomatic wnv infection when the dominant model of inheritance was considered in the analysis. a recent meta-analysis confirmed the role of the ccr5 gene in wnv infection, specifically the association between the ccr5δ32 with severe disease (cahill et al., 2018) . table i summarizes the results of the studies that evaluated the ccr5δ32 genetic variant in the context of human wnv infection. in agreement with studies showing that ccr5δ32 homozygous genotype is a risk factor for symptomatic wnv infection in humans, ccr5-/-wnv-infected mice showed a reduced capacity of viral control, increased disease severity, impaired leukocyte trafficking towards the brain, and high mortality rates than ccr5 wild-type mice. these findings reinforce that ccr5 is a key molecule in the immune response against wnv (glass et al., 2005; durrant et al., 2015) . taking together, these pieces of evidence robustly support the role of ccr5 as a protective molecule on wnv pathogenesis. specifically, ccr5+ leukocytes play a fundamental role in combating wnv in the brain (glass et al., 2005; lim et al., 2006; michlmayr and lim, 2014; durrant et al., 2015) . in this sense, ccr5 plays a specific and non-redundant role in controlling wnv infection (lim and murphy, 2011; durrant et al., 2015; ellwanger et al., 2020a) . based on the data mentioned above, the lack of ccr5 expression linked to ccr5δ32 homozygosis is an important risk factor for increased severity of wnv-associated disease. the opposite effects of the ccr5δ32 genetic variant on both hiv and wnv infections are summarized in figure 5 . hereupon, individuals homozygous for ccr5δ32 and living in endemic areas of the wnv should take additional care to prevent wnv infection (e.g., use of repellents, mosquito nets). also, the use of ccr5 blockers to treat hiv infection may have a negative impact on populations living in wnv-endemic areas. to avoid this negative impact, hiv-infected individuals who live in such areas and who use ccr5 blockers must apply robust measures against mosquito bites lim et al., 2006; lim and murphy, 2011). j o u r n a l p r e -p r o o f influenza infection affects humans seasonally, causing recurrent epidemics and even pandemics in some years. in humans, the infection is caused basically by influenza a and influenza b, both enveloped negative-sense single-stranded rna viruses belonging to orthomyxoviridae family (krammer et al., 2018; petrova and russell, 2018) . influenza a is a zoonotic disease, and influenza b circulates primarily in humans. influenza infection affects mainly the respiratory tract, which can cause mild to severe disease depending on viral and host characteristics. secondary bacterial infection may also occur (krammer et al., 2018) . human co-infection with multiple influenza types is an important neglected problem (gregianini et al., 2019) . influenza is a prevalent infection worldwide, and new vaccines are produced annually, based on strains circulating each year in the northern and southern hemispheres (krammer et al., 2018; petrova and russell, 2018) . antivirals can be used in the treatment of influenza infection (krammer et al., 2018) . investments in new vaccines, antiviral drugs, and surveillance systems are needed to reduce the global burden associated with influenza infection (petrova and russell, 2018) . the severity of influenza infection is related to the intensity of proinflammatory responses and the predominant profile of cytokine production by the host . a body of evidence has shown that both ccl5 and ccr5 participate in the modulation of the immune response to influenza virus infection (matsukura et al., 1998; tyner et al., 2005; sládková and kostolanský, 2006; kohlmeier et al., 2008; oslund and baumgarth, 2011) . of note, ccr5 mediates the recruitment of nk cells to the lungs in influenza a infection (carlin et al., 2018) and participates in neutrophil action in the lungs during influenza pneumonia (rudd et al., 2019) . although flow cytometry data did not indicate significant changes regarding ccr5 expression on the surface of human monocytes after experimental influenza a infection (salentin et al., 2003) , various studies have shown that, in mice, the lack of ccr5 expression is associated with a higher risk of death by influenza infection (dawson et al., 2000; tyner et al., 2005; fadel et al., 2008; tavares et al., 2020) . based on these findings, it was postulated that pharmacological ccr5 blockade may have some undesirable effect on the immune response against the influenza virus in humans (fadel et al., 2008) . importantly, more research on this aspect is needed since this data suggests that, in humans, the ccr5 absence due to the ccr5δ32 polymorphism could affect pathogenesis and the lethality rate of influenza infection. falcon et al. (2015) evaluated the frequency of the ccr5δ32 in pandemic h1n1-infected spanish individuals and revealed an association between the polymorphism with fatal outcome. the ccr5δ32 was also associated with increased disease severity in other studies (keynan et al., 2010; rodriguez et al., 2013) . however, these results should be interpreted with caution since both studies were based on very small sample sizes (keynan et al., 2010; rodriguez et al., 2013) . importantly, other studies addressing humans reported no association between ccr5δ32 and severity of influenza infection (sironi et al., 2014; maestri et al., 2015; matos et al., 2019) . taking together, the body of evidence suggests that ccr5δ32 has little j o u r n a l p r e -p r o o f influence on severity of influenza infection (table 2) . results described by falcon et al. (2015) seem to be specific to that studied population, composed of individuals from 13 regions of spain. human papillomavirus (hpv) is a double-stranded dna virus belonging to the papillomavirus family. hpv is transmitted by direct contact (for example, through sexual intercourse). the hpv infection is quite common worldwide, and is usually controlled by the immune system. viral clearance occurs in most cases within 1-2 years after infection. however, if the infection is not controlled, hpv can cause noncancerous mucosal lesions or different types of malignant lesions such as anal cancer, penile cancer, vulvar cancer, head cancer, neck cancer, and especially cervical cancer. hpv is an oncogenic pathogen because it inhibits the activity of p53 and prb (retinoblastoma protein) tumor suppressor molecules through the action of e6 and e7 viral proteins, respectively. these proteins also exhibit other oncogenic mechanisms. worldwide, between 5-10% of all cancers in women are due to hpv infection (schiffman et al., 2016; sanjosé et al., 2018) . there are several hpv genotypes (>200), and those most associated with cancer are: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and probably 68 (schiffman et al., 2016) . of note, the genotype 16 (hpv16) has a prominent role in cancer development. vaccination is one of the most effective ways to prevent hpv infection, having important positive impacts on multiple aspects of population health. hpv vaccine protects the vaccinated individual from infection per se and from hpv-related cancer (schiffman et al., 2016; sanjosé et al., 2018) . the expression of ccr5 is increased in cervical cancer tissues (sales et al., 2014; che et al., 2016) . also, in vitro growth, proliferation, and invasive capacity of cervical cancer cells can be inhibited through ccr5 downregulation (che et al., 2016) . these findings suggest the involvement of ccr5 in the development of cervical lesions. in a study performed with indian individuals (singh et al., 2008) , the genotype and allele frequencies of ccr5δ32 were