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1、J.gen.Virol.(1981),56,451-455.PrintedinGreatBritain451Keywords:bovineentericcoronavirus/plaqueassayPlaqueAssayforTitrationofBovineEntericCoronavirus(Accepted27May1981)SUMMARYTheplaquingabilityoftwoisolatesofbovineentericcoronavirus(BECV)wasstudiedinHRTI8(humanrectaladeno
2、carcinoma)cellmonolayers.Bothisolateswereabletoinduceplaqueformationwithin2to3days;plaquesappearedasroundopalescentareaswhichremainedcolourlessafterneutralredorcrystalvioletstaining.Agoodcorrelationwasfoundbetweenthetitresasdeterminedeitherbycountingtheplaquesthatwerevis
3、ibletothenakedeyebeforeandafterneutralredstaining,orbyenumeratingfluorescenceorhaemadsorptionfoci.Bovineentericcoronaviruses(BECV)havebeenidentifiedasaetiologicalagentsofneonataldiarrhoeaincalves(Stairetal.,1972;Mebusetal.,1973;Doughri&Storz,1977;Bridgeretal.,1978a;Gouet
4、etal.,1978).Althoughdifferentisolateshavealreadybeenadaptedeithertoorganculture(Bridgeretal.,1978b)ortocellculture(Mebusetal.,1973;Bridgeretal.,1978a;Deaetal.,1980)verylittleisknownaboutstraindifferencesthatmaywellexist.Thisgapinourknowledgeisrelatedtotwomajorproblems:di
5、fficultiesinprimaryisolationofthevirus,andlackofpracticalandreliableprocedurestotitrateandplaque-purifyviruspopulations.NoadequateplaqueassayforBECVhasbeendescribed,althoughacytopathiceffectwasobservedinsomecontinuouscelllines(Deaetal.,1980;lnabaetal.,1976).SinceHRT18(hu
6、manrectaladenocarcinoma)cellshavebeenshowntobehighlysusceptibletoBECV,allowinggoodmultiplicationandimprovedyieldofvirus(Laporteetal.,1980),weusedthesecellstostudytheabilityoftwoFrenchisolatestoinduceplaqueformation.Thecurrentreportdescribestheoptimalconditionsforthisassa
7、y.HRT18cells(Tompkinsetal.,1974)weregrowninRPMI1640mediumsupplementedwith20%foetalbovineserum(FBS),100units/mlpenicillin,and0.1mg/mlstreptomycin(thegrowthmedium).TwoisolatesofBECV(F15andGll0)wereusedthroughoutthisstudy.Bothwereobtainedfromthefaecesofcalvessufferingfromac
8、utediarrhoea.IsolateF15waskindlysuppliedbyDrJ.Laporte.ThisviruswasinitiallyadaptedtoHRT18cellsandsubseq
