63株H9N2亚型禽流感病毒NA基因序列分析

Sequence Analysis of Neuraminidase Genes of 63 H9N2 Subtypes Avian Influenza Viruses

为了从分子水平上掌握我国H9N2亚型禽流感病毒NA基因变异情况和流行规律,作者汇集了我国部分省市的63株H9N2亚型禽流感毒株,采用RT-PCR技术对其NA基因进行扩增、克隆和测序,并对所得序列进行同源性和进化分析。结果显示分离株核苷酸同源性在87.4%~99.5%;52株NA蛋白颈部63—65位点缺失了T、E、I 3个氨基酸,属于Y280-like分支,11株没有发生缺失,属于G9-like分支,没有发现G1-like分支和Y439-like分支的NA基因毒株;唾液酸结合位点（HB）和抗原决定簇的氨基酸发生了明显的变异;酶活性位点处的氨基酸非常保守,没有发生与抗神经氨酸酶抑制剂奥塞米韦和扎那米韦相关的氨基酸突变;36/63毒株失去了402位点处的潜在N-糖基化位点;而28/63毒株新出现了264位点处的潜在N-糖基化位点。本研究提示目前中国大陆地区商业鸡群流行的H9N2亚型禽流感病毒NA基因分为2个差异较大分支——Y280-like分支和G9-like分支,而Y280-like分支仍是我国H9N2亚型禽流感病毒流行的主要类型。

In order to explore the genetic mutations of the neuraminidase and the law of the molecular epidemiology of H9N2 subtype Avian Influenza virus（AIV） in mainland of China,63 H9N2 AIV strains were isolated from some poultry yards in China,the whole NA cDNA fragments of them were amplified by reverse transcription polymerase chain reaction,with primers specific to NA gene.After cloning and sequencing,homology and heredity evolution of NA gene sequences were analyzed.The sequence analysis showed that the homology of NA gene in this study was between 87.4%-99.5%.Neuraminidase of 52 strains had a deletion of 3 amino acid residues at positions 63-65,belong to Y280-like branch;the other 11 strains had no deletion in the stalk,belong to G9-like branch.Amino acid residues at hemadsorbing（HB） sites and antigenic determinants of these isolates exist variation.The amino acid residues in the enzyme active sites of the isolates were highly conserved and showed no mutations associated with resistance to the sialidase inhibitors oseltamivir and zanamivir.36 strains viruses sequenced in this study contained potential glycosylation sites at 264 amino acid residues 28 strains lost potential glycosylation sites at 402 amino acid residues.The results indicated that by now the NA gene of H9N2 AIV in China's Mainland belong to two branches,Y280-like and G9-like,and the most NA gene of the H9N2 isolates in China mainland belong to Y280-like.