贵州省威宁县2015年至2017年外环境H9N2禽流感病毒神经氨酸酶基因分子特征分析

Molecular characteristic analysis of neuraminidase genes of avian influenza virus H9N2 in environments in Weining, Guizhou Province during 2015-2017

目的了解贵州省威宁彝族回族苗族自治县(以下简称威宁县)H9N2禽流感病毒的遗传变异情况,为禽流感病毒的防控提供依据。方法随机选取2015至2017年贵州省威宁县城北活禽市场外环境实时PCR初筛H9N2禽流感病毒阳性标本13株,对13株H9N2禽流感病毒进行基因组的提取、神经氨酸酶(neuraminidase, NA)基因的扩增和测序,运用生物信息学软件对NA基因的同源性、遗传进化、颈部缺失位点、糖基化位点、受体结合关键位点和耐药位点变异情况进行分析。结果13株H9N2禽流感病毒的NA基因核苷酸和氨基酸同源性分别为93.0%~100.0%和92.1%~100.0%,均属于DK/HK/Y280/97系,但遗传来源复杂多样。在NA蛋白颈部39~40位点有3株发生谷氨酰胺(Q)天冬酰胺(N)至Q赖氨酸(K)的突变,13株毒株在63~65位点均缺失苏氨酸(T)谷氨酸(E)异亮氨酸(I)3个氨基酸。13株毒株NA蛋白糖基化位点86、146、200和234较稳定,其余糖基化位点均存在数量和位点的变异。唾液酸结合位点的3个区域均有毒株发生突变,其中结合位点区域399~404变异最为活跃。NA蛋白酶活性位点和关键位点均未发生耐神经氨酸酶抑制剂药物的突变。结论贵州省威宁县2015至2017年H9N2禽流感病毒属于DK/HK/Y280/97系,遗传来源复杂多样,NA基因颈部区域位点、糖基化位点和唾液酸受体结合位点发生了不同程度的变异,需进一步加强对H9N2禽流感病毒的持续监测与防控。

Objective To understand the genetic variations of neuraminidase (NA) genes of avian influenza virus H9N2 in Weining, Guizhou Province, and to provide the scientific evidence for the prevention and control of avian influenza virus. Methods Ribonucleic acids (RNA) were extracted and NA genes were amplified and sequenced from 13 randomly selected H9N2 positive samples from the live poultry market (LPM) environments in north of Weining Yi and Hui and Miao autonomous county (Weining), Guizhou Province during 2015 to 2017. Then the homology, genetic evolution, and sites of stalk deletion areas, potential N-glycosylation, receptor binding regions and drug resistance of H9N2 subtype avian influenza viruses were analyzed by a series of bioinformation software. Results Homology analysis revealed that there were 93.0%-100.0% and 92.1%-100.0% similarity among 13 strains H9N2 avian influenza viruses in nucleotide and amino acid of the NA gene, respectively. All strains belonged to DK/HK/Y280/97 sub-lineage, but their genetic sources were complex and diverse. Thirteen strains had a stalk deletion of 3 amino acid residues TEI at positions 63-65 and 3 isolates had mutation QN to QK at positions 39-40. The potential N-glycosylation sites at amino acid residues 86, 146, 200, and 234 of the NA protein of all strains were highly conserved, while other N-glycosylation sites had quantity and site mutations. There were different mutation types at the three sialic acid binding site areas, especially at 399-404 area. All NA protease activity sites and key sites of the 13 strains had no mutations associated with resistance to the neuraminidase inhibitor drugs. Conclusions All 13 strains H9N2 viruses belongs to DK/HK/Y280/97 sub-lineage in Weining, Guizhou Province during 2015-2017, and their genetic sources are complex and diverse. The mutations on sites of stalk areas, potential N-glycosylation and sialic acid binding site areas are presented at different degrees. Hence, enhancing surveillance and controlling H9N2 avian influenza virus is necessary.