Frontiers | BALB/c Mouse Is a Potential Animal Model ...

Conclusion: BALB/c mice have a great potential for reproducing the process of gt4 HEV infection. The successful establishment of a gt4 HEV small ... Articles ChunfuZheng UniversityofCalgary,Canada PhilipMeuleman GhentUniversity,Belgium ShaoweiLi XiamenUniversity,China Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction MaterialsandMethods Results Discussion Conclusion DataAvailabilityStatement EthicsStatement AuthorContributions Funding ConflictofInterest Acknowledgments SupplementaryMaterial References SuggestaResearchTopic> DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex totalviews ViewArticleImpact SuggestaResearchTopic> SHAREON OpenSupplementalData ORIGINALRESEARCHarticle Front.Microbiol.,16June2020 |https://doi.org/10.3389/fmicb.2020.01156 BALB/cMouseIsaPotentialAnimalModelSystemforStudyingAcuteandChronicGenotype4HepatitisEVirusInfection YunlongLi1†,FeiyanLong1†,ChenchenYang1†,XianhuiHao1†,JianWu2,JianwenSitu1,ShuangfengChen1,ZhongyaoQian1,FenHuang1*andWenhaiYu3* 1MedicalFaculty,KunmingUniversityofScienceandTechnology,Kunming,China 2StateKeyLaboratoryforDiagnosisandTreatmentofInfectiousDiseases,NationalClinicalResearchCenterforInfectiousDiseases,CollaborativeInnovationCenterforDiagnosisandTreatmentofInfectiousDiseases,TheFirstAffiliatedHospital,CollegeofMedicine,ZhejiangUniversity,Hangzhou,China 3InstituteofMedicalBiology,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Kunming,China HepatitisEvirus(HEV)isthemainpathogenofhepatitisworldwide.However,itsinfectionbiologyandpathogenesisremainlargelyunknown.Suitablesmall-animalmodelsarerequiredtoadvancethestudyofHEVinfection.Althoughanefficientmodelofgenotype1(gt1)andgt3HEVinfectionhasbeenestablishedinhumanliverchimericmice,theinfectivityofgt4HEVinfectioninmicehasnotbeencomprehensivelycharacterized.Inthisstudy,immunocompromisedBALB/cnude,immunocompetentBALB/c,andC57BL/6micewereinoculatedwitheithergt3orgt4HEV(19HEVstrains,includinghuman,swine,macaque-adapted,andcowHEVstrains).InfectivitywasidentifiedbyviralRNAandantigendetection,inflammation,andhistopathologicalanalysis.Then,HEV-infectedBALB/cmiceweretreatedwithantiviraldrugs.AcuteHEVinfectionwasestablishedinBALB/cmiceinoculatedwitheightgt4HEVstrains.However,gt3HEVstrainsfailedtoachieveactiveHEVinfection.HEVinfectionwasestablishedinBALB/cnudeandregularmiceinoculatedwithgt4HEVbutnotinC57BL/6mice.Gt4HEVinfectionresultedinrapidviremiaandhightitersinfeces,sera,andreplicationsites.HEVinfectioninmiceshowednogenderpreference.Furthermore,chronicgt4HEVinfectionwaswellimitatedinBALB/cmicefor32weeksandcausedliverfibrosis. Conclusion:BALB/cmicehaveagreatpotentialforreproducingtheprocessofgt4HEVinfection.Thesuccessfulestablishmentofagt4HEVsmall-animalmodelprovidesanopportunitytofurtherunderstandHEVinfectionbiologyandzoonotictransmissionanddevelopanti-HEVvaccine. Introduction HepatitisEvirus(HEV)isaviralpathogenthatcausesacutehepatitisandchronicinfectioninimmunocompromisedpatients(Kamaretal.,2008;AggarwalandJameel,2011).HEVhasoneserotypeandeightgenotypes.Genotype1(gt1)andgt2HEVonlyinfecthumansandaremainlyprevalentinAsia,Mexico,andAfrica.Thesegenotypesareresponsiblefor20millioninfectionsand70,000deathsannually(Reinetal.,2012).Gt3andgt4HEVcanbetransmittedfromseveralanimals,includingswine,deer,andcow,tohumansmainlythroughtheconsumptionofundercookedmeatormilk(Huangetal.,2016a;Rivero-Juarezetal.,2016).HEVinfectionisthoughttobeacuteandself-limiting,butmorechronicinfectionshavebeenreportedrecently(AggarwalandJameel,2011;Aggarwal,2011).ChronicHEVinfections,definedasHEVRNApersistentlypositiveinfecesorserumformorethan3months,havebeenreportedinimmunocompromisedpatients,especiallyinorgan-transplantrecipients,causedpredominatelybygt3andpartiallybygt4(Kamaretal.,2008;Abravaneletal.,2014;Perumpailetal.,2015).However,noantiviralmedicationisavailablebecauseoftheunavailabilityofsuitableanimalmodels.Hence,HEVinfectionbiologyandpathogenesisremainlargelyunknown. ThemostimportanttoolsforHEVinfectionandpathogenesisresearcharesmall-animalmodels.However,thedevelopmentofanimalmodels,particularlytheuseofsmalllaboratoryanimals,hasnotbeenadequatelyexplored.Althoughgt1andgt3HEVchronicinfectionsofhumanliverchimericmicehavebeenestablishedinearly2016toprovethatonlyhumanizedlivercanbeinfectedwithHEV(Allweissetal.,2016;Sayedetal.,2016;vandeGardeetal.,2016),thesemicearefrail,expensive,andcannotreproducehumanizedoffspring.BALB/cnudemouse-basedgt4HEVmodelshavebeensuccessfullyestablishedtosimulateimmunocompromisedpatientsinfectedwithswineHEV(Huangetal.,2009).TheMongoliangerbil-basedHEVmodelisalsosusceptivetogt4swineHEVisolatedinChina(Lietal.,2009;Soomroetal.,2017).However,thequestionofwhethergt4HEVthatisendemicinChinacaninfectmousemustberesolved. NoHEV-specifictreatmentsarepresentlyavailable.Ribavirin(RBV),whichisprimarilyusedtotreathepatitisCandviralhemorrhagicfevers,isthefirst-choicetherapyforchronicgt3HEVinfectionbutitsusehasachievedlimitedsuccess(Todtetal.,2016b;Barragueetal.,2017).Moreover,resistanceandrelapsehavebeenwidelyreportedclinicallywhenRBVmonotherapyisusedinpatientschronicallyinfectedwithHEV(Debingetal.,2014b,2016;Todtetal.,2016b).Asanalternative,interferon-α(IFN-α)(IFN-α1b)hasbeenadministeredaloneorincombinationwithRBVinvitroandinvivo(Debingetal.,2014a;Todtetal.,2016a),butseveresideeffectshavealsobeenreported(Behrendtetal.,2014).Sofosbuvir(SOF),anefficientantiviraldrugforhepatitisCvirus(HCV),isreportedlyapotentialanti-HEVdrugcandidate,butsomestudieshaverejecteditsanti-HEVeffect(Wangetal.,2016).MostchronicHEVinfectioncasesarereportedindevelopedcountrieswithendemicgt1orgt3HEV,andchronicinfectioncausedbygt4HEVhasrarelybeenreported.Therefore,whethergt4HEVinfectionissensitivetotheseantiviraldrugsisunknown. Inthepresentstudy,BALB/c-basedacuteandchronic(HEVRNApersistentlypositivefor32weeks)gt4HEVinfectionsweresuccessfullyestablished.WefoundthatimmunocompromisedandimmunocompetentBALB/corC57BL/6micewerenotsusceptibletogt3HEV.ThesuccessfulestablishmentofacuteandchronicHEVBALB/cmicemodelshasimportantimplicationsforexploitingtheHEVpathogenesismechanismanddevelopingdrugsagainstthisdisease. MaterialsandMethods