H3N8亚型马流感病毒拯救体系的建立

Establishment of reverse genetics system for equine influenza virus H3N8 subtype

利用反向遗传学操作技术,以马流感病毒(EIV)A/Equine/Fuyun/2008/(H3N8)为亲本株,采用RT-PCR技术对该毒株的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,成功拯救出全部基因均来自亲本株的EIVrH3N8,生物学试验结果表明,rH3N8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。以猪流感病毒(SIV)A/Swine/Henan/S4/01(H3N2)内部基因的重组阳性质粒替换rH3N8的相应基因,成功拯救出重组病毒rgH3N8。两拯救毒株经鸡胚一次传代的血凝效价分别为1∶32、1∶64,最高可达1∶1024、1∶2048;接种MDCK细胞54h后的血凝效价最高均可达1∶512。rH3N8亚型EIV的成功拯救及基因的成功替换,为流感病毒突破种间屏障分子机制的研究和流感基因工程疫苗株的筛选奠定了基础。

Eight genes of equine influenza virus（EIV） were amplified from EIV A/Equine/Fuyun/ 2008/（H3N8） strain by RT-PCR, and cloned into pHW2000 vector to construct eight plasmids, respectively. Then the eight plasmids were co-infected into 293T cell co-cultured with MDCK cell to generate recombinant EIV A/Equine/Fuyun/2008/（H3N8）, designated rH3N8, by a plasmid-base reverse genetics. The rescued virus rH3N8 and the wild type virus shared similar biological properties in 50%-embryo infective dose, 50 %-tissue culture infective dose and stability test. It was generated that reassortant virus rgH3N8 containing hemagglutinin and neuraminidase genes of rH3N8 and the other six internal genes from swine influence virus A/Swine/Henan/S4/01（H3N2） strain. The hemagglutination titers of the two rescued virus were 1 ： 32 and 1 ： 64 after the first passage in emhryonated eggs,and the maximum hemagglutination titer were 1 ： 1 024 and 1 ： 2 048,respectively. The maximum hemagglutination titer 1 ： 512 of the two rescued virus derived from MDCK cell were obtained at hour 54 post-infection. The successful rescue of rH3N8 and rgH3N8 established a foundation for studies on the molecular mechanism for EIV trans-species transmission and screening vaccine virus of equine influenza H3N8 subtype.