not different between cervical cancer patients and controls. however, when the patients were stratified by cancer stage (stages 1b to 4), the ccr5 heterozygous genotype was associated with stage 1b of cervical cancer. the hpv positivity rate among the evaluated patients was not described (singh et al., 2008) . the relationship between ccr5δ32, hpv infection, and cervical lesions is addressed in other studies (table 3 ). ccr5δ32 homozygous genotype was associated with increased susceptibility to hpv infection in a study performed with swedish individuals (zheng et al., 2006) . mangieri et al. (2019) investigated the potential influence of ccr5δ32 on susceptibility to hpv infection and cervical lesions in brazilian women. however, the genetic variant was not significantly associated with susceptibility to hpv infection (considering allele frequency, codominant model and dominant model) or hpv-associated lesions (mangieri et al., 2019) . in accordance, previous studies performed with brazilian women did not find an association between ccr5δ32 and susceptibility to hpv infection (suzuki et al., 2008) or between the polymorphism and hpv-related cervical lesions (suzuki et al., 2008; santos et al., 2016) . lastly, ccr5δ32 j o u r n a l p r e -p r o o f did not affect susceptibility to hpv infection in lithuanian individuals with laryngeal cancer (stumbrytė-kaminskienė et al., 2020) . in conclusion, although tissue analysis and evidence obtained in vitro suggest that the ccr5 is potentially involved in the pathogenesis of hpv, most studies point to a lack of involvement of ccr5δ32 in susceptibility to hpv infection or hpv-associated diseases. the the ccr5 and its ligands regulate the action of t cells and other leukocytes in the liver. thus, ccr5 regulates liver inflammation and participates in the local immune response against viruses (ajuebor et al., 2006; sanchooli et al., 2014) . in mice models of hepatitis, ccr5 deficiency was associated with increased liver inflammation, tissue injury, and liver failure (ajuebor et al., 2005; moreno et al., 2005; stevens et al., 2019) . deficiency of ccr5 expression is generally associated with reduced migration of inflammatory cells, which would translate into less inflammation. this reasoning is correct and applies to different situations and tissues (braunersreuther et al., 2007; muntinghe et al., 2009; kaminski et al., 2019a) . however, ccr5 is an immunoregulatory molecule (doodes et al., 2009; dobaczewski et al., 2010; christmann et al., 2011; hütter et al., 2011) and therefore its deficiency can also cause deregulation in the action of various immune cell types (e.g., nk and treg cells), increasing the inflammatory status in some tissues. in humans, multiple evidence has shown the involvement of ccr5 (protein and gene) in distinct aspects of hbv infection (ahn et al., 2006; trehanpati et al., 2009; ahmadabadi et al., 2013; yang et al., 2018) . interestingly, ccr5δ32 and other host genetic factors can affect the immunogenicity of the hbv vaccine (ganczak et al., 2017; ellwanger and chies, 2019b) . considering these aspects, the influence of the ccr5δ32 on susceptibility/resistance to hbv and disease severity is quite plausible. several studies investigated the role of the ccr5δ32 polymorphism in hbv infection. some of them found no association between those variables (arababadi et al., 2010; khorramdelazad et al., 2013; safari et al., 2017; zhang et al., 2018; moudi et al., 2019) . in other studies, the absence of ccr5δ32 allele in the sample, due to ethnic features of the evaluated population, precluded the analysis concerning the potential impact of this genetic variant on hbv infection (ahn et al., 2006; li et al., 2011) . some authors have reported significant influences of ccr5δ32 on hbv infection. suneetha et al. (2006) reported an association between the ccr5δ32 heterozygous genotype and chronic hbv infection. in contrast, thio et al. (2007) found an association between the ccr5δ32 allele and infection recovery in a study that analyzed individuals with persistent hbv infection and individuals who recovered from the infection. subsequently, thio et al. (2008) associated the hbv infection recovery with an epistatic effect between the ccr5δ32 and the rantes-403a promoter polymorphisms. finally, abdolmohammadi et al. (2016) found a protective effect of the ccr5δ32 genetic variant against hbv infection, once the δ32 allele was more frequent in controls compared to hbv-infected individuals. recently, we investigated the frequency of ccr5δ32 in hbv mono-infected and hbv/hiv coinfected brazilian individuals (ellwanger et al., 2020b) . a control group and hiv mono-infected individuals were also evaluated in our study. a total of 1113 individuals were studied, which represents the largest study involving ccr5δ32 and hbv infection to date (see table 4 for comparisons with other studies). we found a significant protective effect of ccr5δ32 on hbv/hiv co-infection, a result probably due to the partial protective effect of ccr5δ32 against hiv infection since no important impact of ccr5δ32 on susceptibility to hbv mono-infection was observed (ellwanger et al., 2020b) . the hepatitis c virus (hcv) was formally described in 1989 (choo et al., 1989) and, currently, hcv infection is one of the most important infectious diseases in terms of global public health burden. hcv is an enveloped single-stranded positive-sense rna virus, has seven genotypes, and belongs to the flaviviridae family, hepacivirus genus (pietschmann and brown, 2019) . it is estimated that 71 million individuals are chronically infected by hcv worldwide (viganò et al., 2019) . similar to the hbv infection, hcv-infected individuals can eliminate the virus naturally or develop chronic infection, which occurs in 55-85% of the cases. chronic infection can cause liver inflammation, cirrhosis, and hepatocarcinoma (lingala and ghany, 2015) . in addition to liver damage, hcv causes a series of immune-mediated extrahepatic manifestations, including rheumatologic, dermatologic, ophthalmologic, renal, pulmonary, neuropsychiatric, cardiovascular, and hematologic manifestations, especially mixed cryoglobulinemia (romano et al., 2018) . hcv therapy using direct-acting antivirals (daas) shows cure rates over 95% (pietschmann and brown, 2019) . early treatment of infected patients decreases death rates from hcv-associated liver disease, reduces disease transmission, and alleviates extrahepatic health problems. focusing the efforts on hcv treatment is extremely important because there is no effective hcv vaccine (viganò et al., 2019) . hcv seropositivity is an important risk factor for hiv infection (zwolińska et al., 2013) . like hiv, hcv is primarily transmitted through blood transfusion and sexual intercourse, and hcv/hiv co-infection is a major problem worldwide. depression of the immune system due to uncontrolled hiv infection may contribute to hcv progression (schlabe and rockstroh, 2018) . both susceptibility to hcv infection and j o u r n a l p r e -p r o o f disease progression are affected by viral and environmental factors and physio-metabolic, immune, and genetic components of the host (ellwanger et al., 2018a) . chemokines and chemokine receptors participate in the recruitment and activity of inflammatory cells in the liver, acting on anti-hcv immune responses and ultimately modifying the rate of inflammation and other histological manifestations observed during infection. based on this rationale, the ccr5 molecule was postulated as having an impact on hcv-induced liver injury, susceptibility to hcv infection, and modulation of the possibilities of viral clearance. the downregulation