Viruses Gt3swineHEV(SAAS-JDY5)isolatedfromShanghaiwasprovidedbyDr.ZhenLi(ShanghaiAcademyofAgriculturalSciences).NineteenGt4HEVstrains,includingswine(KM01),human(LX),chronic-infectedrhesusmacaque-adapted(macKM01),andcowHEV(milk,1#–16#)HEVstrains,wereisolatedfromnineprovincesofChina(Table1).Fecalsuspension(10%[w/v])wascentrifugedat12,000×gat4°Cfor10min,filteredthrough0.22-μmmicrofilters,andtreatedwithpenicillinandstreptomycinfor1h.Viralgenomictitersweredeterminedbyquantitativereal-timepolymerasechainreaction(qRT–PCR),aspreviouslydescribed(Huangetal.,2016a). TABLE1 Table1.HEVstrainsusedinthisstudy. AnimalandViralInoculation SPFBALB/cnude(females,6weeksold,16–18g,n=54),BALB/c(females,n=146;males,n=30;6weeksold,18–20g),andC57BL/6mice(females,6weeksold,18–20g,n=54)werepurchasedfromShanghaiLaboratoryAnimalCenter(China)andmaintainedinapathogen-freeanimalfacility.TheanimalprotocolswereapprovedbytheAnimalCareandUseCommitteeofKunmingUniversityofScienceandTechnology.FecalandserumsampleswerecollectedforHEVRNAdetectionbyqRT–PCRandanti-HEVIgGandIgMdeterminationbyELISApriortotheconductofthestudy,respectively.TheprotocolforHEVRNAdetectionbyqRT–PCRwasdescribedinourpreviousstudy(Huangetal.,2016a).Micenegativetoanti-HEVantibodiesandHEVRNAwereusedinthisstudy. BALB/cmice(females,n=20)wereseparatelyinoculatedwith20HEVstrains(intravenousinjectionwith100μloffecalsuspensionorgavagewith300μlofmilkeachmouse)toscreenwhichstrainofgt3andgt4HEVisinfectious.FeceswerecollectedtwiceperweekforHEVRNAdetection. BALB/cnude,regularBALB/c,andC57BL/6micewereemployedtoassessthesensitivityofthesestrainstogt3andgt4HEV.GiventhattheinfectivityofKM01strainhasbeenconfirmedinrhesusmacaque(Huangetal.,2016a,b),treeshrew(Yuetal.,2016),andBALB/cmice(Figure1),itwasusedtoestablishtheexperimentalinfectionofgt4HEV.Eachstrainofmicewasrandomlydividedintothreegroups.Asnegativecontrol,themiceingroup1(n=6)wereinjectedwith100μlofPBSviathetailvein.Thoseingroup2(n=24)wereinjectedwith100μlofstoolsupernatantofgt4swineHEV(KM01)viathetailvein.Thoseingroup3(n=24)wereinjectedwith100μlofstoolsupernatantofgt3swineHEV(JDY5)bythetailvein.MaleBALB/cmice(n=30)wereusedtodeterminethesexdifferenceingt4HEVinfection. FIGURE1 Figure1.ScreeningtheinfectivityofhepatitisEvirus(HEV)strainsisolatedfromhuman,swine,macaque,andcowinBALB/cmice.BALB/cmicewereseparatelyinoculatedwithgt3orgt4HEV.StoolsampleswerecollectedtwiceperweekanddetectedbyqRT-PCR.Thelimitofquantificationis2.4×103copies/g. Thestoolsupernatantofchronic-infectedrhesusmacaque(HEVRNA+infeceslastedmorethan670days,labeledasmacKM01),whichalsocausedchronicinfectioninanotherrhesusmacaque(seedetailsinourpreviousstudy;Huangetal.,2016b),wasusedastheinoculumtoestablishchronicHEVinfectionmousemodel.BALB/cmice(females,n=36)wereinjectedwith100μlofthestoolsupernatantmacKM01throughthetailvein. Afterviralinoculation,themicewerehousedindividually.StoolsampleswerecollectedtwiceeveryweektotesttheinfectivityofHEVinmice,andbloodsampleswerecollectedweekly.Sixmiceineachgroupwereeuthanizedandnecropsiedat7,14,21,or28dpi.ForchronicHEVinfection,themicewerenecropsiedat14dpior32weekspost-inoculation(wpi,endoftheexperiment).Theliver,spleen,kidneys,intestines,uterus,andbrainwerecollectedforHEVRNAdetectionbyusingreverse-transcriptionnestedPCR(RT-nPCR)andqRT-PCR.HEVantigenswereanalyzedat14dpibyimmunohistochemistry(IHC)andindirectimmunofluorescenceassay(IFA).Histopathologicalanalyseswereperformedat28dpiforacuteHEVinfectionor32wpiforchronicHEVinfection. HEVTreatment RBV,IFN-α(IFN-α1b),andSOFwereappliedingt4HEV-infectedBALB/cmicetoevaluatetheeffectsofantiviralcompoundsonHEVinfection.HEV-infectedBALB/cmice(n=36)wererandomlydividedintosixgroupsaccordingtoviraltiterinthestoolat4dpi.Themiceingroup1(n=6)wereinjectedwithPBS(negativecontrol).Themiceingroup2weretreatedwithRBV(50mg/kg/day,orally)foroneweek.Themiceingroup3weregivenIFN-α1b(30μg/kg,intramuscularinjection).Themiceingroup4weretreatedwithSOF(400μg/day/50kg,orally).Themiceingroup5wereadministeredwithacombinationofSOFandRBV.Themiceingroup6weretreatedwithacombinationofRBVandIFN-α.Stoolsampleswerecollectedtwiceeveryweek,andbloodsampleswerecollectedweekly. VirusDetectionandGeneQuantification TotalRNAwasextractedfromthestool(10%suspension[W/V]),blood,serum,andtissuesbyusingTrizol(Invitrogen,UnitedStates).Reversetranscriptionwasperformedusingareversetranscriptasekit(AMV,Takara,Japan)withHEV-specificnegativeandpositivestrandprimersorrandomprimer.NestedPCRwasconductedaccordingtopreviousstudies(Nandaetal.,1994;Huangetal.,2002,2016a).NegativecontrolwasincludedtoexcludePCRcontamination.HEVviraltiterwasquantifiedusingSYBRGreen-basedqRT-PCRfollowingourpreviousstudy(Huangetal.,2016a). TherelativegeneexpressionofIFN-I,includingIFN-αandIFN-β,wasquantifiedwiththespecificprimersasdescribedinapreviousstudy(Xuetal.,2017).GAPDHwasusedasthehousekeepingcontrolgene.qRT-PCRwasperformedusinganABIPRISM7300Real-TimePCRSystem.Relativegeneexpressionwasdeterminedusingtheformula2–(ΔCtofgene–ΔCtofGAPDH),whereCtisthethresholdcycle. Histopathology,IHC,andIndirectImmunofluorescenceAnalysis Tissuebiopsieswerefixedin10%neutral-bufferedformalinandembeddedinparaffin.Specimenswerecutinto3-to4-μmserialsections.StandardhematoxylinandeosinorMassonstainingwasperformed,andthetissueswereexaminedunderamicroscope. ForIHCandindirectIFAanalysis,tissuesweredeparaffinized,hydrated,heatedinawaterbathforantigenretrieval,andthenblockedwiththeadditionof3%hydrogenperoxidefor15min.Tissuesectionswereincubatedfor2hat37°Cwithprimaryantibodies(HEVORF2,MerckMillipore,Germany;CD45,Abcam,UnitedStates;F4/80,Abcam,UnitedStates),washedwithPBS,andthenincubatedwithHRP-,FITC-,orTRITC-labeledsecondaryantibody,asdescribedinourpreviousstudy(Huangetal.,2018). DetectionofAnti-HEVIgGandIgMAntibodies