2014-2017年盐城地区甲型H1N1流感病毒HA、NA基因变异分析

摘要

目的 研究盐城地区2014-2017年甲型H1N1血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)分子遗传进化特征.方法 将盐城地区2014-2017年流感监测哨点医院以及流感暴发点采集的流感样病例标本进行核酸、病毒分离检测,选取2014-2017年甲型H1N1毒株,采用一步法RT-PCR方法扩增HA1和NA基因,扩增产物经测序,采用相应的生物信息软件进行核苷酸和氨基酸位点变异及基因种系进化特征分析.结果 2014-2017年抽取的17株甲型H1N1毒株HA1和NA基因各自分支的聚类关系基本一致,分为4个进化分支.与疫苗株A/California/07/2009(H1N1)相比,盐城地区毒株HA1基因编码区共涉及3个抗原表位(Ca、Sa、Sb)和6个抗原位点(K154R、S162N、K163Q、S185T、L191I、S203T)变异;受体结合位点220环中有3个位点(D222G/N、G223R和E224K)发生变异,190螺旋中涉及1个位点(L191I)的变异;2017年两株毒株(A/Jiangsu-YC/SWL1540/2017、A/Jiangsu-YC/SWL1545/2017)增加了1个162NQS糖基化位点.抗原表位、受体结合位点、糖基化位点发生了一定程度变异,导致在基因层面上A/California/07/2009(H1N1)疫苗株产生的保护效果有限,而2017年分离的两株毒株(A/Jiangsu-YC/SWL1540/2017和A/Jiangsu-YC/SWL1545/2017)与疫苗株A/Michigan/45/2015(H1N1)具有较好的保护效果.17株分离株未发生NA蛋白酶活性位点以及耐药位点的变化,部分毒株NA基因编码糖基化位点有一定变化.结论 2014-2017年盐城地区流行的甲型H1N1毒株HA1和NA基因正逐渐发生变异,这些变异位点的积累很可能会导致流感病毒发生实质性的抗原性的漂移.%Objective To analyze the genetic characteristics of the hemagglutinin (HA) and neuraminidase(NA) genes of the influenza A/H1N1 (09pdm) viruses isolated in the city of Yancheng in 2014-2017.Methods The throat swab specimens of patients with influenza-like illness (ILI) from sentinel surveillance hospitals and outbreak sites were detected using the method of real time RT-PCR.The influenza A/H1N1 (09pdm) viruses were isolated using M DCK cell culture method in 2014-2017.The strains in 2014-2017 were selected randomly and their sequences of the HA1 and NA genes were amplified through one step RT-PCR method and the PCR products were sequenced.The mutations of genes and acid locus were analyzed and the evolutional trees were generated using bioinformatics software.Results The clustering relationships of the respective branches of HA1 and NA genes of seventeen A/H1 N1 (09pdm) strains isolated in Yancheng area were basically the same and the phylogenetic trees of HA1 and NA genes were respectively clustered into four evolutionary branches.Compared with the vaccine strain A/California/07/2009 (H1N1pdm) in the Northern Hemisphere,a total of three antigen epitopes (Ca,Sa,Sb) in HA1 genes of strains in Yancheng area were involved in six antigenic sites (K154R,S162N,K163Q,S185T,L191I,S203T);there were three mutations (D222 G/N,G223 R,E224K) in the 220 ring and one locus (L191 I) in the 190 helix of the receptor binding sites;the two strains (A/Jiangsu-YC/SWL1540/2017,A/Jiangsu-YC/SWL1545/2017) isolated in 2017 increased the 162NQS glycosylation site.Because the strains of the antigen epitopes,receptor binding sites and glycosylation sites in the HA1 genes had a certain degree of variations in Yancheng area in 2014-2017,the protective effects of vaccine strain A/California/07/2009 (H1N1pdm) was limited at the gene level.The two strains (A/Jiangsu-YC/SWL1540/2017 and A/JiangsuYC/SWL 1545/2017) isolated in 2017 were clustered with vaccine strain A/M ichigan/45/2015 (H1N1 pdm) and had better protective effects.Seventeen A/H1 N1 (09pdm) strains had no mutations in catalytic residues and drug resistant sites of NA genes,but a part of strains had a certain degree of variations in glycosylation sites of NA genes.Conclusions These results indicated the HA1 and NA genes of influenza A/H1N1 (09pdm) viruses circulated in Yancheng area in 2014-2017 changed gradually.The accumulation of these mutations would result in antigenic drift of influenza A/H1N1 (09pdm) viruses.