of ccr5 due to ccr5δ32 may interfere in these processes (ahlenstiel et al., 2004; coenen and nattermann, 2010) . in agreement, several polymorphisms in other immune system genes [especially human leukocyte antigen (hla), mannosebinding lectin (mbl), toll-like receptor (tlr), interleukins (il), and interferon (ifn) gene families] indeed modify both susceptibility to hcv infection and disease progression (ellwanger et al., 2018a) . especially the focus of this review, clinical response to hcv therapy is influenced by ccr5 gene polymorphisms (konishi et al., 2004; omran et al., 2013) . also, there is evidence showing that ccr5 haplotypes can affect susceptibility to hcv infection (huik et al., 2013) . a recent in vitro study suggested that ccr5 blockage could have a beneficial effect on the treatment of hcv infection since ccr5 antagonists (maraviroc and cenicriviroc) inhibit hcv replication (blackard et al., 2019) . the use of ccr5 antagonists in humans is safe (fätkenheuer et al., 2010; gulick et al., 2014; giaquinto et al., 2018) and these drugs have the potential to treat a number of diseases in which ccr5 is involved, including hcv-associated liver disease. although the use of ccr5 antagonists on hcv monoinfection is not yet approved, it can be useful specifically for co-infected hiv/hcv patients, where ccr5 blocking (maraviroc) is already recommended (haïm-boukobza et al., 2013; blackard et al., 2019) . however, the detailed patterns of ccr5 expression in different tissues and at various points in the clinical course of hcv infection are still poorly understood. according to a recent study, the expression of ccr5 in cd8+ t cells is increased in the liver of chronic hcv-infected patients (pirozyan et al., 2019) , but other studies have found mixed results regarding ccr5 expression on t cells in the context of hcv infection (lichterfeld et al., 2002; vincent et al., 2005; larrubia et al., 2007; zahran et al., 2020) . therefore, considering that the role of ccr5 in hcv infection is still uncertain, the potential use of ccr5 blockers to treat hcv mono-infection should be cautious. the influence of the ccr5δ32 variant on hcv infection susceptibility was investigated by several studies. woitas et al. (2002) found a significantly higher frequency of the ccr5δ32 homozygous genotype in hcv-infected individuals compared to controls, hiv-infected and hcv/hiv co-infected individuals, suggesting the ccr5δ32 as a risk factor for hcv infection. in agreement, the δ32 allele was a significant risk factor for infection when the authors compared the hcv-infected group to both controls and hivinfected individuals. moreover, the ccr5δ32 homozygous genotype was associated with increased hcv loads. in their study, it was observed an important deviation from the hardy-weinberg equilibrium in data from hcv-infected individuals; and a high portion of the individuals included in the study was hemophiliac j o u r n a l p r e -p r o o f (woitas et al., 2002) . hemophiliac individuals were at high risk of exposure to hcv and hiv until the mid-1980s (promrat et al., 2003; zhang et al., 2003) . considering that the ccr5δ32 homozygous genotype provides protection against hiv infection, a high frequency of this genotype in an hcv-infected group may be due to hiv resistance, but not to hcv, among individuals highly exposed to both viruses. due to those and other reasons, the results of woitas et al. (2002) were criticized by different authors (klein, 2003; mangia et al., 2003; poljak et al., 2003; promrat et al., 2003; zhang et al., 2003) . in this sense, no influence of ccr5δ32 on susceptibility to hcv infection were reported in studies performed with various populations (glas et al., 2003; mangia et al., 2003; poljak et al., 2003; promrat et al., 2003; zhang et al., 2003; ruiz-ferrer et al., 2004; wald et al., 2004; wasmuth et al., 2004; thoelen et al., 2005; goyal et al., 2006) . reinforcing the observations of those different studies, our group found no association between the ccr5δ32 and susceptibility to hcv infection or hcv/hiv co-infection in a study that evaluated a large number of brazilian individuals (ellwanger et al., 2018b) . bineshian et al. (2018) did not detect the ccr5δ32 allele in any iranian hcv-infected individual and controls included in their study, preventing any conclusion in terms of susceptibility in that population. finally, it is important to mention that in addition to the study by woitas et al. (2002) , a study performed in 2013 also found an association between the ccr5δ32 homozygous genotype with chronic hcv infection in europeans, but the authors of the study mentioned that specific factors regarding selection bias (e.g., co-exposure to hiv) may have influenced their results (suppiah et al., 2013) . taken together, the above-mentioned results called attention for the importance of performing genetic variant studies in different populations, exposed to different social and environmental factors and presenting distinct ethnic backgrounds. considering multiple clinical and histological parameters, two main different results were obtained when the ccr5δ32 genetic variant was evaluated in the context of hcv-related diseases: reduced liver inflammation in δ32 allele carriers (hellier et al., 2003; wald et al., 2004; goulding et al., 2005) ; and no association between the ccr5δ32 and clinical variables (glas et al., 2003; mangia et al., 2003; promrat et al., 2003; goyal et al., 2006; mascheretti et al., 2004; ruiz-ferrer et al., 2004; morard et al., 2014; ellwanger et al., 2018b) . in the context of persistence/resolution of hcv infection and viral control, in the one hand, studies described association of the ccr5δ32 allele with reduced rates of spontaneous viral clearance (nattermann et al., 2011; morard et al, 2014) , higher viral load (yilmaz et al., 2014) , and reduced anti-hcv immune response (ahlenstiel et al., 2009 ). on the other hand, studies have reported association between the ccr5δ32 variant and increased rates of spontaneous viral clearance (goulding et al., 2005; el-moamly et al., 2013) . no significant effect of the ccr5δ32 on viral clearance was reported by other authors (mascheretti et al., 2004) . the potential impact of the ccr5δ32 polymorphism on response to hcv therapy was also evaluated by some authors. ahlenstiel et al. (2003) found an association between the ccr5δ32 allele and reduced response rates to interferon-α monotherapy, but the polymorphism did not affect the response to the combined interferon/ribavirin therapy. this finding shows that the use of more robust therapeutic regimens j o u r n a l p r e -p r o o f compensates the undesirable effects of ccr5δ32 on hcv therapy with interferon-α monotherapy. of note, the effect of ccr5δ32 may be negligible in the context of modern hcv therapies (ahlenstiel et al., 2003) . other studies did not report significant effects of the polymorphism on response to hcv therapy (glas et al., 2003; promrat et al., 2003; goyal et al., 2006; mascheretti et al., 2004; suppiah et al., 2013; morard et al., 2014) . again, it is important to mention that the ethnic distribution of the ccr5δ32 allele could interfere with study results. konishi et al. (2004) , for example, did not detect the ccr5δ32 allele in any japanese individual included in their study focused on host genetic factors involved in the response to interferon therapy. lastly, the polymorphism also does not appear to be associated with any specific hcv genotype (glas et al., 2003; wasmuth et al., 2004; goyal et al., 2006) . ahlenstiel et al. (2004) highlighted that the impact of ccr5 on hcv infection was controversial. in 2020, many controversies