HEVIgGandIgMantibodiesweredeterminedusingacommercialELISAkit(ShanghaiKehuaBio-engineering,KHB,China)onthebasisofrecombinantHEVfusionproteinsaccordingtothemanufacturer’sinstructions,exceptthatthesecondaryantibodieswerereplacedwithHRP-conjugatedantimouseIgMorIgGantibodies(Kirkegaard&PerryLaboratories,KPL,UnitedStates). SerumLiverChemistryProfile Alanineaminotransferase(ALT),aspartateaminotransferase(AST),andtotalbilirubin(T-Bil)activitiesinserumweremeasuredusinganautomatedbiochemistryanalyzer(MindrayBS-200,China). StatisticalAnalysis Datawerepresentedasmean±standarddeviation.GraphPadPrismsoftwarewasusedforstatisticalanalysisandtodeterminep-values.Student’st-testorχ2analysiswasusedtodeterminethesignificanceofdifferencesbetweentwoormoregroups,inwhicha0.05levelofprobability(p<0.05)wasconsideredstatisticallysignificant. Results Genotype4HEVIsInfectiousinBALB/cMicebutNotinEveryStrain Inapreviousstudy,wereportedthatBALB/cnudemicearesensitivetogt4swineHEVinfectionisolatedfromShanghai,China.Moreover,Mongoliangerbils,alsoarodent,aresusceptibletogt4swineHEVisolatedfromYunnan,China(Lietal.,2009;Soomroetal.,2017).Thematterofwhetherallgt4HEVstrainsareinfectiousinmouseanimalmodelsisunsettled.Wescreened20HEVstrains,including1gt4human,1gt3swine,1gt4swine,1gt4macaque,and16gt4cowHEVstrains,isolatedfrom11citiesinnineprovincesofChina.OnlynineofthemwereinfectiousinBALB/cmicewithdetectableHEVRNAsheddinginfeces(Figure1andTable1).However,thequestionwhyBALB/cmicewerenotsusceptibletotheother10gt4HEVstrainsremainsinexplicable,despitethefactthatthesestrainswereisolatedfromthesamevillagewithhigherviraltitersandstoredinthesameenvironment(Table1). GiventhatnoteveryHEVstrainwasinfectiousinBALB/cmice,thequestionofwhetherallrodentspeciesaresensitivetoinfectiousgt4HEVisunresolved.Therefore,wefurtherscreenedtheinfectivityofgt4HEVincommonrodentmodels,includingBALB/cnude,BALB/cregular,andC57BL/6mice. ImmunocompromisedBALB/cNudeMiceAreSensitivetogt4HEVbutNotSusceptibletogt3HEV Inthepresentstudy,nudemicewereintravenouslyinoculatedwithgt4HEV(KM01strain),whoseinfectivityhasbeenconfirmedinrhesusmacaque(Huangetal.,2016a,b),treeshrew(Yuetal.,2016),andBALB/cmouse(Figure1).Similartorhesusmacaqueandtreeshrew,allnudemiceinoculatedwithKM01strainwereinfectedwithHEV,withsheddingofvirusesinstoolsamplesstartingfrom3to7dpiandendingat4wpi(Figure2A).Viremiawasdetectedfrom1to4wpi(Figure2A).NegativestrandofHEVRNAwasdetectedinalltheliverofHEV-infectednudemiceat7dpibyRT-nPCR,whichindicatedthereplicationofHEV.PositivestrandsofHEVRNAweredetectedandquantifiedbyqRT–PCR(Figure2B).Intheotherextrahepaticsites,spleen,kidneys,intestines,uterus,andbrain,HEVRNAwasalsodetectablefrom1to3wpiandsharplydecreasedat4wpi(Figure2B).However,HEVRNAwasundetectableinthestool,serum,ortissuesamplesofnudemiceinoculatedwithPBSorgt3HEVduringtheentireexperiment(Figures2A,B). FIGURE2 Figure2.Profilesofgt3andgt4hepatitisEvirus(HEV)infectioninBALB/cnudemice.HEVRNAwasdetectedinthestoolandserum(A)andtissues(B)ofBALB/cnudemiceinoculatedwithPBS,gt3HEV,orgt4HEV.LOQ,limitofquantification.HEVantigenwasdetectedintheliver,spleen,kidneys,intestines,brain,anduterusofBALB/cnudemicebyimmunohistochemicalmethod(×200;C)andimmunofluorescenceanalysis(×200;D).ThenucleiwerestainedwithDAPI(blue).Immunofluorescentstainingforleukocyte(CD45+,green,E)ormacrophages(F4/80+,green,F)wasperformedintheliverofuninfected(PBS)orgt4HEV-infectedBALB/cnudemice,×400.LOQis1.0×103copies/gorcopies/ml. IHCandindirectIFAclearlyshowedHEVantigensintheseHEVreplicationsitesingt4HEV-infectednudemiceat14dpi(Figures2C,D).Positivestainswereobviouslyobservedintheliver,spleen,kidneys(glomerulus),intestines,uterus,andbrain(conecells)ofnudemiceinoculatedwithgt4HEV(KM01),whereasnostainswereperceivedinnudemiceinoculatedwithPBSorgt3HEV(JDY5)(Figures2C,D).ThepresenceofHEVRNAandHEVantigeninHEVreplicationsitesfirmlyconfirmedthatgt4HEVcanreplicateinimmunocompromisedBALB/cnudemice. AcuteHEVinfectionusuallycausesinflammation.Analysisofinflammationstronglyindicatedthatgt4HEVinfectioncausedacuteinflammatoryresponsewithincreasedCD45+leukocyteandF4/80+macrophageintheliverofHEV-infectednudemice(Figures2E,F).Histopathologicalanalysisalsorevealedmildinflammationintheliver,spleen,andkidneysofgt4HEV-infectednudemice(SupplementaryFigure1A).Nosubstantialdamagewasobservedintheintestines,brain,anduterusofgt4HEV-infectednudemice(SupplementaryFigure1A).NosignificantchangeinALTandASTactivitieswasdeterminedingt4HEV-infectednudemicecomparedwithuninfectedcontrolmice(PBSgroup)ormiceinoculatedwithgt3HEV(gt3HEVJDY5group)(SupplementaryFigures1B,C).Anti-HEVIgMandIgGantibodiesingt4HEV-infectednudemicewerenegative(SupplementaryFigures1D,E).SheddingofHEVprogenyvirusesinthefeces,detectableHEVRNAandHEVantigeninHEVreplicationsites,andmildacuteinflammatoryresponsestablyconfirmedthatgt4HEViscapableofsuccessfullyinfectingimmunocompromisedBALB/cnudemice. Gt3,whichisanotherzoonoticallytransmittedHEV,cannotinfectC57BLmice(Todtetal.,2016a).ItsinfectionresultsinlowHEVRNAlevelinserumandundetectableORF3proteininliverofhumanizedUPA/SCID/beigemice(Allweissetal.,2016).Similarly,BALB/cnudemiceinoculatedwithgt3HEV(JDY5)werenegativeforHEVRNAinallstoolandserumsamples(Figures2A,B).Moreover,HEVRNAandHEVantigenwerenotdetectableintheliver,spleen,kidneys,intestines,brain,oruterus(Figures2B,D).Nosignificantdamagewasobservedinthetissuesofnudemiceinoculatedwithgt3HEVcomparedwithmicetreatedwithPBS(SupplementaryFigure1A).ALTandASTactivitiesexhibitednoobviouschange(SupplementaryFigures1B,C).Anti-HEVIgGandIgMantibodieswerenegative(SupplementaryFigures1D,E).Theseresultsdemonstratedthatgt3HEVcannotinfectBALB/cnudemice.Gt3HEVinfectioninhumansisasymptomatic.Themildcharacteristicsofgt3HEVmayberesponsiblefortheunresponsivenessinmice,eveninimmunocompromisedmice. RegularBALB/cMiceAreAlsoSensitivetogt4HEV