冠状病毒Coronavirusl论文-1991 [Advances in Experimental Medicine and Biology] Coronaviruses and their Diseases Volume 276 __ Sequence Comparisons.pdf

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1、SEQUENCECOMPARISONSOFTHE3'ENDOFTHEGENOMESOFFIVESTRAINSOFAVIANINFECTIOUSBRONCHITISVIRUSEllenW.Collisson1,AnnaK.Williams1,RayVonderHaar2,WangLil,andLoydW.Sneed1lDepartmentofVeterinaryMicrobiology2DepartmentofBiologyTexasA&MUniversityCollegeStation,TXINTRODUCTIONAvianinfectiousbronchitisvirus(IBV)cause

2、sanacute,highlycontagiousrespiratorydiseaseofchickenscharacterizedbytrachealrales,coughingandsneezingl.Thediseasewasfirstdescribedin1931bySchalkandHawnlandsincethattime,manystrainshavebeendefined2,3.Thesestrainsvarywidelyinvirulenceandtissuetropism.Anumberofserologicallydistinguishablestrainsofinfec

3、tiousbronchitisvirushavebeenisolatedfrompoultryintheU.S.A.Ofthestrainsincludedinthisstudy,Beaudette,Conn.andArkDPI75arevaccinestrains,GrayandAustTareknowntobenephropathogeniccausinglimitedrespiratorydisease,andinfectionswithArk99,Mass41andaJapanesestrain(KB8523)aregenerallythoughttoresultinsevereres

4、piratorydiseaseintheabsenceofnephritisandnephrosis4.Inthisstudy,the3'endoftheArk99andGraystrainswerecomparedwiththepublisheddataforMass41andBeaudette5andaJapanesestrain,KB85236.Aninsertinthe3'noncodingregionofthegenomedescribedintheBeaudetteandJapanesestrains,butabsentintheMass41strain,wasalsofoundi

5、ntheArk99andGraystrains.MATERIALSANDMETHODSViralPreparationTheGrayandArk99strainsofIBVwerepurifiedinourlabbythreeterminaldilutioncyclesinembryonatingchickembryos(ECE)andwerepropagatedbyallantoicsacinoculationintoII-dayoldspecificpathogenfree(SPAFAS)ECE.Viruswasprecipitatedwithpolyethylene-glycolandb

6、andedona30-50%glycerol/potassiumtartrategradient.Afterconcentratingbyultracentrifugation,viruswasreconstitutedandthevirionsweredisruptedwithProteinaseKandSDS.TheRNAwasextractedinphenol/chloroform/isoamylandethanolprecipitated?CloningofTheGrayandArk99StrainsFirststrandcDNAsynthesiswascarriedoutbyreve

7、rsetranscriptaseusinganoligodTprimerandsecondstrandsynthesiswithDNAPollandRNaseH8.ThedoublestrandedcDNAwastailedwithdeoxyC'susingCoronavirusesandTheirDiseasesEditedbyD.CavanaghandT.D.K.Brown373PlenumP