H3N2亚型犬流感病毒反向遗传操作系统的建立

摘要

H3N2 Canine influenza virus (CIV) can spread quickly among dogs and even causes death. Moreover, CIV infects other mammals. At present, the molecular mechanism of CIV is not clear. In the present study, RT-PCR technique was used to amplify 8 genes (PB2,PB1,PA,HA,NP,NA,M and NS) of A/Canine/Zhejiang /01/2010(H3N2) (ZJ0110), and the resulting products were then cloned into the two-way expression vector pBD. These 8 recombinant plasmids were co-transfected into 293T cells that were treated with TPCK trypsin at 48 h post-transfection. The supernatant and transfected 293T cells were inoculated into 9-day-old specifi c pathogen free (SPF) chicken embryonated eggs. The rescued viruses were identifi ed in hemagglutination assay (HA). The virus strain (rZJ2010) was sequenced for the above 8 genes and found they were identical to those of its parent virus ZJ2010. The replication of rZJ2010 in the lungs and nasal turbinates of mice were similar to that of ZJ2010. These results indicated that the reverse genetics system of H3N2 subtype Canine infl uenza virus was established as a technological platform for further research on the molecular mechanism, pathogenicity and interspecies transmission of Canine infl uenza viruses.%H3N2亚型犬流感病毒(Canine influenza virus,CIV)可以在犬中迅速的传播,甚至引起犬的死亡,也可感染其他哺乳动物.目前该病毒致病的分子机制尚不清楚.为此,本研究采用RT-PCR技术扩增犬流感病毒A/Canine/Zhejiang/01/2010(H3N2)(简称ZJ2010)的PB2、PB1、PA、HA、NP、NA、M和NS 8个基因片段,并分别克隆至双向表达载体pBD上.采用8质粒系统共转染293T细胞,转染48 h后加入TPCK胰酶作用2 h,将上清和细胞一同接种9~11日龄SPF鸡胚,并检测血凝效价.结果显示,本研究成功拯救获得了病毒株rZJ2010.rZJ2010株的8个基因片段的序列均与亲本ZJ2010株的序列相同.rZJ2010株与ZJ2010株在鼠的肺和鼻中复制水平接近.这些结果证实,本研究已成功建立H3N2亚型犬流感病毒反向株遗传操作系统,为该病毒的致病机理、传播机制以及跨宿主传播机制研究等奠定了技术平台.