Gt4HEVsuccessfullyreplicatesinimmunocompromisedBALB/cnudemiceandcausesclassicalacutehepatitisEinfection.However,theanswertowhetherimmunocompetentregularBALB/cmice,whichhaveanormalimmunesystem,canbeinfectedwithgt4HEVremainselusive.Inthisstudy,gt4HEVwasinoculatedintoimmunocompetentBALB/cregularmice.BothfemaleandmaleBALB/cmicewereusedtodeterminewhethersexdifferenceaffectstheinfectivityofHEV. Gt4HEVwassuccessfullyreplicatedinBALB/cmice.TheprogenyviruseswereshedinthefecesofBALB/cmicefrom3–7dpito25dpi.Viremialastedfrom1wpito4wpiinBALB/cmiceinoculatedwithgt4HEV(Figure3A).Negativeandpositivestrandsweredetectedintheliver,spleen,kidneys,intestines,brain,anduterus(Figure3B).However,nosignificantdifferencewasobservedintheviraltiterinthefeces,blood,andliverbetweenfemaleandmaleBALB/cmice,exceptthatthetiterwashigherinfemalesthaninmalesat4wpi(SupplementaryFigures2A–C).Therefore,femaleBALB/cmicewereusedinthesubsequentexperiments.IHCclearlyshowedHEVantigensintheliver,spleen,kidneys,intestines,brain,anduterusofregularBALB/cmiceinoculatedwithstool-derivedgt4HEV,whereasnopositivestainswereobservedinBALB/cmiceinoculatedwithPBSorstool-derivedgt3HEV(Figure3C).IndirectIFAclearlyrevealedpositivefluorescentparticlesofHEV(red)intheliver,spleen,kidneys,intestines,brain,anduterusofBALB/cmiceinoculatedwithgt4HEV.However,nosignalwasdetectedinBALB/cmiceinoculatedwithPBSorgt3HEV(Figure3D). FIGURE3 Figure3.Profilesofgt3andgt4hepatitisEvirus(HEV)infectioninBALB/cmice.HEVRNAwasdetectedinthestoolandserum(A)andtissues(B)ofBALB/cmiceinoculatedwithPBS,gt3HEV,orgt4HEV.LOQ,limitofquantification.HEVantigenwasdetectedintheliver,spleen,kidneys,intestines,brain,anduterusofBALB/cmicebyimmunohistochemicalmethod(×200;C)andimmunofluorescenceanalysis(×200;D).ThenucleiwerestainedwithDAPI(blue).Immunofluorescentstainingforleukocyte(CD45+,green,E)ormacrophages(F4/80+,green,F)wasperformedintheliverofuninfected(PBS)orgt4HEV-infectedBALB/cmice,×400.ThegeneexpressionofIFN-α(G)andIFN-β(H)wasquantifiedbyqRT-PCRinthebloodofBALB/cmiceinoculatedwithgt3HEVorgt4HEV.LOQofserumis1.2×103copies/ml,LOQofstoolis1.7×103copies/g,andLOQoftissuesis2.5×103copies/g. Similartogt4HEV-infectedBALB/cnudemice,acuteinflammatoryresponsewasobservedinregularBALB/cmiceinoculatedwithgt4HEVwithincreasedCD45+leukocyteandF4/80+macrophageintheliverofHEV-infectedregularBALB/cmice(Figures3E,F).Mildhistopathologicalchangesweredetectedintheliver(enlarged,focalhepatocellularnecrosis),spleen(infiltratinglymphocytesandmacrophages),kidneys(congestion),andintestines(infiltratinglymphocytes)ofBALB/cmiceinfectedwithgt4HEV.However,nodamagewasobservedinBALB/cmiceinoculatedwithPBSorgt3HEV(SupplementaryFigure2D). Toevaluatethedifferenceinimmuneresponsesagainstgt3andgt4HEVinfection,thegeneexpressionoftypeIinterferon(IFN-I,IFN-αandIFN-β),themostimportantantiviralfactorsofhost,weredetermined.Interestingly,theexpressionofIFN-αingt4HEV-infectedmicewas93.77-foldlowerthanthatingt3HEV-inoculatedmice(Figure3G).Similarly,theIFN-βexpressioningt4HEV-infectedmicedecreased5.23-foldthanthatingt3HEV-inoculatedmice(Figure3H).Thesubstantialreducingofhostantiviralresponsescausedbygt4HEVmaycontributetotheefficientinfectioninBALB/cmice. SimilartotheclinicalfeaturesofacuteHEVinfection,gt4HEV-infectedBALB/cmiceshowedsignificantlyincreasedALTandASTactivitiesat1–4wpiandrecoveredsubsequently.However,ALTandASTactivitiesshowednosignificantchangeinBALB/cmiceinoculatedwithPBSorgt3HEV(SupplementaryFigures2E,F).However,adaptiveimmunitywasnotevokedingt4HEV-infectedBALB/cmice(SupplementaryFigures2G,H).ResultsstronglydemonstratedthatBALB/cmicearesusceptibletostool-derivedgt4HEVbutnottogt3HEV. C57BL/6MiceAreInsensitivetogt4HEV ThesusceptibilityofC57BL/6micetoHEVinfectionhasbeenrefutedinapreviousstudyinwhichtheanimalswereinoculatedwithstoolsuspensionsofgt1,gt3,andgt4HEVisolatedfromJapan(Lietal.,2008).Inthepresentstudy,wefurtheridentifiedtheinsensitivityofC57BL/6micetostool-derivedgt4HEVisolatedfromChina.NoHEVRNAwasdetectableinthefeces,blood,ortissues(Figures4A,B),andnoHEVantigenwasobservedinthereplicationsitesofHEVinspiteofgt3orgt4HEVinoculation(Figures4C,D).However,severeimmuneresponseswereobservedintheenlargedspleenwithobviousnecroticfociinC57BL/6miceinoculatedwithstool-derivedgt4HEV(SupplementaryFigure3A).ThisresultmayhavebeencausedbytheoveractiveimmunesysteminC57BL/6miceagainstviralinfectionthanBALB/cmice.ComparedwithBALB/cmice,moreseriousimmuneresponseswereevokedinC57BL/6micewithhigherincreasedCD45+leukocyteandF4/80+macrophageintheliver(Figures4E,F).Meanwhile,theexpressionofIFN-αandIFN-βinC57BL/6miceweresignificantlyexacerbatedthaninBALB/cmiceat7dpi(Figures4G,H). FIGURE4 Figure4.Profilesofgt3andgt4hepatitisEvirus(HEV)infectioninC57BL/6mice.HEVRNAwasdetectedinthestoolandserum(A)andtissues(B)ofC57BL/6miceinoculatedwithPBS,gt3HEV,orgt4HEV.LOQ,limitofquantification.HEVantigenwasdetectedintheliver,spleen,kidneys,intestines,brain,anduterusofC57BL/6miceviaimmunohistochemicalmethod(×200;C)andimmunofluorescenceanalysis(×200;D).ThenucleiwerestainedwithDAPI(blue).Immunofluorescentstainingforleukocyte(CD45+,green,E)ormacrophages(F4/80+,green,F)wasperformedintheliverofuninfected(PBS)orgt4HEV-infectedC57BL/6mice,×400.ThegeneexpressionofIFN-α(G)andIFN-β(H)wasquantifiedbyqRT-PCRinthebloodofgt4HEV-infected/inoculatedBALB/cmiceorC57BL/6miceat7dpi.LOQofstoolandserumis1.7×103copies/gorcopies/ml,andLOQoftissuesis2.1×103copies/g. Moderatehistopathologicalchangeswereobservedintheliverwithincreasedlymphocytesandmacrophages,butfocalnecrosiswasfoundinthespleenofC57BL/6miceinoculatedwithgt4HEV(SupplementaryFigure3B).ALTandASTactivitiesshowedatemporalincreaseinthe1stweekofgt3orgt4HEVinoculationpossiblybecauseofarejectionreactionagainstforeignsubstances,buttheactivitiesrecoveredsubsequently(SupplementaryFigures3C,D).ThepowerfulinnateantiviralimmunityfrustratedHEVinfectioninC57BL/6mice;therefore,humoralresponseagainstHEVwasnotprovoked(SupplementaryFigures3E,F).OurresultsconfirmedagainthatC57BL/6miceareinsensitivetogt3andgt4HEVinfection. SuccessfulEstablishmentofChronicInfectionofgt4HEVinBALB/cMice