冷适应减毒B型流感病毒疫苗候选株的制备及鉴定

Plfl,～：豢翁‰⋯汛 www．pibb．ac．cn ResearchPapersResea DevelopmentandIdentificationofaLive AttenuatedInfluenzaBVirus VaccineCandidate宰 YANGPeng—Hui胛，ANWen—Qil匀“，SHIXin—Fu”，DUANYue—Qiangn，LUODe—Yahn，ZItANGPeng—Feil)， TANGChongn，XINGLi”，ZHANGYu—Jing-2)，LIUXiu—Fan3)”，WANGXi—Lian91)” (1’InstituteofMicrobiology蒯Epidemiology,AcademyofMilit町MedicalScience，StateKeyLaborcaoryofPathogenandBiosecurity, Beo'ing100071，China；4CollegeofAnimalScienceandVeterinaryMedicine，JilinUnwersity,Changchun130000，China； 。Key￡曲or咖，y声rA捌，，lafInfectiousDiseasesofMinistryofAgricuhure，YangzhouUniversity,Yangzhou225009，Chin) AbstractAcold-adapted(c曲，temperaturesensitive伍)，liveattenuatedinfluenzaBvirusstrainB／AnnArbor／I／66waschosenfor influenzavirusrescueresearch，inwhichsixinternalgenesegments，PBl，PB2，队，NP，M，NS，werefullysynthesizedandnineamino acidsubstitIltiomwereartificiallyalterbyhumanintervention．TheresultantB／AnnArbor／1／66plasmidswerenamedaspABl21．PBl． pABl22一PB2，pABl23一PA，pABl24·H～pABl25-NP，pABl26-NA，pABl27-MandpABl28-NS，respectively．Areeombinam influenzaAviruswaspreviouslygeneratedentirelyfromclonedeDNA．AninfeetiousrccombinantinfluenzaBviruswasgenerated here。anddesignatedas订∞V—B，byplasmid-basedreversegenetics．TherMDV-BviruscontainedHAandNAgenesfromall epidemicinfluenzaBvirusswainB／Malaysia／2506／2004inthebackgroundofinternalgenesderivedfrominfluenzaBvirusstrain B／AnnArbor／l／66．HAtiterof岫V-BiIIMDCKcellsandcI]曲rvonatedchickeneggsrangedfroml：64tol：512．Theresultsmay allowalleffectiveliveinfluenzaBvaccinetobeproducedfromasinglemasterstrain,providingamodelforthedesignoffuturelive humaninfluenzavaccines． KeywordsinfluenzaBvirus，reversegenetics，eight-rIlasmidsystem,reassortedinfluenzavirus DoI．103724／SP．J．1206．2008．00542 Influenzaisoneofthemostimportantrespiratory pathogens．ItisreportedbvWHOthatinfluenzais responsibleforapproximately250000～500000deaths eachyearworldwide(http：／／www．ode。gov／)．Influenza Bvirusisoneoftwotypesofinfluenzavirusthat causesubstantialmorbidityandmortalityinhumans， theotherbeinginfluenzaAvirus．InfluenzaAandB viruseseachcontaineightsegmentsofsinglestranded RNAwithnegativepolarity．Theeightgenome segmentsofinfluenzaBencode11proteins．Thethree largestgenescodeforcomponentsoftheRNA polymerase，PB1，PB2，andPA．Segment4encodes theHAprotein．Segment5encodesNP．Segment6 encodestheNAproteinandNBprotein．Bothproteins． NBandN八a!etranslatedfromoverlappingreading framesofa biscistroniemRNA．Segment7 of influenzaBalsoencodestwoproteins：MlandBM2． Thesmallestsegmentencodestwoproteins：NS1 is translatedfromthefillllengthRNA．whileNS2is translatedfromasplicedmRNAvariant． Todate。vaccineisstillaprincipalandeffecfive meansforinfluenzaprophylaxis．Vaccinestoprevent influenzagenerallycontainthesurfacehemagglutinin (HA)andneuraminidase(NA)glycoproteinsfromthe twocurrentlycirculatinginfluenzaAsubtypes(i⋯e H3N2andHlNl1 andonecirculatinginfluenzaB strain．