2009-2017年烟台地区甲型(H1N1)pdm09流感病毒神经氨酸酶基因进化分析

目的 分析2009-2017年烟台地区甲型（H1N1）pdm09流感病毒流行情况及神经氨酸酶（neuraminidase，NA）基因进化特征。方法 收集2009年8月~2017年8月烟台地区两家哨点医院流感样病例咽试子标本10 236份，狗肾细胞（Madin-Darby canine kidney cell，MDCK）分离流感病毒，血凝抑制实验进行病毒分型。选取甲型（H1N1）pdm09流感毒株43株，扩增NA基因全序列并测序，应用分子生物学软件分析其遗传变异特征。结果 系统发生分析表明大部分烟台分离株属于2、7、6C、6B.1和6B.2基因型；NA蛋白分子多个氨基酸位点发生变异，与疫苗株相比，6、7基因型增加了44位糖基化位点；6B.1和6B.2基因亚型，减少了386位糖基化位点；运用固定效应似然比模型和内部固定效应似然比模型发现34和386两个正向选择压力位点，NA蛋白酶活性中心位点及周围辅助位点均未发生变异。结论 2009-2017年烟台地区甲型（H1N1）pdm09流感病毒NA基因持续发生变异，仍对神经氨酸酶抑制剂敏感，未来仍应加强流感流行状况和病原基因特征监测。

Evolution analysis of neuraminidase genes of A(H1N1)pdm09 virus in Yantai from 2009 to 2017

Objective To better elucidate the prevalence and analyze evolution characteristics of neuraminidase (NA) gene of influenza A (H1N1)pdm09 virus circulating in Yantai from August 2009 to August 2017. Methods A total of 10 236 swabs were collected among patients with an influenza-like illness (ILI) admitted to 2 sentinel surveillance hospitals of Yantai from August 2009 to August 2017. All specimen were cultured in Madin-Darby canine kidney (MDCK) cells and identified by hemagglutination inhibition assay. Complete sequences of NA of 43 influenza A (H1N1)pdm09 strains circulating in Yantai were amplified, sequenced and analyzed using molecular and phylogenetic methods. Results The phylogenetic analysis showed that most strains grouped into genetic clades 2,7, 6C, 6B.1 and 6B.2. Compared to the vaccine strain, the amino acid substitution N44S occurred in clades 6 and 7 strains, resulting in an increase in the number of glycosylation sites. The N386K substitution appeared in subclades 6B.1 and 6B.2 strains, contributing to a loss of glycosylation site. Two positive pressure sites (34 and 386)were observed in NA proteins by FEL and IFEL model.The catalytic sites of the NA protein were conserved among all the influenza A (H1N1)pdm09 strains isolated in Yantai area. Conclusions All the influenza A (H1N1)pdm09 virus circulated in Yantai from August 2009 to August 2017 presented continual genetic variation and were still susceptible to neuraminidase inhibitors. Continuous epidemiological and virological surveillance are necessary.