CasesofchronicHEVinfectionarerecentlyincreasing.However,theprogressofHEVchronicityisunclear.Humanliverchimericmicehavebeenrecentlyusedasamodelforstudyinggt3chronicHEVinfections(Allweissetal.,2016).TheyareusefulanimalmodelsfortheinvivostudyofHEVinfectionanddrugevaluation.However,theseimmune-deficienthumanizedmice,whichlackBandTlymphocytesandNKcells,arefragileandexpensive. Giventhatwehadsuccessfullyestablishedacutegt4HEVinfectioninbothimmunocompromisedandimmunocompetentBALB/cmice,weevaluatedthepossibilityofchronicHEVinfectioninBALB/cmousemodelbyusingarhesusmacaque-adaptedgt4chronic-mutatedHEVstrain(macKM01,stoolsamplefromanHEV-persistent-infectedrhesusmacaque;fordetails,seeourpreviousstudy;Yuetal.,2016).TheinfectivityofmacKM01strainhasbeenfurtheridentifiedinanotherrhesusmacaque(Yuetal.,2016).HEVRNAwaspersistentlypositive,lastingfor32weeks(endoftheexperiment)inthestoolandserumsamplesofregularBALB/cmice(Figure5A).HEVwasefficientlyreplicatedinthefirst14weeks,andviraltitersinstoolsampleswerepersistentlyincreasedto3.0×107copies/mlat8–12wpiandslowlydecreasedat22wpibutwerestilldetectableat32wpi(endoftheexperiment,Figure5A).Althoughviraltiterswererelativelylowerinthebloodthaninthestoolsamples,HEVRNAwasstillpersistentlydetectableafter32weeks(Figure5A).HEVRNAwasdetectedintheliver,spleen,kidneys,intestines,brain,anduterusofBALB/cmiceat32wpi(Figure5B).HEVantigenswereclearlyobservedintheseHEVreplicationsites,includingtheliver,spleen,kidneys,intestines,brain,anduterus,at32wpi(Figure5C).BALB/cmiceinoculatedwithchronicgt4HEVstrain(macHEV)exhibitedseverehistopathologicaldamagesintheliver(infiltratinglymphocytes),enlargedspleen(infiltratinglymphocytesandmacrophages),disturbedrenaltubularfunction,andglomerulitis(Figure5D).ExpandedportaltractandproliferativefibrosiswereobservedintheliverofBALB/cmiceinfectedwiththestoolofchronic-infectedrhesusmacaque(macHEV)at32wpi(Figure5E)byusingMassonstaining.SevereimmuneresponseswereevokedwithobviouslyincreasedCD45+leukocyteandF4/80+macrophageintheliverofBALB/cmiceinoculatedwithchronicgt4HEV(macHEV)(Figures5F,G). FIGURE5 Figure5.Profilesofgt4HEV-persistentinfectioninBALB/cmice.HEVRNAwasdetectedinthestoolandserum(A)andtissues(B)ofBALB/cmiceinoculatedwiththestoolsupernatantfromachronicinfectedrhesusmacaque(macKM01).LOQ,limitofquantification.HEVantigenwasdetectedintheliver,spleen,kidneys,intestines,brain,anduterusofBALB/cmicebyimmunohistochemicalmethod(×200;C)inBALB/cmiceinoculatedwithmacKM01,×200.Histopathologicalanalysiswasperformedintheliver,spleen,andkidneysofHEVpersistentinfectedBALB/cmice(H&E),×200(D).LiverfibrosiswasevaluatedbyMassonstaining(E),×200.Immunofluorescentstainingforleukocyte(CD45+,green,F)ormacrophages(F4/80+,green,G)wasperformedintheliverofuninfected(PBS)ormacKM01-infectedBALB/cmice,×400.LOQofstoolis2.8×103copies/g,LOQofserumis1.8×103copies/ml,andLOQoftissuesis2.0×103copies/g. Anti-HEVDrugTreatment Giventhatgt4HEVinfectioninBALB/cmicehadbeensuccessfullyestablished,weusedthissmall-animalmodeltoevaluatetheanti-HEVeffectsofRBV,IFN-α,orSOF.Theviruseswerenotcompletelyclearedinbothstoolandserumsamplesofgt4HEV-infectedmicewitheithermonotherapyofRBV,IFN-α,SOF,orcombinationtherapyfor1week(Figure6).AlthoughRBVmonotherapysuccessfullytreatedsomecasesofgt3HEVchronicinfection(Kamaretal.,2014;Allweissetal.,2016),itfailedtotreatgt4HEV(Figure6).Unsuccessfulresultswerealsoreportedinapatientwithgt4HEVchronicinfectionwhounderwentlivertransplantandtreatedwiththeaforementioneddrugs(Wuetal.,2017)possiblybecauseofthemutationofG1634RinthepolymeraseregionofORF1,anidentifiedsingle-nucleotidevariationresistanttoRBVtreatment.Althoughgt1andgt3HEVinfectionswereclearedfromtheliverandfecesofhumanizedmiceafterpegIFNαinjection,relapsewasidentifiedinhalfoftreatedanimals(vandeGardeetal.,2017).Itwasnotable;viralreplicationwasunaffectedingt4HEV-infectedmicewhentreatedwitheitherRBV/IFN-αmonotherapyorcombinationtherapy(Figures6A–F).SOFisanefficientantiviraldrugforHCVandwasoncerecognizedasapotentialanti-HEVdrugcandidate(Thietal.,2015).However,gt4HEV-infectedmicewereinsensitivetoSOFmonotherapyorincombinationwithRBV(Figures6G–J),consistentwiththeclinicalfailureinpatientswithgt3HEVinfection(HEV/HCVco-infected)whounderwentlivertransplant(WangandPan,2016;Donnellyetal.,2017).Long-termanti-HEVtreatmentwithRBV,IFN-α,orSOFreportedlyonhostimmunesystemsleadstoseveresideeffects,suchaselevatedALT,AST,orT-Bil.However,short-termtreatmentinHEV-infectedmicedidnotaggravateliverdamages(SupplementaryFigure4). FIGURE6 Figure6.Anti-HEVdrugtreatmentofgt4HEV-infectedBALB/cmice.HEVRNAcopynumberwasquantifiedbyqRT-PCRinthestool(A,C,E,G,I)andserum(B,D,F,H,J)ingt4HEV-infectedBALB/cmicetreatedwithanti-HEVdrugs,includingIFN-α,RBV,andSOFmonotherapyorcombination.LOQofstoolis1.7×103copies/g,LOQofserumis1.8×103copies/ml. Discussion HepatitisEvirusisresponsibleforapproximately20millioninfectionsannuallyworldwide.However,animalmodelsforinvivostudiesofHEVinfectionandpathogenesisarelimited.Therefore,developingeffectiveexperimentalmodelsforunderstandingHEVbiology,pathogenesis,andanti-HEVdrugdevelopmentisimportant.AlthoughseveralcellculturesystemsofHEVhavebeenestablished(Okamoto,2011;Meisteretal.,2019),small-animalmodelsareurgentlyneededforstudyingHEVpathogenicityandantiviraldrugdevelopment. Non-humanprimateswereusedintheearlyyearsofHEVresearch(Pandaetal.,2000;Aggarwaletal.,2001).However,theyarenolongeremployedbecauseofethicalconsiderations.Swineisunsuitableasanexperimentalmodelbecauseofitslargebody.Bycontrast,micearethemostcommonlyusedanimalsforresearchpurposes.Asanewmodel,humanliverchimericmicehavebeensuccessfullyestablishedforgt1andgt3HEVinfectionstudies(Allweissetal.,2016;vandeGardeetal.,2016).However