冠状病毒Coronavirusl论文-2016 [Springer Protocols Handbooks] Animal Coronaviruses __ Reverse Genetics of Avian Coronavirus Infectious Bronchitis.pdf

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1、Chapter6ReverseGeneticsofAvianCoronavirusInfectiousBronchitisVirusSarahM.Keep,EricaBickerton,andPaulBrittonAbstractWehavedevelopedareversegeneticssystemfortheaviancoronavirusinfectiousbronchitisvirus(IBV)inwhichafull-lengthcDNAcorrespondingtotheIBVgenomeisinsertedintothevacciniavirusgenomeunderth

2、econtrolofaT7promotersequence.Vacciniavirusasavectorforthefull-lengthIBVcDNAhastheadvantagethatmodiÞcationscanbeintroducedintotheIBVcDNAusinghomologousrecombina-tion,amethodfrequentlyusedtoinsertanddeletesequencesfromthevacciniavirusgenome.Here,wedescribetheuseoftransientdominantselectionasametho

3、dforintroducingmodiÞcationsintotheIBVcDNA;thathasbeensuccessfullyusedforthesubstitutionofspeciÞcnucleotides,deletionofgenomicregions,andtheexchangeofcompletegenes.InfectiousrecombinantIBVsaregeneratedinsitufollowingthetransfectionofvacciniavirusDNA,containingthemodiÞedIBVcDNA,intocellsinfectedwit

4、harecombinantfowlpoxvirusexpressingT7DNAdependantRNApolymerase.KeywordsTransientdominantselection(TDS),Vacciniavirus,Infectiousbronchitisvirus(IBV),Coronavirus,Avian,Reversegenetics,Nidovirus,Fowlpoxvirus,T7RNApolymerase1IntroductionAvianinfectiousbronchitisvirus(IBV)isagammacoronavirusthatisthee

5、tiologicalagentofinfectiousbronchitis(IB);anacuteandhighcontagiousdiseaseofpoultrycharacterizedbynasaldischarge,snicking,trachealciliostasisandrales[1].IBVreplicatesprimarilyintherespiratorytractbutalsoinmanyotherepithelialsurfacesincludingoviducts,entericsurfacesandkidneys[2Ð5].Followinginfectio

6、nwithIBV,eggproductionandqualitymaybeimpairedinlayersandweightgaininbroilersisreduced[6].Infectedbirdsarepredisposedtosecondarybacterialinfectionssuchascolibaccilosisandmortalityinyoungchicksisnotuncommon.Fecalexcretionofthevirusisaconsequenceofreplicationintheintestinaltract;however,thisdoesnotn

7、ormallyresultinclinicaldisease.InfectiousbronchitiswasÞrstdescribedintheUSAinthe1930s[7Ð9]andisprevalentinpoultryfarmingacrosstheworldduetoLeyiWang(ed.),AnimalCoronaviruses,SpringerProtocolsHandbooks,DOI10.1007/978-1-4