云南边境地区禽流感H5N1亚型病毒NS基因进化分析

Genetic evolution of non-structural gene among avian influenza H5N1 viruses isolated from the boundary of Yunnan province

目的阐明云南边境地区禽流感H5N1亚型病毒NS1、NS2基因变异特征及遗传进化关系。方法在云南边境地区采集境外家禽和野生鸟类棉拭子样品，经H5N1亚型特异性多重RT-PCR检测，阳性样品对病毒NS基因进行扩增，克隆至pMD18-T载体测序，获得NS1、NS2基因序列，并与已知参考毒株序列进行序列比对及系统发育分析。结果自1240份样品中检出H5N1亚型阳性样品71份，阳性率为5．72％；30份代表性阳性样品病毒NS基因测序获得17种序列，存在3个不同进化（亚）分支（Ⅰ-1、Ⅰ-2、Ⅱ）；NS1／NS2基因与病毒血凝素（HA）基因呈现不同进化关系；NS1蛋白涉及核定位信号区、RNA结合区、效应区及其他致病性相关的关键性氨基酸位点存在替代或突变。结论云南边境地区H5N1亚型病毒NS1／NS2基因具有遗传差异，2010年以来NS基因进化分支Ⅰ-2、Ⅱ已成为当地流行的优势毒株。

Objective To elucidate the characteristics of variation and the genetic evolution of non-structural protein （NS1, NS2） genes related to avian influenza subtype H5N1 viruses isolated from the boundary region of Yunnan province. Methods Swab samples were collected from foreign poultry and wild birds in the boundary regions of Yunnan province and screened by H5/N1 subtype- specific multiplex RT-PCR. The NS segment of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. The alignment and phylogenetic analysis on those available NS1, NS2 genes were performed with sequences of the known reference strains. Results 71 positive samples were identified from 1240 samples, with the positive rate as 5.72%. Fourteen different NS segment sequences were obtained from 30 representative positive samples and could be divided into 3 distinct clades or sub-clades （ I -1, I -2 and II ） , by phylogenetic analysis. The NS1/NS2 genes and Hemagglutinin （HA） genes of H5N1 viruses from the boundary regions of Yunnan province showed different relationships regarding the characteristics on genetic evolution. The substitution or mutation of key amino acids sites had been noticed in the nuclear location signal domains, effect domain, and other pathogenicity markers. Conclusion NS genes of H5N1 subtype viruses in boundary region of Yunnan province showed genetic divergence and the virus ofclade I -2 and II had become dominant epidemic strains in this region since 2010.