,gt4HEVisdifferentfromgt1orgt3HEV;theclinicalpresentationsaremoresevereingt4HEVthaningt3HEV(Jeblaouietal.,2013).Furthermore,humanliverchimericmiceareexpensive,fragile,andinfertile.Thus,establishingaregularornormalmice-basedgt4HEVmodelisimportantforstudyingHEVinfectionandpathogenesis. Wehavesuccessfullyestablishedagt4swineHEV(isolatedfromShanghai,China)mousemodelbyusingBALB/cnudemice(Huangetal.,2009).TheimmunodeficientnatureofnudemiceprovidesanadvantageforinvestigatingHEVinfectioninimmunodeficientpatients,suchasorgan-transplantrecipientsorpatientswithHIVinfection.Inthisstudy,wesuccessfullyinfectedBALB/cnudemicewithgt4stool-derivedswineHEVstrain(KM01)isolatedfromYunnanProvince,China(Figure2andSupplementaryFigure1).However,mostHEV-infectedpatientsareimmunocompetent.Thus,regularBALB/cmice,whichhaveacompetentimmunesystem,arethemostsuitableanimalmodelsforexploringHEVpathogenesis. Inthepresentstudy,only9of20HEVstrainswereinfectiousinBALB/cmice.ThediversityofviralgenomemayhavecontributedtotheinfectivityinBALB/cmicebecausesomeuniqueaminoacidsinORF2werefoundinthesestrains,resultinginefficientinfection(SupplementaryFigure5).However,furtherexperiments,suchasmetagenomicsequencingtoidentifypotentiallycriticalpolymorphismswithinthesevirusgenomesandconstructionofinfectiouscDNAclonesbyusingreversegeneticstoidentifythesecriticalmutations,shouldbeconducted.BALB/cmiceweresuccessfullyinfectedwithgt4HEV(KM01strain).HEVRNAwasdetectedinfecesstartingfrom3to7dpi,similartoourgt4swineHEVstraininoculatedinBALB/cnudemice(Huangetal.,2009)butearlierthanthatinoculatedinpigswithgt4swineRNAtranscripts(7dpi)(Cordobaetal.,2012)andinSDratinoculatedwithgt4swineHEV(Zhuetal.,2013).HEVRNAwasdetectableintheHEV-replicatedsites,includingtheliver,spleen,kidneys,intestines,brain,anduterus,andthisresultstablyconfirmedtheextrahepaticreplicationsitesofHEV.HEVreplicationhasrecentlybeenidentifiedinthekidneys(Gengetal.,2015;Pischkeetal.,2017),brain(Salimetal.,2017;Zhouetal.,2017),andreproductiveorgans(Soomroetal.,2017;Huangetal.,2018)ofpatientsinfectedwithHEV.HEVRNAandantigenswerealldetectableinthesereplicationsitesandwillthusfacilitatestudiesonHEVpathogenesisandtissuetropism. Bycontrast,gt3HEVisdullinmicenotonlyinimmunocompromisedSCID(Allweissetal.,2016)orBALB/cnudemicebutalsoinimmunocompetentBALB/corC57BL/6mice.ThecapsidproteinisthemajorcomponentofHEVvirionstorecognizehostreceptor(s).Thedistinctdifferencesincapsidproteinbetweengt3andgt4HEVmaycontributetothesusceptibilityinBALB/cmice.Moregt3HEVstrainsshouldbeassessedinBALB/cmicetoevaluatetheinfectivity.Inaddition,gt3isprevalentindevelopedcountries,whereasgt4ismainlyreportedinAsiaandbelievedtobemorepathogenic(Jeblaouietal.,2013).Consistentwithpreviousreports,C57BL/6micewerenotpermissiveforbothgt3andgt4HEVinfection.Stronghostinnateimmunesystemorspecifichostfactorexpressionmayhavecontributedtothefailureofinfection. ChronicHEVinfectionisincreasinglybeingreportedinimmunosuppressedpatientswithHIVinfection,hematologicalmalignancy,andorgan-transplantrecipients.CirrhosisandliverfailurepostchronicHEVhavebeenreported(Kamaretal.,2014;Barragueetal.,2017).However,themechanismofchronicHEVinfectionremainsunclear.Immunosuppressionmaybethemaincauseofpersistentinfection,andviralmutationalsocannotbedismissed.Wesuccessfullyestablishedamacaque-adaptedHEVchronicinfectionmousemodelwithliverfibrosisandpersistentsheddingofvirusinserumandstoolfor32wpi(8months).ThismodelwillexpandourknowledgeofHEVchronicinfection.However,norobustcellculturesystemforlarge-scalepropagationofHEVhasbeenestablished,limitingin-depthresearchonHEV.Wefoundamacaque-adaptedHEVstrainthatcanpersistentlyinfectrhesusmacaquesandBALB/cmice.EstablishinganewandpromisingcellculturesystemforHEVresearchisnowpossiblebecauseastableHEV-infectedanimalmodelhasbeenachieved. Hitherto,nospecifictreatmentforHEVhasbeenapproved.AlthoughRBV,whichisanoff-labeldrug,ispromisingbecauseitcaninhibitHEVreplicationbydepletingintracellularGTPpool,G1634RmutationisreportedlyassociatedwithresistanceofRBVtherapyinacuteandchronichepatitisEpatients(DaltonandKamar,2016).Thus,anewspecificantiviraltherapyforcuringchronicHEVinfectionmustbedeveloped.Inthepresentstudy,RBV,IFN-α,SOFmonotherapy,orcombinationtherapywasusedtotreatgt4HEVinfectioninthemousemodel.Noneoftheseantiviraldrugsworkedsatisfactorily.Short-term,low-dosetreatmentofantiviraldrugsmayberesponsibleforthenegativity.Highercompoundconcentrationandlongertreatmentshouldbeperformedinthefurtherstudy.Thus,themousemodelshouldbeusedtodevelopsafeandeffectivetreatmentmodalitiesforHEVinfection. Conclusion Wesuccessfullyestablishedamousemodelforacuteandchronicgt4HEVinfectioninimmunocompromisedBALB/cnudeandimmunocompetentBALB/cmice.Althoughonly50%ofnaturalgt4HEVstrainscouldestablishsuccessfulinfectioninBALB/cmice,BALB/cmiceisapotentialmodelsystemforstudyingacuteandchronicgt4HEVinfection.Thissystemcanbeusedtostudythepathogenesisofgt4HEVanddevelopanti-HEVdrugs. DataAvailabilityStatement Alldatasetsgeneratedforthisstudyareincludedinthearticle/SupplementaryMaterial. EthicsStatement TheanimalstudywasreviewedandapprovedbytheAnimalCareandUseCommitteeofKunmingUniversityofScienceandTechnology. AuthorContributions YL,FL,CY,andXHperformedtheexperiment.JW,JS,SC,andZQcontributedtokeeptheanimalsandsamplescollection.FHandWYdesignedandwrotethemanuscript.Allauthorscontributedtotheanalysisandinterpretationofdata. Funding ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(81660338)toFH,theNaturalScienceFoundationofYunnanProvince(2017FA036toFHand2018FB132toWY),theChineseAcademyofMedicalSciencesInnovationFundforMedicalSciences(2019-I2M-1-004),KunmingScienceandTechnologyProject(2020-1-N-036),andPUMCYouthFund(3332019008)toWY. ConflictofInterest Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. Acknowledgments WewouldliketothankDr.ZhenLifromShanghaiAcademyofAgriculturalSciencesforgenerouslyprovidingtheJDY5strainandJiahongLifromKunmingUniversityofScienceandTechnologyforconductingthehistologicalanalysis. SupplementaryMaterial TheSupplementaryMaterialforthisarticlecanbefoundonlineat:https://www.frontiersin.org/articles/10.3389/fmicb.2020.01156/full#supplementary-material References Abravanel,F.,Lhomme,S.,Chapuy-Regaud,S.,Mansuy,J.M.,Muscari,F.,Sallusto,F.,etal.