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1、CULTIVATIONTECHNIQUESFORANIMALCORONAVIRUSES:EMPHASISONFELINEINFECTIOUSPERITONITISVIRUS,CANINECORONAVIRUS,TRANSMISSIBLEGASTROENTERITISVIRUS,ANDPORCINEHEMAGGLUTINATINGENCEPHALOMYELITIS~RUSRogerD.WoodsandRonaldD.WesleyUSDA-ARS,NationaIAnimalDiseaseCenter,P.O.Box70,Am
2、es,Iowa50010SUMMARY:Techniquesaredescribedforthegrowthandcharacterizationofsomemammaliancoronaviruses.Becauseofthefastidiousnatureoftheirgrowthrequirements,mostwillreplicateonlyincellsderivedfromthenaturalhostoracloselyrelatedspecies.FetalcatceilsareusedtogrowFIPV
3、,andporcineceilsareusedtogrowTGEVandHEV.However,CCVwillreplicateinbothfelineandcaninecells.Althoughallfourofthesevirusesprefertoreplicateinacellinthestationaryphaseofgrowth,FIPVisabletoreplicateinanactivelygrowingcell.Eachviruscausesacytopathiceffectinmonolayercel
4、lculturesunderagarormedia18to72hpostinfection.Primaryisolationofeachvirusfromfieldspecimensisdifficult,althoughmostcanusuallybeisolatedafter1to3blindpassagesinthecellculture.Keywords:entericcelllines;isolation;growthrequirements;coronavirus.I.INTRODUCTIONMHVandCCV
5、arecapableofgrowingtohightitersinmorethanonecellline114).WithinthelastfewyearsTheCoronaviridaefamilyofviruseshasaworldwideseveralmembersofthecoronavirdaefamily(MHV,distribution.Thesevirusescauseeconomicallyim-IBV,TGEV)havebeenstudiedintensivelyandserveasportantdis
6、easesinmanandindomesticandlaboratorymodelsforthemolecularbiologyofthegroup(7,20,32).animals{33).Atthepresenttimethegroupconsistsof14recognizedvirusesand4ormoreunclassifiedisolates.ThevirusesandtheirabbreviationsarelistedinII.MATERIALSTable1.Thesevirusescomprisefou
7、rdistinctantigenicgroupsplusamiscellaneousgroup(35).GenerallytheseA.EquipmentvirusesinfectepithelialcellsoftherespiratorytractWaterjacketedincubator,model3158,Forma~[humancoronavirus(HCV),infectiousbronchitisvirusCentrifuge,modelUV,IEC2(IBV),ratcoronavirus(RCV),po
8、rcinerespiratoryLaminarflowhood,Labgardmodel,NuAire3coronavirus(PCV)]andepithelialcellsofthePipet-aid,tissueculturemodel,DrummondScientific4gastrointest
