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1、Chapter6ReverseGeneticsSystemfortheAvianCoronavirusInfectiousBronchitisVirusEricaBickerton,SarahM.Keep,andPaulBrittonAbstractWehavedevelopedareversegeneticssystemfortheaviancoronavirusinfectiousbronchitisvirus(IBV)inwhichafull-lengthcDNAcorrespondingtotheIBVgenomeisinsertedintothevacc
2、iniavirusgenomeunderthecontrolofaT7promotersequence.Vacciniavirusasavectorforthefull-lengthIBVcDNAhastheadvantagethatmodificationscanbeintroducedintotheIBVcDNAusinghomologousrecombination,amethodfrequentlyusedtoinsertanddeletesequencesfromthevacciniavirusgenome.Here,wedescribetheuseof
3、transientdominantselectionasamethodforintroducingmodificationsintotheIBVcDNAthathasbeensuccessfullyusedforthesubstitutionofspecificnucleotides,deletionofgenomicregions,andexchangeofcompletegenes.InfectiousrecombinantIBVsaregeneratedinsitufollowingthetransfectionofvacciniavirusDNA,cont
4、ainingthemodifiedIBVcDNA,intocellsinfectedwitharecombinantfowlpoxvirusexpressingT7DNA-dependantRNApolymerase.KeywordsTransientdominantselection(TDS),Vacciniavirus,Infectiousbronchitisvirus(IBV),Coronavirus,Avian,Reversegenetics,Nidovirus,Fowlpoxvirus,T7RNApolymerase1IntroductionTheCor
5、onaviridaeformpartoftheorderNidovirales,whichcom-prisestwosubfamilies,theCoronavirinaeandTorovirinae.Therearefourgeneraofcoronaviruses,alpha-,beta-,gamma-,anddelta-coronaviruses[1],whichweresonamedfortheirvisualresem-blancetothecoronaofthesuninnegativelystainedpreparations[2].Coronavi
6、rusesareenvelopedviruseswithasingle-strandedpositive-senseRNAgenomeof26–32kb,thelargestgenomesofallRNAvirusescurrentlyknown,thatreplicateinthecytoplasmofinfectedcells.Thegenomeassociateswiththenucleoprotein(N)formingahelicalnucleocapsidwithinthevirusparticles.Althoughcommonamongnegati
7、ve-senseRNAviruses,coronavirusesaretheonlypositive-senseRNAvirusestopossesshelicalnucleocapsids,whichareenclosedwithinlipidenvelopescontainingthespike(S)glycoprotein,membrane(M)protein,andenvelope(E)protein.DanielR.Perez(ed.),ReverseGeneticsofRNAViruses:MethodsandProtocols,MethodsinMo
8、lecul
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