(2014).HepatitisEvirusreinfectionsinsolid-organ-transplantrecipientscanevolveintochronicinfections.J.InfectDis.209,1900–1906.doi:10.1093/infdis/jiu032 PubMedAbstract|CrossRefFullText|GoogleScholar Aggarwal,R.(2011).ClinicalpresentationofHepatitisE.VirusRes.161,15–22.doi:10.1016/j.virusres.2011.03.017 PubMedAbstract|CrossRefFullText|GoogleScholar Aggarwal,R.,andJameel,S.(2011).HepatitisE.Hepatology54,2218–2226. GoogleScholar Aggarwal,R.,Kamili,S.,Spelbring,J.,andKrawczynski,K.(2001).ExperimentalstudiesonsubclinicalHepatitisEvirusinfectionincynomolgusmacaques.J.Infect.Dis.184,1380–1385.doi:10.1086/324376 PubMedAbstract|CrossRefFullText|GoogleScholar Allweiss,L.,Gass,S.,Giersch,K.,Groth,A.,Kah,J.,Volz,T.,etal.(2016).HumanliverchimericmiceasanewmodelofchronicHepatitisEvirusinfectionandpreclinicaldrugevaluation.J.Hepatol.64,1033–1040.doi:10.1016/j.jhep.2016.01.011 PubMedAbstract|CrossRefFullText|GoogleScholar Barrague,H.,Condat,B.,Petitdidier,N.,Champagne,E.,Renou,C.,Izopet,J.,etal.(2017).ChronicHepatitisEvirusinfectioninacirrhoticpatient:acasereport.Medicine96:e7915.doi:10.1097/md.0000000000007915 PubMedAbstract|CrossRefFullText|GoogleScholar Behrendt,P.,Steinmann,E.,Manns,M.P.,andWedemeyer,H.(2014).TheimpactofHepatitisEinthelivertransplantsetting.J.Hepatol.61,1418–1429.doi:10.1016/j.jhep.2014.08.047 PubMedAbstract|CrossRefFullText|GoogleScholar Cordoba,L.,Feagins,A.R.,Opriessnig,T.,Cossaboom,C.M.,Dryman,B.A.,Huang,Y.W.,etal.(2012).Rescueofagenotype4humanHepatitisEvirusfromclonedcDNAandcharacterizationofintergenotypicchimericvirusesinculturedhumanlivercellsandinpigs.J.Gen.Virol.93,2183–2194.doi:10.1099/vir.0.043711-0 PubMedAbstract|CrossRefFullText|GoogleScholar Dalton,H.R.,andKamar,N.(2016).TreatmentofHepatitisEvirus.Curr.Opin.Infect.Dis.29,639–644. GoogleScholar Debing,Y.,Emerson,S.U.,Wang,Y.J.,Pan,Q.,Balzarini,J.,Dallmeier,K.,etal.(2014a).RibavirinInhibitsinvitroHepatitisEvirusreplicationthroughdepletionofcellularGTPpoolsandismoderatelysynergisticwithalphainterferon.Antimicrob.AgentsChemother.58,267–273.doi:10.1128/aac.01795-13 PubMedAbstract|CrossRefFullText|GoogleScholar Debing,Y.,Gisa,A.,Dallmeier,K.,Pischke,S.,Bremer,B.,Manns,M.,etal.(2014b).AmutationintheHepatitisEvirusRNApolymerasepromotesitsreplicationandassociateswithribavirintreatmentfailureinorgantransplantrecipients.Gastroenterology147,1008–1011. GoogleScholar Debing,Y.,Ramiere,C.,Dallmeier,K.,Piorkowski,G.,Trabaud,M.A.,Lebosse,F.,etal.(2016).HepatitisEvirusmutationsassociatedwithribavirintreatmentfailureresultinalteredviralfitnessandribavirinsensitivity.J.Hepatol.65,499–508.doi:10.1016/j.jhep.2016.05.002 PubMedAbstract|CrossRefFullText|GoogleScholar Donnelly,M.C.,Imlach,S.N.,Abravanel,F.,Ramalingam,S.,Johannessen,I.,andPetrik,J.(2017).Sofosbuviranddaclatasviranti-viraltherapyfailstoclearHEVviremiaandrestorereactiveTCellsinaHEV/HCVco-infectedlivertransplantrecipient.Gastroenterology152,300–301.doi:10.1053/j.gastro.2016.05.060 PubMedAbstract|CrossRefFullText|GoogleScholar Geng,Y.,Zhao,C.,Huang,W.,Harrison,T.J.,Zhang,H.,Geng,K.,etal.(2015).DetectionandassessmentofinfectivityofHepatitisEvirusinurine.J.Hepatol64,37–43.doi:10.1016/j.jhep.2015.08.034 PubMedAbstract|CrossRefFullText|GoogleScholar Huang,F.,Li,Y.,Yu,W.,Jing,S.,Wang,J.,Long,F.,etal.(2016a).ExcretionofinfectiousHepatitisEvirusintomilkincowsimposeshighrisksofzoonosis.Hepatology.64,350–359.doi:10.1002/hep.28668 PubMedAbstract|CrossRefFullText|GoogleScholar Huang,F.,Yang,C.C.,Zhou,X.Y.,Yu,W.,andPan,Q.(2016b).RhesusmacaquespersistentlyinfectedwithHepatitisEshedvirusintourine.J.Hepatol.64,1446–1447.doi:10.1016/j.jhep.2015.12.026 PubMedAbstract|CrossRefFullText|GoogleScholar Huang,F.,Long,F.,Yu,W.,Situ,J.,Fu,L.,He,Z.,etal.(2018).HighprevalenceofHepatitisEvirusinsemenofinfertilemaleandcausestestisdamage.Gut67,1199–1201.doi:10.1136/gutjnl-2017-314884 PubMedAbstract|CrossRefFullText|GoogleScholar Huang,F.,Zhang,W.,Gong,G.,Yuan,C.L.,Yan,Y.J.,Yang,S.X.,etal.(2009).ExperimentalinfectionofBalb/cnudemicewithHepatitisEvirus.BMCInfect.Dis.9:93.doi:10.1186/1471-2334-9-93 PubMedAbstract|CrossRefFullText|GoogleScholar Huang,F.F.,Haqshenas,G.,Guenette,D.K.,Halbur,P.G.,Schommer,S.K.,Pierson,F.W.,etal.(2002).Detectionbyreversetranscription-PCRandgeneticcharacterizationoffieldisolatesofswineHepatitisEvirusfrompigsindifferentgeographicregionsoftheUnitedStates.J.Clin.Microbiol.40,1326–1332.doi:10.1128/jcm.40.4.1326-1332.2002 PubMedAbstract|CrossRefFullText|GoogleScholar Jeblaoui,A.,Haim-Boukobza,S.,Marchadier,E.,Mokhtari,C.,andRoque-Afonso,A.M.(2013).Genotype4HepatitisEvirusinfrance:anautochthonousinfectionwithamoreseverepresentation.Clin.Infect.Dis.57,E122–E126. GoogleScholar Kamar,N.,Izopet,J.,Tripon,S.,Bismuth,M.,Hillaire,S.,Dumortier,J.,etal.(2014).RibavirinforchronicHepatitisEvirusinfectionintransplantrecipients.N.Engl.J.Med.370,1111–1120. GoogleScholar Kamar,N.,Selves,J.,Mansuy,J.M.,Ouezzani,L.,Peron,J.M.,Guitard,J.,etal.(2008).HepatitisEvirusandchronicHepatitisinorgan-transplantrecipients.N.Engl.J.Med.358,811–817. GoogleScholar Li,T.C.S.Y.,Ami,Y.,Tsunemitsu,H.,Miyamura,T.,andTakeda,N.(2008).MicearenotsusceptibletoHepatitisEvirusinfection.J.Vet.Med.Sci.70,1359–1362. GoogleScholar Li,W.,Sun,Q.,She,R.,Wang,D.,Duan,X.,Yin,J.,etal.(2009).ExperimentalinfectionofMongoliangerbilsbyagenotype4strainofswineHepatitisEvirus.J.Med.Virol.81,1591–1596.doi:10.1002/jmv.21573 PubMedAbstract|CrossRefFullText|GoogleScholar Meister,T.L.,Bruening,J.,Todt,D.,andSteinmann,E.