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1、JournalofVirologicalMethods,43(1993)5344Q1993ElsevierSciencePublishersB.V.Allrightsreserved/0166-0934/93/$06.00JournalofVirologicalVIRMET01491MethodsDevelopmentandoptimizationofplaqueassaysforratcoronavirusesD.J.Gaertner,D.F.Winograd,S.R..Compton,F.X.PaturzoandA.L.SmithSectionofComparativeMedici

2、ne,YaleUniversitySchoolofMedicine,NewHaven,CT(USA)(AcceptedI6December1992)SummaryPlaqueassaysunderSephadexoragaroseoverlaysaredescribedforratcoronaviruses(RCVs)growninL2mousefibroblasts.AplaqueassayusingSephadexwassimple;however,viableplaquescouldnotbecollectedforpropagation,andfixationwasnecess

3、arybeforeevaluation.Plaqueformationunderagarosewasoptimizedusingdiethylaminoethyl-dextran(DEAE-D)inthepre-treatmentandabsorptionmediaandtrypsinaddedtotheabsorptionmediaandagaroseoverlay.TheuseofDEAE-Dalone,trypsinaloneortrypsincombinedwithDEAE-Dsignificantlyincreasedplaquenumbersandvisibility.Pl

4、aquenumberswerehighestwhenpre-treatmentmediacontainedDEAE-D,absorptionmediacontainedDEAE-Dandtrypsin,andtheagaroseoverlaycontainedtrypsin.Theassaywasusefulforplaqueisolationandquantificationofsialodacryoadenitisvirus(SDA),Parkersratcoronavirus(PRCV)andothercoronavirusisolatesfromratsanditsspecif

5、icitywasdemonstratedbyplaque-reductionneutralizationtesting.ThesemethodswillfacilitateproductionofclonedvirusstocksforstudyofRCVbiologyandvirusquantificationforinvitroandinvivostudiesofRCVs.Sialodacryoadenitisvirus;Ratcoronavirus;Plaqueassay;DEAE-D;Trypsin;Agarose;SephadexCorrespondenceto:D.J.Ga

6、ertner,SectionofComparativeMedicine,YaleUniversitySchoolofMedicine,333CedarSt.,P.O.Box3333,NewHaven,CT06510,USA.54IntroductionRatcoronaviruses(RCVs)infectlaboratoryratsathighprevalenceandinterferewithresearchusingrats(Jacoby,1986).Relativelylittleisknownaboutthebasicbiologyandbiochemistryofthese

7、viruses,largelyduetotheirfastidiousgrowthrequirementsinvitro.RecentimprovementsinmethodsforcultivationofRCVs,includingtheuseoftheL2mousetibroblastcellline(Percy,1989;Percy,1990)supplementedwithtrypsinanddiethylaminoethyl-dex