remain, although some points are better defined. table 5 summarizes the main findings of the studies that evaluated ccr5δ32 on hcv infection. although some studies indicate an influence of the polymorphism on susceptibility to infection, most studies indicate that the δ32 allele has little (or no) influence on hcv susceptibility. the impact of the ccr5δ32 on hcv-related liver disease is quite variable and context-dependent. finally, available data suggest some benefit of ccr5 antagonists for the treatment of hcv mono-infection. however, these data are still limited and further studies evaluating this topic are needed. poliovirus (pv) is a single-stranded rna enterovirus of the family picornaviridae. there are three types of pv: wild pv type 1 (wpv1), type 2 (wpv2), and type 3 (wpv3). the pv replicates in the tonsils and intestinal tract. in few infection cases (~1%), the virus invades the cns and can cause poliomyelitis resulting in paralysis. poliomyelitis is a condition characterized by inflammation of the gray matter of the spinal cord and muscle paralysis unleashed by pv replication in motor neurons (racaniello, 2006; kew and pallansch, 2018; keohane et al., 2020) . polioencephalitis can also occur and is characterized by the pv outbreak occurred in that country (table 6 ). the authors found no statistically significant effect of the ccr5δ32 on pv infection; only a trend of association between the δ32 allele and increased risk of pv infection was observed (rosenberg et al., 2013) . however, this study had a very small sample size (only seven cases of severe pv infection were evaluated) and therefore the results were not conclusive. in addition, due to the declining number of pv infection cases in the world, the effect of ccr5δ32 will be increasingly difficult to be assessed in population-based studies. dengue virus (denv) is a single-stranded rna virus that belongs to the flaviviridae family, flavivirus genus. there are four denv serotypes, being all transmitted to humans by aedes mosquitoes (aedes aegypti and aedes albopictus) (guzman et al., 2016) . denv infection is a global health problem with huge impacts on public health systems, especially in tropical countries (bhatt et al., 2013) , being considered the most common arbovirosis in the world (stanaway et al., 2016) . globally, the incidence of symptomatic denv infection is within the range of 50 to 100 million cases per year, resulting in ~10,000 deaths each year (stanaway et al., 2016) . clinically, dengue illness is divided into three basic phases: acute febrile phase, critical phase, and recovery (convalescent) phase. dengue disease occurs with/without warning signs or severe dengue. warning signs (suggestive signs or symptoms of important fluid loss, capillary leakage, and shock, such as severe abdominal pain and mucosal bleeding; observed at the end of the febrile phase) allow the rapid identification of patients who need more clinical attention and supportive therapy, in an attempt to avoid severe dengue. when severe disease occurs, this condition can lead to serious organ involvement, shock, and hemorrhage, among other signals and symptoms (guzman et al., 2016) . infection with a denv serotype triggers long-term immunity to that specific serotype (homotypic denv). immunity to heterotypic denv also occurs, but it is transitory. therefore, an individual can have dengue disease more than once. severe dengue occurs more frequently in recurrent infection with a different viral serotype (murphy and whitehead, 2011; st john and rathore, 2019 ). an immune response mediates denv clearance and the resolution of dengue diseases, but it is also involved in the disease pathogenesis (murphy and whitehead, 2011) . some evidence suggests the participation of ccl5/ccr5 axis in the protection against denv (sierra et al., 2014) , as well as in the pathogenesis of dengue disease (islam et al., 2019) . indeed, denv infection is associated with increased frequency of human ccr5+ t cells (de-oliveira-pinto et al., 2012; badolato-corrêa et al., 2018) . in a study performed by marques et al. (2015) , lower viral replication was found in macrophages treated with ccr5 blockers. in the same study, ccr5-/-mice were protected from denv infection. these findings suggest that ccr5δ32 could be a protective factor against denv infection. however, xavier-carvalho et al. (2013) found no statistical difference in the frequency of ccr5δ32 polymorphism between brazilian children with j o u r n a l p r e -p r o o f severe denv infection and healthy controls. in the same direction, no effect of ccr5δ32 on susceptibility to denv infection was found in a small sample-size study performed with individuals from western australia (brestovac et al., 2014) and recent data suggested no important involvement of ccr5 gene or ccr5 polymorphisms in denv infection (cahill et al., 2018; ornelas et al., 2019) . finally, the ccr5δ32 allele was not identified in a small group of indian denv-infected individuals (islam et al., 2019) . table 6 shows some details of the studies involving ccr5δ32 and denv infection. taking together, the data mentioned above indicate that the effects of ccr5 on denv infection are very different between humans and rodents. however, it should be noted that the approach of each mentioned study is quite particular, and we cannot exclude some potential effects of ccr5 and ccr5δ32 on denv infection in humans. cmv is also linked to cancer development, once several cmv proteins (e.g., pul122, pul123, pus28, pul83, pul111a) activate pro-oncogenic pathways, including angiogenesis, escape of immune control and tumor suppressors, tumoral inflammation, invasion and metastasis, genome instability, and increased cell survival and proliferation (herbein, 2018) . poor socioeconomic condition favors cmv infection. antibodies indicating past cmv infection are found in ~60% of adults from high-income countries. in low-income countries, the rate of past infections can reach 100% (griffiths et al., 2015) . cmv can manipulate the immune system producing virokines (virus-encoded cytokine/chemokine homologs) and viroceptors (virus-encoded cytokine/chemokine receptor homologs), molecules that enable the virus to evade host immune defenses. such molecules can also facilitate viral replication (lucas et al., 2001; froberg, 2004; vomaske et al., 2012) . importantly, cmv-encoded proteins can interact with ccr5, (johnson et al., 2015) . however, mixed results were reported regarding the effect of cmv on ccr5 expression since there is evidence indicating that cmv infection may reduce ccr5 expression in various cell types (lecointe at al., 2002; varani et al., 2005) . interestingly, these mixed results may not be contradictory. cmv-infected cells may indeed exhibit decreased ccr5 expression, limiting hiv infection in these cells. however, cmv-infected cells release cmv-associated soluble factors that increase ccr5 expression in non-infected bystander cells, then facilitating hiv replication in such cells and, consequently, contributing to hiv pathogenesis (king et al., 2006) . there is evidence showing that variants in the ccr5 gene can influence multiple aspects of cmv infection (loeffler et al., 2006; sezgin et al., 2011) . for example, the ccr5 promoter polymorphism rs1800023 affects cmv replication (bravo et al., 2014; corrales et al., 2015) . in a study evaluating children, kasztelewicz et al. (2017) found no influence of ccr5δ32 on susceptibility to congenital cmv infection, severity of congenital cmv disease, or cmv-related sensorineural hearing loss at birth. as an individual genetic factor, ccr5δ32 was not statistically associated with the progression of