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1、ArchivesofVirology50,339342(1976)~bySpringer-Verlag1976ReplicationofBovineCoronavirnsinCellLineBEK-ICultureBriefReportByY.INABA1,K.SATe1,H.KIJROOI1,E.TAKAHASHI:t,Y.ITO1,T.OMO~I1,Y.GOTO2,andM.MAT1JMOTO3NationalInstituteofAnimalHealth,Kodaira,Tokyo,Japan2NationalVeterinaryAssayLaboratory,Kokubun
2、ji,Tokyo,Japan8InstituteofMedicalScience,UniversityofTokyo,Minato-ku,Tokyo,JapanWith1FigureAcceptedDecember4,1975SunlmaryBovinecoronavirusreadilymultipliedandinducedmarkedcytopathiceffectinBEK-1cultures,thusprovidingasensitive,practicalassaysystemforviralinfectivityandneutralizingantibodyandas
3、atisfactorysourceofthevirus.g~1V[~iBusandhisassociates(4--6)havedemonstratedanagentwithmorphologicfeaturesofcoronavirusbyelectronmicroscopyinfractionspreparedbydensitygradientultracentrifugationfromdiarrhealfecesofcalveswithnaturalandexperi-mentallyproducedneonataldiarrhea.Theagentmultipliedbu
4、tfailedtoinducereadilyrecognizablecytopathiceffectinbovineembryonickidneycellcultures(3).Thevirushasbeenassayedthereforeintheseculturesbyarathercumbersomemethodinwhichthepresenceoftheviruswasdeterminedbymicroscopicexamina-tionforsyncytiaofculturesstainedwithgentianviolet,orbyimmunofluorescentt
5、echnique(3).Thispaperdescribesbrieflyourrecentobservationthatthevirusreadilyreplicatesandinducesamarkedcytopathiceffectinculturesofacontinuouscellline,BEK-1,derivedfrombovineembryonickidney(2),thusprovidingasensitive,practicalassaymethodandasatisfactorysourceofthevirus.BEK-1cellsweregrownat37°
6、CinEagle'sminimumessentialmedium(MEM)containing10percenttryptosephosphatebroth(Difco)(TPB),10percentbovineserum,10Ounits/m]penicillin,100~zg/mlstreptomycin,and1~xg/mlfungizone.Thebovinecorona~drus(3),atthe29thpassagelevelinbovineembryonickidneycells,waskindlysuppliedbyDr.C.A.Mebus,Universityof
7、Nebraska,andwaspassagedtwiceinprimarycalfkidneycellculturesinourlaboratorybeforeuseinthepresentstudy.BEK-1cellculturespreparedin10×110mmtubeswere340Y.INABAetal.:inoculatedwith0.1mlamountsofvirusdilution,andincubatedinarollerdrumat34°C.T
