冠状病毒Coronavirusl论文-2008 [Methods in Molecular Biology] SARS- and Other Coronaviruses Volume 454 __ Titration of Human Coronaviruses, HCoV-2.pdf

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1、8TitrationofHumanCoronaviruses,HCoV-229EandHCoV-OC43,byanIndirectImmunoperoxidaseAssayFrancineLambert,HélèneJacomy,GabrielMarceau,andPierreJ.TalbotAbstractCalculationofinfectiousviraltitersrepresentsabasicandessentialexperimentalapproachforvirologists.

2、Classicalplaqueassayscannotbeusedforvirusesthatdonotcausesignifi-cantcytopathiceffects,whichisthecaseforstrains229EandOC43ofhumancoronavirus(HCoV).Analternativeindirectimmunoperoxidaseassay(IPA)ishereindescribedforthedetectionandtitrationoftheseviruses

3、.Susceptiblecellsareinoculatedwithserialloga-rithmicdilutionsofsamplesina96-wellplate.Afterviralgrowth,viraldetectionbyIPAyieldstheinfectiousvirustiter,expressedasTissueCultureInfectiousDose(TCID50).Thisrepresentsthedilutionofavirus-containingsampleatw

4、hichhalfofaseriesoflabo-ratorywellscontainreplicatingvirus.ThistechniqueisareliablemethodforthetitrationofHCoVinbiologicalsamples(cells,tissues,orfluids).Keywords:humancoronavirus;HCoV-229E;HCoV-OC43;cellandtissuesamples;titration;immunoperoxidaseassay

5、;TCID501.IntroductionHCoVwerefirstisolatedinthemid-1960sfrompatientswithupperrespi-ratorytractdisease(13).AsdescribedbyMcIntosh(4),theidentificationofcoronavirusesinclinicalsampleswasaverydifficulttaskuntilthe1970s,asHCoVinducedsubtleornonexistentcytop

6、athiceffects,andmanycellstypeswerenotsusceptibletothevirus.Commonly,HCoV-OC43(ATCC:VR-759)From:MethodsinMolecularBiology,vol.454:SARS-andOtherCoronaviruses,Editedby:D.Cavanagh,DOI:10.1007/978-1-59745-181-98,CHumanaPress,NewYork,NY9394Lambertetal.wasgr

7、ownandmaintainedonBS-C-1(ATCC:CCL-26),RD(ATCC:CCL-136)orHRT-18(alsocalledHCT-8;ATCC:CCL-224)celllines.Ontheotherhand,HCoV-229E(ATCC:VR-740)wasgrownandmaintainedonWI-38(ATCC:CCL-75),MRC-5(ATCC:CCL-171)orL-132(ATCC:CCL-5)celllines.Inter-estingly,cellsfro

8、mthecentralnervoussystem(CNS)werereportedtobehighlysusceptibletoHCoVreplication.TheSK-N-SH(ATCC:HTB-11)neuroblas-tomaandH4(ATCC:HTB-148)neurogliomacelllineswerehighlysuscepti-bletoinfection,aswellasastrocytomacelllinesU-87MG(ATCC:HTB-14