11株H9N2亚型禽流感病毒神经氨酸酶基因遗传分析

Genetic analysis of neuraminidase genes of 11 H9N2 subtype avian influenza viruses

[目的]本文旨在了解H9N2亚型禽流感病毒神经氨酸酶（NA）基因的遗传特点和变异情况。[方法]选择2008—2013年从江苏、安徽和浙江省家禽中分离的11株H9N2亚型禽流感病毒,用RT-PCR方法对NA基因片段进行扩增、克隆和序列测定,并对其核苷酸序列及推导的氨基酸序列进行同源性及遗传进化分析。[结果]11株毒株的NA基因核苷酸和推导的氨基酸同源性分别为86.2%~99.5%和86.9%~99.1%,均属于欧亚分支。其中9株鸡源毒株属于Y280-like亚系,在NA蛋白颈部63~65位点缺失了3个氨基酸;2株鸭源毒株属于G1-like亚系,在颈部39~40位点缺失了2个氨基酸。11株分离株除了69、146、200、234位点处潜在的N-糖基化位点比较保守以外,其余的糖基化位点都出现了变异情况。红细胞结合位点（HB）在431~433位点区域比较保守,但在366~373和399~404位点区域的氨基酸发生了明显的变异。抗原决定簇中部分氨基酸序列变异也较为活跃,推测与使用疫苗后的抗体选择有关。NA蛋白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相关的氨基酸突变。[结论]江苏、安徽、浙江等省养殖鸡群中的H9N2亚型禽流感病毒仍以Y280-like分支较为常见,鸭群中出现G1-like分支应引起人们的注意。NA基因存在一定的变异,应加强对该类病毒的分子流行病学监测。

[Objectives]The aim of the paper is to study the evolutionary characteristics and the genetic mutations of H9N2 subtype avian influenza virus（ AIV） in China. [Methods]11 H9N2 AIVs were isolated from domestic fowl in Jiangsu,Anhui and Zhejiang Provinces during 2008 to 2013. The neuraminidase（ NA） genes of the isolates were amplified by reverse transcription polymerase chain reaction. After cloning and sequencing,homology and phylogenetic analysis of nucleotide and deduced amino acid sequences of NA were performed. [Results]The phylogenetic analysis results showed that the NA genes of the isolates shared nucleotide and deduced amino acid homologies that ranged from 86. 2% to 99. 5% and 86. 9% to 99. 1%,respectively. All the 11 strains belonged to Eurasian lineage,and 9 chicken-origin isolates which had a stalk deletion of 3 amino acid residues at positions 63- 65 were derived from Y280-like sublineage,and 2 duck-origin isolates which had a stalk deletion of 2 amino acid residues at positions 39- 40 were derived from G1-like sublineage. The potential N-glycosylation sites at amino acid residues at enzyme active sites of the isolates were highly conserved and had no mutations associated with resistance to the neuraminidase inhibitors. [Conclusions]The results indicated that Y280-like sublineage is still the common H9N2 subtype AIV in chickens in Jiangsu,Anhui and Zhejiang Provinces,and the emergence of G1-like sublineage in ducks in this area should be paid attention to. The continuing evolution of NA gene of H9N2 subtype AIV in East China emphasizes the importance of surveillance throughout this region.