H3N2亚型猪流感病毒的拯救及HA与NA基因的替换

Rescue of H3N2 subtype swine influenza virus and substitution of Hemagglutinin,Neuraminidase

【目的】为研究流感病毒突破种间屏障分子机制,筛选流感基因工程疫苗株。【方法】本试验以猪流感病毒A/Swine/Henan/S4/01(H3N2)为亲本株,利用反向遗传学操作技术,采用RT-PCR技术对该病毒的8个基因片段分段进行扩增,通过与双向转录载体pHW2000连接,重组质粒转染293T和MDCK共培养细胞,拯救出全部基因均来自于亲本株的猪流感病毒rgH3N2,并分别以人流感病毒A/PR/8/34(H1N1)、禽流感病毒A/Duck/Nanchang/4-165/2000(H4N6)、马流感病毒A/Equine/Fuyun/2008/(H3N8)的HA和NA基因替换A/Swine/Henan/S4/01的相应基因。【结果】生物学试验结果表明rgH3N2在鸡胚半数感染量、组织培养半数感染量、稳定性试验等方面都与亲本株保持一致。rgH3N2经鸡胚多次传代后血凝价最高可达到1∶256,接种MDCK细胞60h后,血凝价可以达到1∶64。基因替换后成功拯救出的重组病毒rgH1N1、rgH4N6和rgH3N8在鸡胚和细胞上均具有较高的增殖能力。【结论】病毒的成功拯救为流感病毒突破种间屏障分子机制,HA、NA基因在流感病毒跨种属传播中所扮演角色的研究和流感基因工程疫苗株的筛选奠定了基础。

[ Objective] To study the mechanisms of trans-species transmission of influenza virus for developing novel vaccine of influenza in future. [Methods] We rescued H3N2 subtype swine influenza virus strain A/Swine/Henan/S4/01 successfully by a plasmid-base reverse genetics. Eight gene segments were synthesized by reverse transcriptase-PCR and cloned into bidirection expression vector pHW2000. We cotransfected 8 recombinant plasmids into 293T and MDCK cells and got the rescued virus rgH3N2. Then we replaced Hemagglutinin, Neuraminidase of rgH3N2 by Hemagglutinin, Neuraminidase gene from Human influenza virus, Avian influenza virus, Equine influenza virus. [Results] The rescued virus rgH3N2 and the wild type virus shared similar biological properties such as in titers of 50 % embryo infective, 50 % tissue culture infective dose and stability tests. The rescued virus titer in MDCK cell culture was measured by hemagglutination assay and the maximum virus titre of 1 ： 64 hemagglutination unit was obtained after infection of MDCK cell for 60 h, The hemagglutination titre was 1：256 after several passages in embryonated eggs. With various combinations of HA, NA genes, we successfully generated high-yield reassortant viruses rgH1N1, rgH4N6 and rgH3N8 in embryonated eggs and MDCK cells. [ Conclusion] The successful rescue of reassortment viruses establish the foundation for the molecular mechanism research on how the swine influenza virus breakthrough the intermediate barriers and the function of HA, NA during transmitting among species, Also it is feasible to be used for developing novel vaccine of H3N2 subtype Swine influenza in future.