Theinabilitytoprovidelastingprotectionto humansagainstinfluenzavirusinfectionisdue。inpart， toantigenicdriftorantigenicshiftintheHA．Hence． +Thiswork啷supportedbyagrantfix)mChin雠NationalPrograms 斯IIighTechnologyResearchandDevelopmentf2006AA02ZA50)． ’。YANGPeng-HuiandANWen-Qicontributedequallytothiswork． ’’’Correspondingauthor． WANGXi-Lian8．Tel：86—10-66948678，E-mail：xiliangw@yahoo．com LIUXiu-Fan．Tel：86-514-7991416，E-mail：liuxioufan@yahoo．17,0111 Received：July29，2008Accepted：October20，2008 万方数据 2009；36(3) 杨鹏辉等：冷适应减毒B型流感病毒疫苗候选株的制备及鉴定 "359" thevaccinesmustbefrequentlyupdatedtoensure coverageagainstvirusstrains． F1uMistisa liveattenuatedinfluenzavacclne (LArV)composedofthethreedominantcirculating strainsofhumaninfluenzavirus．Thisvaccinehas beenshowntobee伍caciousandsafefordeliveryin adultsandchildrenandhasbeenlicensedforuseinthe UIlitedStatesin2003[“．Inrecentyears．rgvgrsc—genetics systemsfortherescueofrecombinantinfluenzavirus haveproventobeofgreatvalueforinfluenzavirus researchandvaccinedevelopment． Inerrpreviousstudy,areversegeneticssystem forthegenerationofrecombinantinfluenzaAvirus waswellestablishedthatfacilitatesthegenerationof vaccineviruseswithouttheneedfortime．consuming CO．infectionandselectionprocedurescurrently requiredtoproducereassortants．Moreover，thesystem successfullycontributestovaccineproduction．Here． thestrategyweappliedisbasedonthatdescribedfor recoveryofrecombinantinfluenzaAvirus．Inthis report,wedescribedtheestablishmentofan eight—plasmidsystemthatenablesthegenerationof infectious，recombinantinfluenzaBvaccinecandidate byreversegenetics． 1 Materialsandmethods 1．1 Viruses,cellsandeggs InfluenzavirusstrainB／Malaysia／2506／2004were receivedfromChinaCDCandpropagatedin 10·-day-oldspecificpathogen··freeembryonated researchgradeeggs(Centeroflaboratoryanimal， Beijing)．COS-1cellsandMadin．Dardycaninel【idnev (MDCK)cellswereobtainedfromYangzhou Universityandmaintainedinessentialmedium (DMEM，sigma)containing10％fetalbovineserurfl (FBS)at37℃inanatmospherewith5％C02． 1．2 VirusRNAextraction,PCRamplification． andgenecloning Acold-adapted(c曲，temperaturesensitive∞)， liveattenuatedinfluenzavirusstrainB／AnnArbor／l／66 Wasusedasthemasterdonorvirus(MDV)forvirus rescue．inwhichsixinternalgenefragmentsworefully synthesized．Meanwhile，nineaminoacidsubstitutions atpositions，PB2锄($630R)，PA铂1(V431M)，Ⅳ一 (Y497H)，NP55(T55A)，NPll4Ⅳ114A)，NP410(P410H)， NP510(A510T)，M1159(H159Q)，M1183(M183Ⅵ，have beenartificiallyalteredbyhumaninterventionandsix parrsspecificpnmersweredesignedaccordingto referencestZ3J(Tablen．111e5’．endshaverecognition sequencesfor therestrictionendonucleases BsmBI(Bm)orBsaI(Ba)(NewEnglandBiolabs)． B／Malaysia／2506／2004Waspropagatedin10一day-old embryonatedchickeneggsandconcentratedbydensity gradientcentrifugationonsucrose．