甲型H1N1流感病毒株的分离鉴定以及PR8株的相关基因对重组病毒增殖能力的影响

Isolation of an HIN1 influenza A virus and identification of genes influencing on the replication of reassortant virus with PR8 backbone

为分离流感病毒,本研究将疑似甲型流感患者的咽喉拭子接种SPF鸡胚尿囊腔,经检测收获的鸡胚尿囊液具有血凝活性。采用流感病毒通用引物进行PCR扩增出8个基因节段,经测序与GenBank中登录的序列比对后确定该病毒株为2009年爆发的甲型H1N1流感病毒,并命名为A/Harbin/LM/2009(H1N1),简称LM株。由于LM分离株在鸡胚中增殖能力比较弱,为提高其在鸡胚中的增殖能力,我们利用反向遗传技术将LM株的HA和NA基因与高增殖特性的人流感病毒细胞高产株PR8的另外6个节段进行"6+2"重配,获得拯救病毒,但该重配病毒血凝价并未显著提高。为进一步鉴定影响病毒增殖能力的基因,本研究将LM株所有节段以"7+1"的方式逐一与PR8的7个片段搭配构成完整的流感病毒基因组,分别得到8个重配病毒。结果显示分离株的PB1、PA、HA基因显著降低了重配病毒在鸡胚细胞中的增殖能力。

In this study, a virus was isolated from an adult throat swab in Harbin and identified as an H1N1 influenza virus by hemagglutination test and gene sequencing, designated A/Harbin/LM/2009 （H1N1）. To improve the replication capacity of the isolate in embryoed eggs, we utilized reverse genetics technique to construct ＂6＋2＂ reassortant virus, which included HA and NA segments of the isolate and the remaining segments of high-yield influenza virus PR8, as a result the HA titer of the reassortment virus was not significantly improved. To further identify which segment was critical to replication capacity of reassortant virus. A total of 8 reassortment viruses were constructed with 7 segments from PR8 and one segment from the isolate as a ＂7＋1＂ strategy. The results showed that PB1, PA and HA segments of the isolate dramatically reduced the replication capacity of reassotant virus with PR8 backbone.