冠狀病毒Coronavirusl論文-2017 [Methods in Molecular Biology] Reverse Genetics of RNA Viruses Volume 1602 __ Reverse Genetics System for the Avian.pdf

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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