TotalRNAwas extractedfrominfectedallantoicfluidwiththeRNeasy kit(Qiagen)inaccordancewithmanufacturer’S instructions．ReversetranscriptionWascarriedoutwith theuni9primer(5’AGCAG八AGC3’)[41andAMV reversetranscriptase(Invitrogen)．ExpandHighFidel时 PCRSystem(RoChe)wasusedand8～10cloneswere sequencedinordertoconfirmtheauthenticityof5’ and3’ends．Finally,wesplicedthe8 wholegene Table1 RT-PCRprimersforamplificationoftheeightvRNAsofinfluenzac口B／AA／1／66 万方数据 "360- 生物化学与生物物理进展Prog．Biochem．Biophys． 2009；36(3) sequencescontainingthenon。codingregionof3’and 57endsbyBL八ST．eDNAfragmentswithBsmBIand BsaIofB／AnnArbor／1／66andB／Malaysia／2506／2004 wereclonedbetweenthetwoBsmBI sitesofthe vectorpAD3000faderivativeofpHW2000which allowsthetranscriptionofnegativesensevRNAand positivemRNA)．Eighttranscription／expression plasmidswel'eobtainedandnamed嬲pABl21-PBl． pABl22一PB2，pABl23．PA。pABl24-HA，pABl25- NP。pAB126-NA,pAB127．MandpAB128一NS， respectively．Sitesofthetworestrictionendonucleases insixintemalgeneswere嬲followed(Table2)．A1l clonedwereconfirmedbyfull—lengthsequencing． Table2 SitesofthetworestrictionendonucleasesinsixInternalgenes 1．3 Transfectionandgenerationofvirus TherescueofinfectionvirusfromclonedeDNA wasdoneunderGMPconditions．MDCKandCOS．1 cellswereco—culturedin6一wellplateataratioofl：1 andthecellswereusedfortransfectionataconfluency ofapproximately70％"-80％．PlasmidDNAtransfeetion wasperformedusingPolyFecttransfectionreagent (Qiagen)bymixing0．2斗gofeachoftheeight recombinantplasmids(BPB2，BPB1，BPA，BHA， BNP，BNA，BM，BNS)with10tdofPolyFectdiluted in100It,lDM匣M．TheDN觚andtransfectionreagent mixturewereincubatedatroomtemperaturefor5，一 10minfollowedbyadditionof500“lDMEM．Tlle transfectionmixturewasthenaddeddropwisetothe co-culturedMDCK／COS．1cells．111etransfectioncells wereincubatedat33℃for48h．1mlofDMEM containingl mg／LTPCK-trypsin(PIElK：E1wasadded tothecells．At96hpost-transfection．1mlofDMEM containing1meCLTPCK-trypsinwasaddedtothe cells．Sixdayspost-transfection，transk；ctedculture supernatantswereharvested．Therecoveredviruswas thenamplifiedinconfluent加CKcellsordirectly amplifiedinembryonatedchickeggs．Theamplified viruseswerestoredat一80℃．200IJ,loftheclarified supernatantwasinjectedintotheallantoiecavityof individual10一dq—oldembryonatedchickeneggs． After72hincubationat33℃．eacheggwascandled todetermineembryoviabilitybeforechillingat4℃． Allantoicfluidwasharvestedfromeacheggandwas testedforhaemagglutinationactivity． 1．4 Identificationandanalysisofrecombinant virus 1．4．1 RT—PCR．RT．PCRwasperformedtomapthe genotypesoftherecoveryviruses．VirusRNAwas isolatedfromtheinfectedeellculturesupematantor allantoicfluidfromthesecondpassageusingthe RNeasyminikitandtheeightinternalgenesegments wereamplifiedbyRT-PCRusingspecificprimersof eachMDV-Bgenesegment(Tablel)． 1．4．2 Electronicmicroscope．Theallantoicfluidfrom thesecondpassagewasconcentratedandpurifiedby densitygradientcentrifugationonsucrose．