cmv retinitis, a condition that cmv can cause in immunocompromised individuals (sezgin et al., 2011) (table 6 ). bunyaviridae. cchfv circulates in africa, europe, middle east, and asia countries, and can infect a variety of domestic animals and wild species, but without causing symptomatic illness. humans are accidental hosts of cchfv, for which the virus is transmitted mainly by tick-bites (especially ticks of the genus hyalomma), although other routes of transmission also exist, such as exposure to blood of infected animals. most cchfv-infected individuals have no symptoms or have mild nonspecific febrile syndrome. however, in some individuals, the infection can cause the crimean-congo hemorrhagic fever, a severe disease characterized by fever, myalgia, hemorrhage, among other manifestations. neurological complications can also occur, being the spectrum and intensity of the disease quite variable (ergönül, 2006; bente et al., 2013; garrison et al., 2019) . hemorrhagic fever (ergönül, 2006; saksida et al., 2010; bente et al., 2013; garrison et al., 2019) . in a study performed by arasli et al. (2015) , expression of the ccr5 ligands ccl2, ccl3, and ccl4 was increased in cchfv-infected adults compared to controls. considering these same chemokines in cchfv-infected children, only ccl4 was significantly increased compared to pediatric controls (arasli et al., 2015) . engin et al. (2009) evaluated the ccr5δ32 in 15 turkish cchfv-infected individuals and observed the wild-type homozygous genotype in all cases. in a subsequent study evaluating the turkish population, rustemoglu et al. (2017) found a protective effect of ccr5δ32 heterozygous genotype and δ32 allele on cchfv infection, since the genotype and allele frequencies were higher in controls than in cchfv-infected individuals. conversely, the wild-type genotype (normal ccr5 expression) was prevalent among infected j o u r n a l p r e -p r o o f individuals. these findings suggest that ccr5 contributes to susceptibility to cchfv infection and that ccr5 down-regulation due to ccr5δ32 results in some protection against the infection. however, further studies are needed to explain the mechanisms by which ccr5 participates in cchfv infection. in the same study, the ccr5δ32 was not significantly associated with disease severity, clinical parameters, or mortality rate (rustemoglu et al., 2017) . together, these findings indicate that the effect of ccr5δ32 is given specifically on resistance against cchfv infection, without affecting the pathogenesis/outcome of crimean-congo hemorrhagic fever. the genus enterovirus is composed of non-enveloped, positive-stranded rna viruses, belonging to the picornaviridae family. enteroviruses (ev) can infect the gastrointestinal tract, cns, and other organs, including heart (tapparel et al., 2013) . cardiomyopathy is a common consequence of ev infection in the heart. ev infection is associated with myocardial inflammation (myocarditis) and other damages to heart tissues (badorff et al., 2000; cooper, 2009; sagar et al., 2012; tapparel et al., 2013; weintraub et al., 2017) . damage heart tissues. viral persistence in individuals with enteroviral cardiomyopathy is associated with an increased mortality rates (kühl et al., 2005; lassner et al., 2018) . some studies suggest that ccr5 influences different aspects of the pathogenesis of viruses belonging to the picornaviridae family, including encephalomyocarditis virus (christmann et al., 2011; shaheen et al., 2015) , coxsackievirus b3 (valaperti et al., 2013) , and rhinovirus (muehling et al., 2017) , once ccr5 participates in the regulation of the host immune response during infection by these viruses. considering the effects of the genetic variant ccr5δ32 on ccr5 expression and ccr5-related immune responses, it is possible that ccr5δ32 also shows some impact on ev-related diseases. in a german study that evaluated patients with enteroviral (chronic/inflammatory) cardiomyopathy (lassner et al., 2018) , the ccr5δ32 was strongly associated with spontaneous viral clearance and better clinical outcome (reduced mortality rate) ( table 6 ). these findings indicate a critical involvement of the ccr5 molecule in the pathogenesis of ev cardiomyopathy. it was suggested that the ccr5δ32 genotyping could be used to assist in the prediction of the clinical progression of enteroviral cardiomyopathy: the δ32 allele as a predictor of a better prognosis, without the need of antiviral interferon-β (ifn-β) therapy; and the wild-type genotype as a predictor of a worse prognosis and immediate need of antiviral ifn-β therapy (lassner et al., 2018) . the clinical use of ifn-β is effective to eliminate the virus, avoid irreversible cardiac injury, and reduce mortality rates, but it is also associated to numerous adverse effects (kühl et al., 2012; lassner et al., 2018) . considering the prognostic value of the ccr5δ32 on the clinical course of enteroviral cardiomyopathy, it is necessary to evaluate the relationship between the ccr5δ32 and the disease in different human populations, mainly through genetic association studies. if this association is confirmed in other populations, the ccr5δ32 genotyping will be a broad-spectrum clinical tool, enhancing and driving the treatment of enteroviral cardiomyopathy. jev is the etiological agent of most cases of viral encephalitis in many countries, reaching ~30% mortality rate (van den hurk et al., 2009; tiwari et al., 2012; le flohic et al., 2013) . some evidence obtained in laboratory conditions (in vitro analysis and murine models) showed that jev infection induces the up-regulation of ccr5 gene (gupta and roa, 2011; pereek et al., 2014; zhang et al., 2019) . also, increased infiltration of ccr5 + cd8 + t cells was observed in the brains of jev-infected mice (zhang et al., 2019) . in this context, using a mouse model of japanese encephalitis, larena et al. (2012) showed that ccr5 protects the host against jev infection in the cns, being essential for disease recovery. ccr5-deficient mice showed higher viral loads in the brain and spinal cord as well as increased mortality rate, as compared to control mice (larena et al., 2012) . also using a mouse model of japanese encephalitis, kim et al. (2016) demonstrated that ccr5 controls the infiltration of cd4 + foxp3 + t regulatory cells (treg) in the cns, contributing to protection against japanese encephalitis. the infiltration and action of inflammatory cells in the brain are important to limit neuroinvasive infections, processes that are regulated by multiple factors, especially cytokines, chemokines and their receptors, including ccr5. however, the uncontrolled action of inflammatory cells can cause damage to the cns. of note, cd4 + foxp3 + treg cells regulate the immune responses, avoiding undesirable or excessive action of inflammatory cells (bardina and lim, 2012; veiga-parga et al., 2013; simonetta and bourgeois, 2013; campbell, 2015; kim et al., 2016) . according to these pieces of evidence, ccr5-mediated action of treg cells is critical for the control of japanese encephalitis. however, the role of ccr5 in jev infection may be more complex than it appears at first. liu et al. (2018) demonstrated that the use of the ccr5 antagonist maraviroc reduces the jev-induced inflammation in mice brain, increasing survival rate. this result suggests that potential deleterious effects of ccr5-mediated inflammation in the brain may override the effects of ccr5-mediated action of treg cells and, therefore, the use of ccr5 antagonists to treat japanese encephalitis may be beneficial. based on these complex and contradictory results, it can be concluded that ccr5 indeed has an important