(2019).CellculturesystemsforthestudyofHepatitisEvirus.AntiviralRes.163,34–49.doi:10.1016/j.antiviral.2019.01.007 PubMedAbstract|CrossRefFullText|GoogleScholar Nanda,S.K.,Panda,S.K.,Durgapal,H.,andJameel,S.(1994).DetectionofthenegativestrandofHepatitisEvirusRNAintheliversofexperimentallyinfectedrhesusmonkeys:evidenceforviralreplication.J.Med.Virol.42,237–240.doi:10.1002/jmv.1890420306 PubMedAbstract|CrossRefFullText|GoogleScholar Okamoto,H.(2011).HepatitisEviruscellculturemodels.VirusRes.161,65–77.doi:10.1016/j.virusres.2011.01.015 PubMedAbstract|CrossRefFullText|GoogleScholar Panda,S.K.,Ansari,I.H.,Durgapal,H.,Agrawal,S.,andJameel,S.(2000).Theinvitro-synthesizedRNAfromacDNAcloneofHepatitisEvirusisinfectious.J.Virol.74,2430–2437.doi:10.1128/jvi.74.5.2430-2437.2000 PubMedAbstract|CrossRefFullText|GoogleScholar Perumpail,R.B.,Ahmed,A.,Higgins,J.P.,So,S.K.,Cochran,J.L.,Drobeniuc,J.,etal.(2015).FatalacceleratedcirrhosisafterimportedHEVgenotype4infection.Emerg.Infect.Dis.21,1679–1681.doi:10.3201/eid2109.150300 PubMedAbstract|CrossRefFullText|GoogleScholar Pischke,S.,Hartl,J.,Pas,S.D.,Lohse,A.W.,Jacobs,B.C.,VanderEijk,A.A.,etal.(2017).HepatitisEvirus:infectionbeyondtheliver?J.Hepatol.66,1082–1095. GoogleScholar Rein,D.B.,Stevens,G.A.,Theaker,J.,Wittenborn,J.S.,andWiersma,S.T.(2012).TheglobalburdenofHepatitisEvirusgenotypes1and2in2005.Hepatology55,988–997.doi:10.1002/hep.25505 PubMedAbstract|CrossRefFullText|GoogleScholar Rivero-Juarez,A.,Frias,M.,Rodriguez-Cano,D.,Cuenca-Lopez,F.,andRivero,A.(2016).IsolationofHepatitisEvirusfrombreastmilkduringacuteinfection.Clin.Infect.Dis.62:1464. GoogleScholar Salim,O.J.,Davidson,A.,Li,K.,Leach,J.P.,andHeath,C.(2017).BrainstemencephalitisandacutepolyneuropathyassociatedwithHepatitisEinfection.BMJCaseRep.2017:bcr2017220799.doi:10.1136/bcr-2017-220799 PubMedAbstract|CrossRefFullText|GoogleScholar Sayed,I.M.,Verhoye,L.,Cocquerel,L.,Abravanel,F.,Foquet,L.,Montpellier,C.,etal.(2016).StudyofHepatitisEvirusinfectionofgenotype1and3inmicewithhumanisedliver.Gut66,920–929.doi:10.1136/gutjnl-2015-311109 PubMedAbstract|CrossRefFullText|GoogleScholar Soomro,M.H.,Shi,R.,She,R.,Yang,Y.,Wang,T.,Wu,Q.,etal.(2017).MolecularandstructuralchangesrelatedtoHepatitisEvirus(HEV)antigenanditsexpressionintestisinducingapoptosisinMongoliangerbilmodel.J.Viral.Hepat.24,696–707.doi:10.1111/jvh.12690 PubMedAbstract|CrossRefFullText|GoogleScholar Thi,V.L.D.,Debing,Y.,Neyts,J.,Moradpour,D.,andGouttenoire,J.(2015).SofosbuvirinhibitsHepatitisEvirusreplicationinvitroandresultsinanadditiveeffectwhencombinedwithribavirin.J.Hepatol.62,S289–S290. GoogleScholar Todt,D.,Francois,C.,Anggakusuma,Nitsche,A.,Behrendt,P.,Suneetha,P.V.,etal.(2016a).AntiviralactivitiesofdifferentinterferontypesandsubtypesagainstHepatitisEvirusreplication.Antimicrob.AgentsChemother.60,2132–2139.doi:10.1128/aac.02427-15 PubMedAbstract|CrossRefFullText|GoogleScholar Todt,D.,Gisa,A.,Radonic,A.,Nitsche,A.,Behrendt,P.,Suneetha,P.V.,etal.(2016b).Invivoevidenceforribavirin-inducedmutagenesisoftheHepatitisEvirusgenome.Gut65,1733–1743.doi:10.1136/gutjnl-2015-311000 PubMedAbstract|CrossRefFullText|GoogleScholar vandeGarde,M.D.,Pas,S.D.,vanderNet,G.,deMan,R.A.,Osterhaus,A.D.,Haagmans,B.L.,etal.(2016).HepatitisEvirus(HEV)genotype3infectionofhumanliverchimericmiceasamodelforchronicHEVinfection.J.Virol.90,4394–4401.doi:10.1128/jvi.00114-16 PubMedAbstract|CrossRefFullText|GoogleScholar vandeGarde,M.D.B.,Pas,S.D.,vanOord,G.W.,Gama,L.,Choi,Y.,deMan,R.A.,etal.(2017).Interferon-alphatreatmentrapidlyclearsHepatitisEvirusinfectioninhumanizedmice.Sci.Rep.7:8267. GoogleScholar Wang,W.P.M.,andPan,Q.(2016).TargetingviralpolymerasefortreatingHepatitisEinfection:howfararewe?Gastroenterology.150:1690.doi:10.1053/j.gastro.2016.01.045 PubMedAbstract|CrossRefFullText|GoogleScholar Wang,W.S.,Hakim,M.S.,Nair,V.P.,deRuiter,P.E.,Huang,F.,Sprengers,D.,etal.(2016).DistinctantiviralpotencyofsofosbuviragainstHepatitisCandEviruses.Gastroenterology151,1251–1253.doi:10.1053/j.gastro.2016.09.061 PubMedAbstract|CrossRefFullText|GoogleScholar Wu,C.H.,Ho,C.M.,Tsai,J.H.,Sun,H.Y.,Hu,R.H.,Lee,P.H.,etal.(2017).Firstcasegenotype4HepatitisEinfectionafteralivertransplant.Exp.Clin.Trans.15,228–230. GoogleScholar Xu,L.,Wang,W.,Li,Y.,Zhou,X.,Yin,Y.,Wang,Y.,etal.(2017).RIG-Iisakeyantiviralinterferon-stimulatedgeneagainstHepatitisEvirusregardlessofinterferonproduction.Hepatology65,1823–1839.doi:10.1002/hep.29105 PubMedAbstract|CrossRefFullText|GoogleScholar Yu,W.H.,Yang,C.C.,Bi,Y.H.,Long,F.,Li,Y.,Wang,J.,etal.(2016).CharacterizationofHepatitisEvirusinfectionintreeshrew(Tupaiabelangerichinensis).BMCInfect.Dis.16:80.doi:10.1186/s12879-016-1418-1 PubMedAbstract|CrossRefFullText|GoogleScholar Zhou,X.H.F.,Xu,L.,Lin,Z.,deVrij,F.M.,Ayo-Martin,A.C.,vanderKroeg,M.,etal.(2017).Virusinfectsneuronsandbrains.J.Infect.Dis.13,379–393. GoogleScholar Zhu,Y.M.,Yu,X.M.,Zhang,Y.S.,Ni,Y.Y.,Si,F.S.,Yu,R.S.,etal.(2013).Infectivityofagenotype4HepatitisEviruscDNAclonebyintrahepaticinoculationoflaboratoryrats.Vet.Microbiol.166,405–411.doi:10.1016/j.vetmic.2013.06.021 PubMedAbstract|CrossRefFullText|GoogleScholar Keywords:hepatitisEvirus,genotype,BALB/cmice,infectivity,animalmodel Citation:LiY,LongF,YangC,HaoX,WuJ,SituJ,ChenS,QianZ,HuangFandYuW(2020)BALB/cMouseIsaPotentialAnimalModelSystemforStudyingAcuteandChronicGenotype4HepatitisEVirusInfection.Front.Microbiol.11:1156.doi:10.3389/fmicb.2020.01156 Received:09February2020;Accepted:06May2020;Published:16June2020. Editedby: ChunfuZheng,FujianMedicalUniversity,China Reviewedby: ShaoweiLi,XiamenUniversity,China PhilipMeuleman,GhentUniversity,Belgium Copyright©2020Li,Long,Yang,Hao,Wu,Situ,Chen,Qian,HuangandYu.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)andthecopyrightowner(s)arecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:FenHuang,[email protected];[email protected];WenhaiYu,[email protected] †Theseauthorshavecontributedequallytothiswork COMMENTARY ORIGINALARTICLE Peoplealsolookedat SuggestaResearchTopic>