Then, morphologyoftherescuedvirusandwild．typevirus strainwereobservedbyelectronicmicroscope． 1．4．3 Titrationofviralinfectivityineggsand MDCK．Infectivityineggs(EID茹wasdeterminedby inoculatingeggswith0．2mlof10．folddilutionsof allantoicfluidr4eggsperdilution)．Afterincubationat 33℃for72h．thepresenceofviruswasdeterminedby hemagglutination．Thehemagglutinationtiter(HA titer)wasmeasuredbystandardmethodusing1．0％ chickenredbloodcells(RBC)inphosphate—buffered saline(PBS，pH7．2)(Palmereta1．，1975)．Thevalues ofHAtitersweredetermined勰anaveragefrom4 eggs．TodeterminevirusinfectivityinMDCK (TCID50)cellsgrowntoconfluencein96-wellplates werewashedwithPBSandinoculatedwithseriallO folddilutionsofvires(eightreplicatesforeach sample)．Afteronehourat33℃．MDCKcellswere washedandDM匣Mcontainingl mg／LTPCK-trypsin wasadded．TheendpointforcalculationofTCD∞was cytopathiceffect(CPE)aftertheincubationofthe inoculatedcellsfor3"--4daysat33℃inall atmospherewi也5％C02．TCID卯titerswerecalculated bythemethodofReedandMuenchf1938)． 1．4．4 SDS．PA(讯．Toverifythemaincomponentof therescueinfluenzaBvirus，theaUantoicfluidfrom thesecondpassage，afterconcentratedandpurified wereanalyzedby15％SDS-PAGEusingconventional techniques． 万方数据 2009；36(3) 杨鹏辉等：冷适应减毒B型流感病毒疫苗候选株的制备及鉴定 ·361· 1．4．5 Stabilitytestingineggs．Inordertotestthe stabilityofthevaccinevirusOllpropagation,ten consecutivepassagesofthevirusweremadein embryonatedchickeneggs．Theviruswasadjustedto 10‘3dilutionwithPBS．and0．2mlofthesolutionWas injectedintotheallantoiccavitiesoffour10-day-old embryonatedchickeneggsandthenreinjectedinto anotherfoureggs．Toconfirmthegeneticstabilityof therescuevirus，wecomparetheHA，NAandNPgene sequencesoftherescuedvirusfromthesecond，fourth， sixth，eighthandtenthpassage． 2 Reslllts 2．1 GenerationofrecombinantinfluenzaBvirus fromeightplasmids Sixdayspost．transfection。culturesupernatants witheiightrecombinantplasmidsincorporatingthe6 MDV—Binternalgenes．andHAandNAderivedfrom B／Malaysia／2506／2004(6：2reassortant,rMDV-B)， wereusedtoinfectfreshMDCKcells．Then．the infectedcellswereincubatedat33℃for3～4daysin DMEMwith1 mg／LTPCK-trypsin．Thecytoplasmic effectoftherecombinantvirusoninfectedMDCK cellsWasobservedusingamicroscope．Expressionof viralhemagglutininwasmonitoredusingastandard hemagglutinationassay(HA)．HAtitersweredetected t0be1：64approximatelyfromtheamplified6：2 reassortantvirusinthefirstpassage．Meanwhile，no viruswasdetectedinsupernatantsderivedfromcells transfectedwithonlysevenplasmids．These transfectionreactionswereasfollows(Table3)． Table3 Generationofinfectiousinfluenza Bvirusfromeightplasmids 2．2 RT-PCR AsshowninFigure1，，PCRproductswere generatedforallsegments．Afterasubsequentpassage onMDCKcells．RT．PCRofthesupernatantof infectedcellswasusedtoconfirmtheauthenticityof 也egeneratedvirus．RT．PCRwasperformedwith segmentspecificprimersforalleightsegments．