神经氨酸酶茎部氨基酸缺失对H5N1亚型禽流感病毒生物学特性

464200684ActaMicrobiologicaSinica46(4):5425464August2006:(05KJA23016)3Tel:[phone];E-mail:[e-mail]:(1980-),,,,H5N1E-mail:wangquzhi@1631com:2005-12-28;:2006-01-23;:2006-03-26H5N1,,,,3(225009):H5N1(AIV)(NA)1520,AIV,5NAH5N1PPR8AIV(HA),NA,NA20,5SPF,EID50MDT;NA,;NA1520MDCK,10100,610h,MDCKAIVNA,NA1520AIV,H5N1AIV5H5N1PPR8,AIVNAMDCK,:H5N1;;;:Q78:A:000126209(2006)0420542205,H5N1AIVNA,,,NA,,NA,N-,,,[1]NA[2][3]NA[4,5]APGoosePJiangsuP220P2003(H5N1)APGoosePHuadongP1P2000(H5N1)17H5N1AIVNAcDNA:APGPJSP03NA16460APGPHDP00APPRP8P34(H1N1)15[4]Hoffmann[6]8,,RNA,AB[7,8]8,H5N1AIV2HA,NAPR86PB2,PB1,PA,NP,M,NS,NA,COS-1H5N1PPR85,NAH5N1AIV111111111:APGoosePHuadongP1P2000(H5N1),APGoosePJiangsuP220P2003(H5N1)COS-1MDCK10%DMEMSPFSPF11112:RNASangonExpendHighFidelityPCRSystem,dNTPsRocheAgroseGelDNAPurificationKitTaKaRaM-MLVT4DNARNasin(RNA)PromegapCR210vectorInvitrogenBsmBFermantasPCR(Biometra),BIO-RAD3000Xi(Eppendorf)11288St.JudeRobertWebstercDNApHW2000,APPRP8P348cDNApHW191-PB2,pHW192-PB1,pHW193-PA,pHW194-HA,pHW195-NP,pHW196-NA,pHW197-M,pHW198-NS113DANStar410(EditSeqMegAlignMapDraw):DNAStarInc.Madison,WI153715,USA114RT-PCRAPGPJSP03RNAM-MLVcDNAHoffmann[8]HA,HApHW2000BsmB,DH5,PCR,pHW644-HA(644),,NA,pHW2000,,pHW646-NA(646)APGPHDP00NA,pHW506-NA(506),PCRAPGPHDP00NA60,MutantpHW506-NA(m506-)APGPJSP03NA60,MutantpHW646-NA(m646+),1155646,644PR8(646+644+PR8)m646+,644PR8(m646++644+PR8)506,644PR8(506+644+PR8)m506-,644PR8(m506-+644+PR8)644PR87(644+PR8)5COS-148h116,10SPF,012mL,2,35,,,(HA)(HI)[10]HA11000,SPF4,HA,[11]11711711(EID50):[12,13],10,10SPF,012mL,35,24h96h,116,Reed-MuenchEID5011712MDCK(PFU):[14],MDCK60mmdishes10,1mL375%CO2MDCK,45min,PBS(018%,2%DMEM)375%CO248h(018%,2%DMEM,011%)12h,PFU11713:[3]5HA1128,4,37,,,11714MDCK:MDCK,,,2211,,H5345:H5N1.P(2006)46(4)5506m506-646m646+19611Table1TheoriginofrecombinantsgenefragmentsVirusesAPGPHDP00APGPJSP03PR8506NAHAInternalgenesm506-NA(20aadeleted)HAInternalgenes646HA,NAInternalgenesm646+HA,NA(20aaadded)Internalgenes196HANA,Internalgenes21221211(EID50):5646,m646+,506,m506-196EID5010-71625P012mL,10-7P012mL,10-715P012mL,10-7.67P012mL10-8P012mL22(EID50)Table2EID50,MeanHAandMDTVirulenceindexRecombinant646m646+506m506-196EID50(P012mL)10-7162510-710-71510-7.6710-8MeanHA291332913291632919281375MDT(h)48471744461344181(A)(B)Fig.1Viruseselutionfromchicken(A)andguineapig(B)erythrocytes.21212:506m646+7215h,2015m506,64619624h7,(12A,B)21213MDCK:m506-,646196506m646+,610h;,10100(3)3MDCKPFUPmLTable3ThefirsttimeprogenyvirusparticlesdetectedandthehighestPFUPmLVirusesHoursafterinfectionHighestPFUPmL506(Long-Stalked)12105m506-(Short-Stalked)4107646(Short-Stalked)6107m646+(Long-Stalked)17106196(Short-Stalked)610721214-:5MDCKPFUPmL,646106,4107,MDCK506m646+,m506-,646196,0121mm(4)4MDCKPFUPmLTable4PFUPmLandplaquesizeofrecombinantsallantoicfluidsinMDCKcellsVirusesPFUPmLPlaqueSize(ave.)506(Long-Stalked)2115107pinpointm506-(Short-Stalked)515751070151mm646(Short-Stalked)718351060151mmm646+(Long-Stalked)114107pinpoint196(Short-Stalked)21727107012mm3NA40[2],NA1520,HA,445WANGQu-zhietal.PActaMicrobiologicaSinica(2006)46(4)--,,37,NA,[1]NA1520H5N11520NA,NA,,MDCK,HA,,,,,NA--[15],,,,MDCKMDCK(4),,NA,,NA,,,,MDCK,(3)5EID50MDT,(2),PFU,(4),NAMDCK(34),H5N1,NAH5N1AIV,H5N1AIV[5],NAAIVNA;5H5N1PPR8,;AIVNAMDCK,[1],,..:,2000,11[2]ElsMC,AirGM,MurtiKG,etal.An18-Aminoaciddeletioninaninfluenzaneuramindase.Virology,1989,142:241-2471[3]CastrucciMR,KaWaoKaY.BiologicimportanceofneuraminidasestalklengthininfluenzaAvirus.JournalofVirology,1993,67(2):759-7641[4]BlockJ,AirGM.BlockdeletionintheNeuraminidasegenesfrominfluenzaAvirusesoftheN1subtype.Virology,1982,118:229-2341[5]MatrosovichM,ZhouN,KawaokaY,etal.ThesurfaceglycoproteinsofH5influenzavirusesfromhumans,chickens,andwildaquaticbirdshavedistinguishableproperties.JournalofVirology,1999,73(2):1146-11551[6]HoffmannE,NeumannG,KawaokaY,etal.ADNAtransfectionsystemforgenerationofinfluenzaAviruseightplasmids.ProceNatlAcadeSciUSA,2000,97:6108-61331[7]HoffmannE,NeumannG,HobomG,etal.AmbisenseapproachforthegenerationofinfluenzaAviruse:vRNAandmRNAsynthesisfromonetemplate.Virology,2000,267:310-3171[8]HoffmannE,ZHOUN,WesterRG.Eight-plasmidrescuesystemforinfluenzaAvirus.InternationalCongressSeries,2001,1219:1007-10131[9]HoffmannE,StechJ,GuanY,etal.Universalprimersetforthefull-lengthamplificationofallinfluenzaAviruses.ArchVirol,2001,146(12):2275-22891[10]...:,20021[11],,,.8H9N2PWSNA.,2005,21(1):48-531[12],...:,19971[13],,,..:,19841[14]WagnerR,WolffT,HerwigA,etal.IndependenceofHemagglutininglycosylationandNeuraminidaseasregulatorsofinfluenzavirusgrowth:astudybyreversegenetics.JournalofVirology,2000,74:6316-63231[15]BaigentSJ,McCauleyJW.Glycosylationofhaemagglutininandstalk-lengthofneuraminidasecombinetoregulatethegrowthofavianinfluenzavirusesintissueculture.VirusRes,2001,79:177-1851545:H5N1.P(2006)46(4)BiologicalsignificanceofaminoacidsdeletioninNAstalkofH5N1avaininfluenzavirusWANGQu-zhi,LONGJin-xue,HUShun-lin,WUYan-tao,LIUXiu-fan3(KeyLaboratoryofAnimalInfectiousDiseases,MinistryofAgriculture,YangzhouUniversity,Yangzhou2