influence on japanese encephalitis, but it is not yet possible to state its specific roles, once they are varied and appear to be context-dependent. also, the results obtained in animal models may not fully represent the disease course in humans. indeed, some evidences suggest the involvement of ccr5 in the pathogenesis of japanese encephalitis in humans. in indian individuals with mild cases of japanese encephalitis, a significant dowregulation of the ccr5 gene was observed as compared to healthy controls (chowdhury and khan, 2019) . however, the ccr5δ32 does not have a relevant influence on the disease. deval et al. (2019) investigated the effect of various genetic variants, including ccr5δ32, on the development of japanese encephalitis in individuals from north india (table 6 ). no statistically significant difference was found when the ccr5δ32 (as an individual factor) was compared between cases and controls (deval et al., 2019) . considering that both studies mentioned above were performed in the indian population, studies evaluating the potential effects of ccr5 and ccr5δ32 on japanese encephalitis in other populations are necessary. puumala virus (puv) infection is a zoonosis endemic in europe. puv is an enveloped singlestranded rna virus, belonging to the bunyaviridae family, genus hantaviridae. most puv infections are mild or subclinical. in some infected individuals, puv is responsible for the development of nephropathia epidemica, a milder form of hemorrhagic fever with renal syndrome, a condition typically caused by other hantaviruses (settergren, 2000; mustonen et al., 2013; lebecque and dupont, 2019 ). an in vitro study found increased ccr5 gene expression in primary monocytes infected by hantaviruses (hantaan virus, sin nombre virus and andes virus) (markotić et al., 2007) . in this sense, host genetic factors and the immune responses affect different aspects of puv infection and progression of nephropathia epidemica (mustonen et al., 2013) , although the role of ccr5 in this disease is little known. (table 6 ). of note, in their study, the authors stated hantavirus infection as the cause of nephropathia epidemica, not specifying the puv detection. no statistical difference was observed in ccr5δ32 genotype frequencies between cases and controls, indicating that ccr5δ32 does not influenced the susceptibility to hantavirus infection in the studied population. considering only nephropathia epidemica cases, the study revealed an association between the wild-type homozygous genotype (functional ccr5) and increased severity of the disease. conversely, the heterozygous genotype was considered a protective factor against increased disease severity ( in 2019, the multiple roles of ccr5 in physiological and pathological conditions gained the attention of the scientific community and lay public due to ccr5 gene editing in human embryos, the "crispr babies", performed by a chinese biophysicist. the researcher claimed to have used crispr gene editing technology to edit the ccr5 of germline cells to mimic the effects of ccr5δ32, aiming to generate babies resistant to hiv infection. the ethical, safety, and legal aspects related to this procedure have caused an intense and broad discussion in the media and scientific literature (cohen, 2019; daley et al., 2019; greely, 2019; rosenbaum, 2019) , and this case culminated in a three years prison sentence for the biophysicist responsible for the procedure (cyranoski, 2020). also, the consequences of the ccr5 absence have brought many concerns to light, once the ccr5 protein participates in tissue development processes, control of immune responses, and several other physiological processes. considering these concerns, our group and others described some undesirable effects associated to natural ccr5 absence (due to the ccr5δ32 homozygous genotype) and ccr5 editing (ellwanger et al., 2019; wang and yang, 2019; xie et al., 2019) . besides gene editing techniques, the expression of ccr5 can be artificially modulated through the use of pharmacological antagonists (e.g. maraviroc), chemokine ligands, and monoclonal antibodies (fantuzzi et al., 2019) . the use of ccr5 antagonists is well established for the treatment of hiv infection and is currently being tested for the treatment of many other diseases, such as cancer (pervaiz et al., 2019; huang et al., 2020a) , stroke (joy et al., 2019), and cocaine addiction-related disorders (hall et al., 2020; nayak et al., 2020) . taking together, it is increasingly clear that the specific inhibition of ccr5 expression through different techniques is gaining pace in different clinical contexts. the pharmacological ccr5 blockade has many benefits in different clinical situations, particularly in the treatment of hiv infection. also, the potential of gene editing (especially in somatic cells) for the treatment of genetic diseases is very promising. however, considering viral infections, two aspects must be considered, as follows: i) the non-redundant characteristics of the ccr5 should be taken into consideration when studying the ccr5 protein and the effects of ccr5δ32 on viral infections: the traditional concepts of redundancy and robustness of the chemokine system consider that the absence of a specific chemokine or chemokine receptor is adequately compensated by other molecules. although these concepts are generally correct for some chemokines/receptors and for some physiological conditions, the lack of ccr5 expression may affect the protection against some specific infectious diseases once the ccr5 absence is not perfectly compensated for by other receptors (ellwanger et al., 2020a) . the nonredundancy of ccr5 is relevant especially for infections by neuroinvasive flaviviruses (bradina and lim, 2012) . for example, the absence of ccr5 due to ccr5δ32 is considered deleterious in wnv infection lim et al., 2008) and in some aspects of tbev infection (ellwanger and chies, 2019a) . the non-redundant role of ccr5 is also likely in jev infection (larena et al., 2012) . it is possible that those individuals using ccr5 antagonists (e.g., for the treatment of hiv infection) and living in areas endemic for neuroinvasive viruses, especially wnv and tbev, may be at increased risk of j o u r n a l p r e -p r o o f developing viral encephalitis-related problems. although, it is still necessary that studies consistently evaluate this assumption, since the available evidence does not support risks for the use of ccr5 antagonists in endemic areas of flaviviruses (martin-blondel et al., 2016) . as mentioned in the topic addressing wnv in this review, it is prudent to recommend to individuals using ccr5 antagonists to adopt measures to minimize the risk of neuroinvasive infections, such as the use of mosquito repellents and mosquito nets (considering the risk of wnv infection), and to limit their exposure to tick-infested areas (considering the risk of tbev infection). concerns regarding the use of ccr5 antagonists in areas of jev circulation have also been raised by other authors (kim et al., 2016; larena et al., 2012) . if the connection between the use of ccr5 antagonists and increased risk of neuroinvasive infections is confirmed in methodologically wellcontrolled studies, these recommendations should be considered of essential importance for users of ccr5 antagonists. "extracellular vesicles" (evs) is a general term used to designate different membranous vesicles released by various cell types. evs include microparticles, microvesicles, nanovesicles, nanoparticles, ectosomes, exosomes, exovesicles, exosome-like vesicles, oncosomes, among other vesicle types. evs act in the transport of different