11坞 m11．1engthsequenceoftherecombinantvirusafter passageintoembryonatedchickeneggsWasidentical tothatoftheinputplasmids．Eightdesiredfragments wereamplifiedbyRT．PCRfromthesecondpassage， theywere2．3kb，2．3kb，2．3kb，1．8kb，1．8kb，1．5kb， 1．1kband1．0kb．respectively．However．HAandNS genecouldn'tbeamplifiedfromallantoicfluidexclude contaminationofplasmids． Fig．1EightgenesegmentsofrMDV-BamplifiedbyPCR MI：DLl5000marker；M2：DL2000marker；J～8：PB2，PBI，队HA， NP，NA,M，NS；CK：Negtiveeon仃01． 2．3 Electronicmicroscope Influenzaviralparticlewithcapsulecouldbe observedintwosamples(Figure2)．Thevirion morphologyoftherescuedviruseswasverifiedtobe identicaltothatofthewild．typeinfluenzaBvirus strainB／Malaysia／2506／2004．Therewerenoobvious differencesbetweeninfluenzaAandBstraininvirion morphology． Fig．2MorphologyofinfluenzaBviruses (a)RccovercdrMDV-Breassortant(x30000)．(b)Wild-typeB／Malaysia／ 2506／2004(x93000)．Atthebottomofpicture(a)Or(b)isasingle virion(×65ooo)． 2．4 SDS-PAGE AfterrMDV·BWasconcentrated,therewere manybandsappearedanddosageincreaseobviously． 万方数据 "362- 生物化学与生物物理进展 Frog．Biochem．Blophys． 2009；36(3) Evenafter舳V—Bwaspurified．threebandsstill existinlaneone．Accordingtothemolecularmass， theyweretheNP．NAandMproteinrespectively (Figure31．The∞resultsshowedthatthemain componentofinfluenzavLrusdidnotloseduringthe processing． tag．3SDS-PAGEofrMDV-B J：PurificdrMDV-B；2：ConcentratedrMDV—B：了：rMDV-B；Jlf： Molecularn麟marker． 2．5 StabmtvofrMDV-B FromtheH九NAandNPgenesequenceof differentpassages，wefoundvirustiterrangedfrom 1：64to1：512．Moreover。thehomologyofHA，NA andNPgenewereabove99．99％intheallantoicfluid ofthesecond，fourth，sixth，eighthandtenthpassages． Thisresultdemonstratesthatpassageof舳V—Bdoes notleadtochangesintheHA，NAandNPgene． 3 Discussion Influenzaisundoubtedlythemostimportant humanrespiratorypathogenworldwide，causing considerablemorbidityandalloften．underestimated mortality．Moreover,itimposesahugeburdenonthe economy．Onlyaneffecfiveandsafevaccineoffersthe possibilityofprotectingthehumanpopulationfrom thevagariesofalmostyearlyepidemic．Twiceayear， theWorldHealthOrganization(WHO)meetsto updatethevaccinestrainstomatchthestrainsinthe communityineachhemisphere．Recentdiscoveriesin influenzapathogenicityandreversegeneticshavethe potentialtorevolutionizethewaypandemicand interpandemicinfluenzavaccinesarepreparedand manufactured．Sofar，attenuatedlivevaccineplaysan importantroleforinfluenzacontr01． Since1977，influenzaA subtypeshave co·circulatedalongwithinfluenzaBVIruses【“．Alive attenuatedvaccine(FluMismwaslicensedin2003in theUSA．Thevaccineviruseswereproducedby selectingHAandNAsegmentsfromcurrently circulatingvirusesandallowingthemtoreassortwith allinternalgenesegmentsfromeithermasterdonor virus(cold．adaptedA／AnnArbor／6／60fortypeA (H1N1andH3N2)andB／AnnArbor／1／66fortypeB viruses)嘲．