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1、Coronaviruses:Propagation,UNIT15E.1Quanti?cation,Storage,andConstructionofRecombinantMouseHepatitisVirusJulianLeibowitz,1GiliKaufman,1andPinghuaLiu11TexasA&MCollegeofMedicine,CollegeStation,TexasABSTRACTThefocusofthisprotocolismousehepatitisvirus(MHV),withoccasionalrefe
2、rencestoothercoronaviruses.Manyoftheseprotocolscanbeeasilyadaptedtoothercoronaviruses.ProtocolsforpropagatingMHVinDBTand17CL-1cells;thestorageandtitrationofviralstocks;puri?cationofMHVonsucrosegradients;andthegenerationofrecombinantvirusesbyacDNAassemblymethodandbytarge
3、tedrecombinationwillbepresented.ProtocolsarealsoincludedforthepropagationofDBT,17CL-1,andL2cellsusedforgrowingandtitratingMHV,andforthegrowthofBHK-RcellsandFCWFcells.ThelattertwocelllinesareusedforregeneratinginfectiousMHVbyaninvitrocDNAassemblyprotocolandbyatargetedrec
4、ombinationprotocol,respectively,allowingreversegeneticmanipulationoftheseviruses.AnadditionalprotocolforthemaintenanceofthelargeplasmidsusedforgeneratingrecombinantMHVswillalsobepresented.Curr.Protoc.Microbiol.21:15E.1.1-15E.1.46.C2011byJohnWiley&Sons,Inc.Keywords:coro
5、navirusreversegeneticsplaqueassayviruspuri?cationtargetedrecombinationmousehepatitisvirusINTRODUCTIONThecoronavirusesencompassagroupofenvelopedRNAvirusesthatarewidespreadinnatureandinfectawidevarietyofanimalsaswellashumans,mostcommonlycausinggastrointestinalorrespi
6、ratoryillnessesintheinfectedhost,althoughmoresystemicinfectionscanalsooccur.Beforetheoutbreakofsevereacuterespiratorysyndrome(SARS)in2002to2003,themajorityofcoronavirusesthathadbeenisolatedwererecoveredfromhumansanddomesticatedanimalspecies,includinglaboratoryrodents,an
7、dwereregardedprimarilyasveterinarypathogens.Therodentcoronaviruses,suchasthevariousstrainsofMHV,providedrodentmodelsforvarioushumandiseases.ThedemonstrationthattheagentthatcausedSARSwasanovelcoronavirus,andthesubsequentinvestigationsoftheoutbreakthatindicatedthattheSARS
8、-coronavirus(SARS-CoV)almostcertainlyenteredthehumanpopulationthroughzoonoticspreadinwildanimalmarketsinChina,
