成都某部一起感染Yamagata系乙型流感病毒血凝素基因分析

目的 分析成都某部感染乙型流感病毒遗传进化与血凝素（HA）基因突变位点。方法 通过犬肾上皮细胞（MDCK细胞）体外分离患者咽拭子标本流感病毒毒株，用PCR获取乙型流感病毒HA基因并测序，与NCBI数据库在线比对并利用MEGA 6.06软件构建系统发育进化树，分析突变位点。结果 通过MDCK细胞接种，分离出1株乙型流感病毒株，以感染病例咽拭子核酸及分离毒株的核酸为模板进行PCR扩增得到1 755 bp全长HA基因，获得的序列提交至GenBank数据库，获得基因登录号为MH236281。通过在线比对及系统发育树构建，该病例感染的病毒为Yamagata系乙型流感病毒。与乙型流感病毒Yamagata系的代表毒株Influenza B/Yamagata/16/88（GenBank No.M36105）相比，HA基因点突变碱基为57个；与世界卫生组织（WHO）推荐的疫苗株Influenza B/Utah/08/2014（GenBank No.KU592766）相比，突变碱基数为20个。进一步对HA1氨基酸突变位点进行分析，与四川地区往年流行株相比均发生了不同程度的突变，其中与2010年四川温江的分离株（GenBank No.KP461138）相比突变位点较少，仅有4处点突变；与WHO推荐的疫苗株Influenza B/Utah/08/2014相比，有2个氨基酸位点发生了变异，分别为L176Q和M255V，但突变没有处于HA1上的抗原决定簇区域。其中176位点是一个全新的突变，以往四川流行毒株、Yamagata系的代表毒株Influenza B/Yamagata/16/88及WHO推荐的疫苗株Influenza B/Utah/08/2014的HA1 176位点均为亮氨酸（L），而本研究病例感染的乙型流感毒株HA1 176位点突变为谷氨酰胺（Q）。结论 成都地区驻地部队2017-2018流行季感染的乙型流感病毒HA基因已发生一些突变，但突变尚未造成抗原性的改变。

Hemagglutinin genetic analysis of a Yamagata influenza B virus infected in a military camp in Chengdu

Objective To analyze the genetic evolution and hemagglutinin (HA) gene mutation sites of influenza B virus in Chengdu. Methods Influenza virus was isolated from patient''s throat swab samples using Madin-Darby canine kidney (MDCK) cells in vitro. The HA gene of influenza B virus was obtained by PCR and was sequenced. The phylogenetic tree was constructed and mutation sites were analyzed by online comparison with NCBI database and MEGA 6.06 software. Results One strain of influenza B virus was isolated by MDCK cells, and 1 755 bp full-length HA gene was obtained by PCR amplification using the nucleic acids of the throat swab and the isolated strain as templates. The obtained sequence was submitted to the GenBank database, and the gene accession number was MH236281. By online alignment and phylogenetic tree construction, the virus infected in this case was confirmed to be Yamagata type B influenza virus. There were 57 HA point mutation bases as compared with Influenza B/Yamagata/16/88 (GenBank No. M36105). There were 20 mutated bases in HA as compared with the World Health Organization (WHO)-recommended vaccine strain Influenza B/Utah/08/2014 (GenBank No. KU592766). HA1 amino acid mutation site was further analyzed. Compared with the epidemic strains in Sichuan in recent years, HA1 amino acid sites have undergone varying degrees of mutations, of which there were only 4 point mutations compared with Wenjiang/2010 isolate (GenBank No. KP461138). Compared with the WHO-recommended vaccine strain Influenza B/Utah/08/2014, two amino acid sites mutated (L176Q and M255V), but the mutation was not located in the antigenic determinant region of HA1. Among them, site 176 was a new mutation. The amino acid was leucine (L) at site 176 of HA1 epidemic strains in Sichuan, Influenza B/Yamagata/16/88, and the WHO-recommended vaccine strain Influenza B/Utah/08/2014, whereas the amino acid was glutamine (Q) in the influenza B strain isolated in this study. Conclusion Mutations have occurred in the HA gene of influenza B virus infected in Chengdu during 2017-2018, while these mutations have not yet caused antigenic changes.