Theresultantvaccineviruseswerethen combinedandsprayedintothenostrilsofvaecinees． Theclinicalstudiesshowedthatthesevaccinesare attenuated．safeande珩caciousinadultsandchildren． In2002，David，et01．andErich，eta1．reportedthe rescueofinfluenzaBvinlsentirelyfromcDNA respectivelyB31．Recently,Seo．eta1．[61publishedthe developmentofrecombinantinfluenzaBvirusby reversegeneticsinSouthKorea．However,itisablank fieldinChina．、Ⅳepreviouslygeneratedarecombinant influenzaAvirusentirelyfromclonedcDNA．Herewe describedthegenerationandidentificationof reassortantinfluenzaBvirusesthatcarriedfunctional HAandNAfromanepidemieinfluenzaBvirusstrain B／Malaysia／2506／2004inthebackgroundofintemal genesderivedfrominfluenzaBvirusstrainB／Ann Arbor／1／66，yieldingrMDV．B． Asforinfluenzavaccineproduction，highvirus yieldisacriticalfactor．becauseitcanreducecostand sustainlongerimmuneprotectionperiodsbyproviding enoughantigens【7’柳．Vaccineproductionprocedures mustallowthevirusproducedtoretaintheantigenic propertiesoftheparentviruses．Anybenefitfroman increaseinyieldwouldbenegatedbychangesinthe antigenicityofavaccine．Therescuedviruseswere antigenicallyindistinguishablefromthecorresponding wild-typeviruses．Toconfirmthegeneticstabilityof therescuedviruses．theHAgenesequencesofthe rescuedandwild—typeviruseswerecompared．Andwe foundthehomologyofHA．NAandNPgenesequence wereabove99．99％duringeachpassageof recombinantvirus．Inaddition，theinjectedeggsgavea positiveHAtiterrangingfrom1：64tol：512wim passageincreasing．ARerrescuedviruswas concentratedandpurified，themaincomponentdidnot lose． Inprinciple，plasmid-onlysystemsallowthe manipulationofthenon-codingandcodingregionsof theviralItNA．Thenon．codingregionscontain c／s·actingsignalsfortheregulationoftranscriptionand replicationofviralRNA．ComparedwithinfluenzaA virus，theNCRsofinfluenzaBvirusesarerelatively 万方数据 2009；36(3) 杨鹏辉等：冷适应减毒B型流感病毒疫苗候选株的制备及鉴定 "363" large，extendingupto100nt．Abasemutationmay causefailureforgenerationofrecombinantvirus．So。 thesesequencesplayanimportantroleinrescuingof recombinantvirus．Inordertoassuretheauthenticity andaccuracyofgenefragment,eachrecombinant plasmidmustbeidentifiedandsequencerepeatedly． Insummary,wehaverescueda cold．adapted recombinantinfluenzaBvirusbyCO．transfectingeight transcriptionplasmidsentirelyfromcDNA．The plasmidrescuesystemforinfluenzaBwillenable moleculargeneticstudiesofinfluenzaBviruses．In addition．theestablishmentofcold．adaptedinfluenza virusrescuesystembvRGwillcontributeto developmentofcold．adaptedhumaninfluenzavirus vaccinecandidatesandmucosalimmunemechanisms againstinfluenza． AcknowledgementsWethankDr．RobertG．Webster forprovidingUSwitllthebi·directionaltranscription／ expressionvectorpHW2000andeight-plasmidsystem． I HuberVC．McCullersJA．Liveattenuatedinfluenzavaccineissafe andimmunoganicinimmunocompromisedferrets．JInfectious Diseases．2006．193：677～684 2 DavidJ，AndrewC，Thomasz，eta1．