molecules (e.g. micrornas, mrnas, proteins) between cells and tissues in a regulated and precise manner. evs promote the maintenance of physiological processes, also participating in the pathogenesis of various diseases, such as cancer and inflammatory diseases (colombo et al., 2014; théry et al., 2018) . besides, the regulation of multiple aspects of the immune system is strongly influenced by evs (o'neill and quah, 2008; colombo et al., 2014; ellwanger et al., 2016) . the multiple roles of evs in viral infections began to be studied more intensively in recent years, representing an emerging topic in the field of infectious diseases. currently, the relationship between evs and viral infections has already been explored (to a greater or lesser extent) in the context of the following . evs participate in immune evasion processes, ultimately allowing viruses to bypass host defenses (kaminski et al., 2019b) . for example, hiv is likely to usurp the exosome biogenesis pathway in such a way that enhances its infectivity, while increasing its evading strategies from the host immune defenses (ellwanger et al., 2017a) . regarding tbev, exosomes were indicated as important participants in both viral infection and pathogenesis; in this case, tick-derived exosomes facilitate tbev transmission to the host. also, exosomes derived from tbev-infected host neurons can facilitate the spread of the virus in the cns . exosomes have shown multi-dimensional roles in denv life cycle. they can modulate negatively or positively denv pathogenesis, where they are suggested as instrumental for dengue j o u r n a l p r e -p r o o f hemorrhagic fever development in reason of the transportation of specific micrornas (mishra et al., 2019) . since hcv can be found inside exosomes (liu et al., 2014) , it is not suprising that blood-derived exosomes from hcv-infected patients can transmit the virus to other cells (bukong et al., 2014) . these findings suggest that exosomes and other evs assist in the transport of hcv particles/components between cells, ultimately facilitating viral infection. of note, evs can transport multiple viral components or molecules from virus-infected cells that end up facilitating the spread of the virus in the host. on the other hand, evs can act in favor of the host, transporting molecules that assist host defenses in the elimination or control of infections (kaminski et al., 2019b) . evs can transport a range of cytokines and chemokines, such as il-1, il-2, ifn-α, ifn-β, ccl2, ccl3, ccl4, and cxcl10 (chen et al., 2011; konadu et al., 2015; hosseinkhani et al., 2018; kodidela et al., 2018; gao et al., 2019a; gao et al., 2019b; aiello et al., 2020; chiantore et al., 2020) . besides, evs and microparticles also transport chemokine receptors (shen et al., 2016; liang et al., 2018) , including ccr5 tokarz et al., 2019) . interestingly, evs from ccr5 wild type cells can deliver ccr5 molecules to ccr5 δ32/δ32 cells, making them susceptible to hiv infection . a similar phenomenon has been reported involving microparticles, hiv, and cxcr4 (rozmyslowicz et al., 2003) . ccr5 expression is also influenced by the release of evs, specifically microparticles (renovato-martins et al., 2017) . therefore, evs have the potential to attribute greater complexity to ccr5 roles in viral infections. it is possible that the presence of ccr5 in cells is not only dependent on mechanisms of membrane expression and internalization of the receptor. it can also be dependent on ccr5 delivery mediated by evs. however, the actual impact of evs on ccr5-mediated immune response in infections remains to be determined and deserves further investigation. finally, a truncated ccr5 soluble form (tsimanis et al., 2005) , as well as soluble cxcr4, have also been reported in the plasma of humans (malvoisin et al., 2011) . of note, the truncated ccr5 soluble form has ~22 kda (half the size of the ccr5 found on cell membranes) and is truncated at the end of the second extracellular loop (the third extracellular loop and the subsequent three transmembrane regions are absent) (tsimanis et al., 2005) . however, the existence of cell-free soluble ccr5 is still controversial and does not represent a consensus in the scientific community. such doubts occur mainly in consideration of the structure of the receptor, which is highly characteristic of a cell membrane-associated molecule. we believe that evscontaining ccr5 can explain potential (misleading) detections of soluble ccr5. the importance of circulating evs-containing ccr5 on viral infections represents an additional and interesting open question to be investigated. chávez et al. (2013) (ellwanger et al., 2017b) . specifically, using a mouse model of rocv-associated encephalitis, ccr5-deficient mice showed increased survival rate and reduced levels of brain inflammation compared to mice expressing ccr5, indicating the participation of ccr5 in rocv-associated encephalitis (chávez et al., 2013) . although other studies discussed in this review have shown an important involvement of ccr5 in infection by flaviviruses [especially as an important molecule for the resolution of neuroinfection, in opposition to results of chávez et al., (2013) ], the role of the ccr5 in rocv infection represents a neglected topic. this knowledge gap should be addressed in further studies since the reemergence of rocv in brazil is a concern in terms of public health (figueiredo, 2007; ellwanger et al., 2017b) . although no other rocv outbreaks have been reported in brazil after the 1980s, there is evidence of rocv circulation in animals (casseb et al., 2014; pauvolid-correa et al., 2014; silva et al., 2014) . ccr5 expression on t cells. of note, zikv is another mosquito-borne flavivirus that recently reemerged in many countries, affecting brazil in particular. zikv infection is associated with microcephaly and other human development problems (baud et al., 2017) . zachova et al. (2019) showed that epstein-barr virus (ebv)-infected b cells have increased ccr5 expression compared to ebv-non-infected cells. also, recent evidence points to a pivotal role of the ccr5 in the attenuation of rhinovirus-associated inflammation, by controlling the activity of cd4 + foxp3 + treg cells (hossain et al., 2020) . altered levels of cytokines and chemokines are associated with several aspects of zikv (barros et al., 2018; naveca et al., 2018; khaiboullina et al., 2019; lima et al., 2019) , ebv (piovan et al., 2005; ehlin-henriksson et al., 2009; cohen et al., 2017) , and rhinovirus infections (rajan et al., 2014; shelfoon et al., 2016; hansel et al., 2017) . however, the potential role of the chemokine receptor ccr5 in the pathogenesis of zikv, ebv, and rhinovirus represents open questions in the field of ccr5 research. coronaviruses are positive-sense rna viruses that belong to the coronaviridae family (li et al., 2020) . coronaviruses infect a wide range of species, including humans. until 2019, humans have faced two major outbreaks of high pathogenic coronaviruses, the severe acute respiratory syndrome coronavirus (sars-cov) outbreak and the middle east respiratory syndrome coronavirus (mers-cov) outbreak (fung and liu, 2019) . in 2019, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in wuhan (hubei province, china) and rapidly spread to more than 180 countries/regions (interactive dashboard of global covid-19 cases -johns hopkins university: https://arcg.is/0fhmtx ; dong et al., 2020) . of note, sars-cov-2 infection causes the "coronavirus disease 2019" and therefore is also called "covid-19". considering the ongoing sars-cov-2 pandemic and the clinical variability observed in the affected individuals, ranging