2016年～2018年山东地区25株H9N2亚型禽流感病毒分子分析

Molecular analysis of 25 H9N2 subtype avian influenza viruses in Shandong from 2016 to 2018

H9N2亚型禽流感(AI)持续在我国鸡群中广泛流行,为了明确山东及周边养殖场H9N2亚型禽流感病毒(AIV)的分子进化情况,本研究对2016年~2018年间从山东及周边养殖场病鸡中分离鉴定得到的25株H9N2亚型AIV分别纯化后进行全基因组测序和遗传演化分析,并分析序列中重要氨基酸位点的变异情况。全基因组序列分析显示:25株H9N2 AIV 8个基因节段均属于欧亚谱系,但8个基因节段的分子进化关系呈现出基因多样性:HA、NA基因来自Y280-like,25株分离株HA基因之间的同源性为92.8%~99.9%,NA基因之间的同源性为91.0%~99.9%;PB2基因来自G9-like;PB1、PA、NP、NS基因来自F98-like;M基因来自G1-like。HA蛋白的受体结合位点149、198、234和235位点变异最多,25株分离株HA均出现Q234L突变(相当于H3第226位氨基酸);21株分离株在HA蛋白218~300位糖基化位点缺失,所有分离株均在313~315位出现潜在的糖基化位点;仅3株分离株的NA蛋白在402位出现糖基化位点,而NA蛋白在264位有11株分离株出现了潜在糖基化位点;5株分离株在PB2蛋白的627位点发生变异,1株在PB2蛋白的714位点发生变异。以上结果表明,近3年山东周边地区流行的H9N2 AIV是由不同进化方式而来,部分重要氨基酸位点出现了变异,具有潜在感染哺乳动物的可能性,因此需加强H9N2 AIV的分离鉴定,密切监测其遗传变异情况。

H9N2 subtype avian influenza(AI) continues to be widely prevalent in China’s chicken flocks. In order to clarify the molecular evolution of H9N2 subtype avian influenza virus(AIV) in Shandong and surrounding farms, twenty-five chicken-origin H9N2 subtype AIV were isolated and identified from 2016 to 2018. The viruses were purified for genome sequencing and genetic evolution analysis, and the mutations of important amino acid were analyzed. Whole genome sequence analysis showed that eight gene fragments of 25 H9N2 AIV belonged to the Eurasian lineage, but the molecular evolution of the eight gene fragments showed genetic diversity. HA and NA genes were from Y280-like. The homology between HA genes of 25 isolates ranged from 92.8 to 99.9%, and the homology between NA genes ranged from 91.0 to 99.9%. PB2 genes were from G9-like. PB1, PA, NP and NS genes were derived from F98-like. M genes were derived from G1-like. Four positions at 149, 198,234 and 235 in the receptor binding domain of HA protein had the highest mutation frequency. The amino acid at position 234 of HA protein of 25 isolates(corresponding to the amino acid at position 226 of HA protein of H3 subtype) were all changed from Q to L. The glycosylation sites at position 218 to 300 of HA protein of 21 strains were lost, and all 25 isolates showed potential glycosylation at position 313 to 315 of HA protein. Only 3 isolates had potential glycosylation site at position 402 of NA protein,while 11 isolates showed glycosylation at position 264 of NA protein. Five isolates mutated at position 627 in PB2 protein, while one isolate mutated at position 714. The results showed that the H9N2 AIV circulated in Shandong and surrounding areas in the past three years were derived from different evolutionary ways. Some key amino acid sites were mutated, which indicated the viruses may have the potential to infect mammals. It is necessary to strengthen the isolation and identification of H9N2 AIV and its genetic variation analysis. This study has public health implications.