Areversegeneticsapproachfor recovel"yofrecombinantinfluenzaBvims髓entirelyfrom战瞄kl virology，2002，76(22)：11744～11747 3 EfichH．KumbuddinM。YangCF，et以．RescueofinfluenzaBvirus fromeightplasmids．ProcNailAcadSciUSA,2002,99(17)： 1141l～11416 4 ZouSM．Apracticalapproachtogeneticscreeningforinfluenza virusvariants．JClinicalMicrobiology,1997．35(10)：2623～2627 5 HofimotoT，1watsuki-HofimotoKHattaM，eta1．InfluenzaA virusespossessingtypeB hemagglutininandnettraminidase： potentialasvaccineeomlxments．MicrobesandInfection,2004．6 (6)：579～583 6 SeoSU，ByunYH，LeeEY，et口f．Developmentand characterizationofaliveattenuatedinfluenzaBvirusvaccine candidate．V∞cine，2008，26(7)：874～88l 7 ShiH，“uXF，Zhang)('eta1．Generationof强attenuatedH5N1 avianinfluenzavirusvaccinewithalleightgenesfromavianviruses． Vaccine，2007，25(42)：7379～7384 8 YangPH，YeY，WangXL，et01．Constructionofattenuated influenzavirusvaccinesof2006～2007．ProgressinBiochemistry andBiophysics，2008，35(3)：312～319 冷适应减毒B型流感病毒疫苗候选株的制备及鉴定 杨鹏辉1)”安文琪1，2)“石辛甫1’段跃强1’罗德炎1) 张鹏飞1) 唐冲1’ 邢丽1)张玉静∞ 刘秀梵3)．．‘王希良1)“。 (，)军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室，北京100071； ≈吉林大学畜牧兽医学院，长春130000：∞扬州大学农业部畜禽传染病学重点开放实验室，扬州225009) 摘要 以冷适应、温度敏感、减毒的B／AnnArbor／l／66流感病毒株作为重配病毒骨架，对其6个内部基因片段进行了全基因 合成，同时人工引入9个氨基酸突变．构建了8个基因的拯救载体，经测序获得序列准确的拯救质粒，命名为： pABl21．PBl。pABl22．PB2，pABl23．PA，pABl24-}【A’pABl25-NP，pABl26-NA，pABl27-M和pABl28-NS．在成功拯救冷适应 A型流感病毒的基础上，利用反向遗传学技术成功获救了具有感染性的重配B型流感病毒株，命名为rMDV．B．该重配病毒 株以B／AnnArbor／1／66为病毒骨架，其中HA和NA来源于2006～2007年当年流行株B／Malaysia／2506／2004．rMDV-B在鸡 胚尿囊液和MDCK细胞中的HA效价可达1：64～l：512．实验结果暗示：从单一供体病毒株可以产生有效的减毒活B型 流感病毒疫苗候选株，能够为将来人用流感疫苗的设计提供可借鉴的模型． 关键词B型流感病毒，反向遗传学，8质粒拯救系统，重组流感病毒 学科分类号R183．3，R392．1 DOh10．3724／SPA．1206．2008．00542 ·国家高技术研究发展 计划 项目进度计划表范例计划下载计划下载计划下载课程教学计划下载 资助项I；1(863X2006AA022450)． ．．共同第一作者． ¨·通讯联系人． 王希良．Tel：010-66948678，E-mail：xiliangw@yahoo．eom 刘秀梵．Tel：0514-7991416，E-mail：liuxioufan@yahoo．eonl 收稿日期：2008-0％29，接受日期：2008·10·20 万方数据 冷适应减毒B型流感病毒疫苗候选株的制备及鉴定 作者： 杨鹏辉， 安文琪， 石辛甫， 段跃强， 罗德炎， 张鹏飞， 唐冲， 邢丽， 张玉静， 刘 秀梵， 王希良， YANG Peng-Hui， AN Wen-Qi， SHI Xin-Fu， DUAN Yue-Qiang， LUO De-Yan， ZHANG Peng-Fei， TANG Chong， XING Li， ZHANG Yu-Jing， LIU Xiu-Fan， WANG Xi-Liang 作者单位： 杨鹏辉,石辛甫,段跃强,罗德炎,张鹏飞,邢丽,王希良,YANG Peng-Hui,SHI Xin-Fu,DUAN Yue-Qiang,LUO De-Yan,ZHANG Peng-Fei,XING Li,WANG Xi-Liang(军事医学科学院微生物流 行病研究所病原微生物生物安全国家重点实验室,北京,100071)， 安文琪,AN Wen-Qi(军事 医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京,100071;吉林大 学畜牧兽医学院,长春,130000)， 唐冲,张玉静,TANG Chong,ZHANG Yu-Jing(吉林大学畜牧 兽医学院,长春,130000)， 刘秀梵,LIU Xiu-Fan(扬州大学农业部畜禽传染病学重点开放实 验室,扬州,225009) 刊名： 生物化学与生物物理进展 英文刊名： PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS 年，卷(期)： 2009，36(3) 被引用次数： 0次 参考文献(8条) 1.Huber V C.McCullers J A Live attenuated influenza vaccine is safe and immunogenic in immunocompromised ferrets 2006 2.David J.Andrew C.Thomas Z A reverse genetics approach for recovery of recombinant influenza B viruses entirely from c