from mild to severe respiratory disease j o u r n a l p r e -p r o o f 2020b), the following question arose: does ccr5 have any clinically relevant influence on sars-cov-2 or other coronaviruses infections? recent data indicated that sars-cov-2 binds to the ace2 receptor (andersen et al., 2020) , using this receptor for entry into human cells. therefore, it is unlikely that ccr5 or ccr5δ32 have some significant effect on the susceptibility or resistance to sars-cov-2 infection. however, ccr5 may have some impact on the clinical course of sars-cov-2 infection. the pattern and intensity of the human immune responses regulate the clinical progression and even the outcome of infections by coronaviruses (li et al., 2020) , including sars-cov-2 (qin et al., 2020; shi et al., 2020) . sars-cov-infected mice showed increased expression of ccr5 mrna in lungs along with the production of ccl2, ccl3 and ccl5 chemokines, the major ccr5 natural agonists (chen et al., 2010) . intracranial infection of mice with mouse hepatitis virus (mhv, a murine coronavirus) induces an increased ccr5 expression, which contributed to mhv-induced demyelination through ccr5-mediated macrophage recruitment to the cns (glass et al., 2001) . subsequent studies using mhv-infected mice showed that ccr5 participates in the regulation of cd4 + and cd8 + t cell activities against the virus in the cns (glass and lane, 2003a; glass and lane, 2003b) . also, sars-cov-infected human monocyte-derived dendritic cells showed increased ccr5 expression in vitro (law et al., 2009) . taking together, the findings mentioned above suggest that ccr5 could have some influence on the clinical course of sars-cov-2 infection. however, future studies addressing humans are needed to clarify this suggestion. in this sense, we emphasize that so far, there is no evidence showing a clear involvement of ccr5 in sars-cov-2 human infection. the ccr5δ32 is a highly penetrating polymorphism, and exerts a robust phenotypic effect on ccr5. however, the expression of ccr5 is regulated by other polymorphisms in addition to ccr5δ32 (mehlotra, 2019) . also, the ccr5 expression is influenced by non-coding genetic elements. the effect of the genetic backround of the host can also be exacerbated or diminished depending on the environmental conditions to which the individual is exposed (ellwanger et al., 2018c; kulkarni et al., 2019) . taking together, these factors help to explain why many of the effects exerted by ccr5 on a given disease are specific to a certain population, ethnic group, or individual. research involving ccr5 and hiv began in the mid-1990s. since then, many achievements in the field of ccr5/hiv research have been made, such as the identification of ccr5δ32 as a strong protective factor against hiv infection and the development of ccr5 antagonists for the treatment of hiv infection. currently, these drugs are being tested for the treatment of other inflammatory and infectious diseases. in this context, the use of ccr5 antagonists has raised some concerns, since the blockade of ccr5 can affect j o u r n a l p r e -p r o o f or even weaken the host defenses against certain infections, especially neuroinvasive infections by flaviviruses. however, to date, there is no strong evidence indicating that the use of ccr5 antagonists has increased the susceptibility of individuals to problematic flaviviruses infections. the frequency of ccr5δ32 is quite varied among human populations, and therefore the effects of the allele in a particular population may not apply to another population. moreover, the effects of ccr5 and ccr5δ32 are disease-specific. for instance, the ccr5δ32 does not significantly affect susceptibility to wnv infection. however, the effect of the ccr5 and ccr5δ32 on wnv disease progression is very robust. some animal-derived findings suggest the involvement of the ccr5 in the pathogenesis of denv infection. however, the effects of ccr5 on denv infection are very different between humans and rodents. of note, ccr5δ32 does not significantly affect susceptibility to denv infection or disease progression. moreover, the effect of ccr5δ32 on hbv-related disease is population-specific. interestingly, cmv release virokines, which are molecules that can manipulate the host immune response and affect the ccr5 function. the examples cited above highlight the varied impacts of ccr5 and ccr5δ32 on viral infections. the few studies involving cchfv, ev, and hantavirus infection have indicated important effects of ccr5δ32 on these conditions. based on these findings, further studies should investigate the role of ccr5δ32 and ccr5 protein in such infections, considering populations with distinct genetic backgrounds. some evidence suggested the participation of ccr5 in infections by zikv, ebv, and rhinovirus. also, a mouse model of rocv-associated encephalitis suggested an important role for ccr5 in host immune responses against the virus. however, the roles of ccr5 and ccr5δ32 in infections by zikv, ebv, rhinovirus, and rocv are still poorly understood and need to be investigated in future studies. the role of evs in the transport of ccr5 between cells indicates that the expression of ccr5 on the cell surface may also depend on the release of evs containing ccr5. also, the transport of host molecules and viruses through evs adds complexity to the topics covered in this review and should be taken into account in future studies that investigate the role of ccr5 in viral infections. gene editing technologies have the potential to be used to treat different diseases, mainly when applied to somatic cells. however, ccr5 gene editing in human embryos presents a number of ethical problems. besides, the absence of ccr5 can have deleterious effects in certain conditions, such as increased susceptibility to symptomatic wnv infection. finally, this article showed that the participation of ccr5 in different viral infections is complex and varied and, therefore, cannot be generalized. this article also pointed out neglected gaps in knowledge involving ccr5 that should be addressed in future studies. the authors declare no conflicts of interest. ccr5 polymorphism as 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mimicry by hiv exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral rna and proteins to the vertebrate neuronal cells chemokines: a new classification system and their role in immunity protective effect of ccr5-δ32 against hiv infection by the heterosexual mode of transmission in a polish population american 395 symptomatic wnv+ individuals; 145 symptomatic but noninfected individuals; 1318 controls (blood donors)the ccr5δ32 homozygous genotype was strongly associated with increased risk of symptomatic wnv infection glass et al. (2006) american 224 symptomatic wnv+ individuals; 1318 controls (blood donors)corroborating data from glass et al. (2006) , the ccr5δ32 homozygous genotype was strongly associated with increased risk of symptomatic wnv infection lim et al. (2008) american 634 wnv+ individuals; 422 non-infected individuals ccr5δ32 homozygous genotype was associated with clinical symptoms of wnv infection; no association between ccr5δ32 and susceptibility to wnv infection lim et al. (2010) american, canadian 385 symptomatic wnv+ individuals; 328 asymptomatic wnv+ individuals; 1318 controls [blood donors from glass et al. (2006)] no association of ccr5δ32 and wnv infection considering symptomatic and asymptomatic wnv+ individuals; considering a dominant model of inheritance of ccr5δ32 and using controls from glass et al. (2006) , the ccr5δ32 was associated with symptomatic wnv infection and infection risk bigham et al. (2011)