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1、VIROLOGY213,569–580(1995)ExperimentalEvidenceofRecombinationinCoronavirusInfectiousBronchitisVirus1SANNEKEA.KOTTIER,DAVIDCAVANAGH,andPAULBRITTONDivisionofMolecularBiology,InstituteforAnimalHealth,Compton,Newbury,Berkshire,RG207NN,UnitedKingdomReceivedAugust8,1995;acceptedSe
2、ptember6,1995Embryonatedeggswerecoinfectedwithtwostrainsofthecoronavirusavianinfectiousbronchitisvirus(IBV),IBV-BeaudetteandIBV-M41,toinvestigatewhetherrecombinationbetweenthetwostrainswouldoccur.VirionswereisolatedfromtheallantoicfluidofthecoinfectedeggsandputativehybridRN
3、Asweredetectedbypolymerasechainreaction(PCR),usingstrain-specificoligonucleotides.PCRproducts,oftheexpectedsizes,wereobtainedaspredictedfrompotentialrecombinationeventsbetweenthenucleoprotein(N)geneandthe3*-untranslatedregionofthetwoIBVgenomes.Sequenc-ingconfirmedthattheyco
4、rrespondedtohybridRNAs.VirusproducedasaresultofthemixedinfectionwastreatedwithanM41-specificneutralizingmonoclonalantibodyandpassagedinVerocells,inwhichIBV-Beaudette,butnotIBV-M41,replicated.HybridRNAwasstilldetectableafterthreeserialpassages.SincenoIBV-M41wasdetectablethis
5、confirmedthatinfectiousrecombinantgenomeshadbeenproducedintheembryonatedeggs.Thesefindingsnotonlysupportthecircumstantialevidence,fromsequencingstudiesofIBVfieldstrains,thatrecombinationoccursduringreplicationofIBVandcontributestothediversityofIBV,butalsoshowthatcoronavirus
6、RNArecombinationisnotlimitedtomousehepatitisvirus.q1995AcademicPress,Inc.INTRODUCTIONArecombinationmap,usingtemperature-sensitiveMHVmutants,revealedthefrequencyofrecombinationtobeInfectiousbronchitisvirus(IBV)isamemberoftheapproximately1%per1300nucleotides.AssumingthatCoron
7、aviridae(forreviewsseeLai,1990;Cavanaghetrecombinationoccursrandomly,thefrequencyofrecom-al.,1994)andcontainsasingle-strandedpositive-sensebinationintheentiregenomewasestimatedtobeRNAgenomeof28kb(Boursnelletal.,1987).Itisaaround25%(Baricetal.,1990).highlyinfectiousandcontag
8、iouspathogenofchickensAlthoughseveralsequencingstudiesonfieldisolateswhichreplicat
