Genetic characterization of VP1 region of enterovirus group A 71 circulating in Weifang, Shandong, 2014−2019
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摘要:
目的 了解2014—2019年山东省潍坊市流行的肠道病毒A组71型（EV71）VP1区基因遗传特征、进化关系及VP1区密码子所处的选择性压力。 方法 随机选取2014—2019年潍坊市EV71感染手足口病患者的阳性标本，经人横纹肌肉瘤细胞分离培养后提取病毒总RNA，采用反转录聚合酶链式反应对VP1区基因扩增后进行序列测定，并分析与EV71各基因型代表株的核苷酸及氨基酸的同源性，构建2008年以来我国不同省份EV71及本地EV71 VP1区系统进化树。 通过Datamonkey 在线服务器采用单似然祖先计数（SLAC）及固定效应可能性模型（FEL）方法对C4a亚型 VP1区密码子进行选择性压力分析。 结果 共获得的41株本地EV71毒株，均与C4a基因亚型的同源性最高，核苷酸同源性为94.9%～98.2%，氨基酸同源性为98.3%～99.6%。 系统进化树显示本地EV71毒株均属C4a基因亚型，形成2个明显的支系，每一分支中既有成簇毒株也有散在分布毒株，并与多地毒株亲缘关系较近。 采用SLAC和FEL分别鉴定出136和190个密码子为负向选择性压力位点，不存在正向选择性压力位点。 结论 2014—2019年潍坊市流行的EV71毒株属于C4a基因亚型，存在多条传播链，VP1区强烈的负选择压力表明 VP1区抗原的稳定性，VP1蛋白的变异不是由宿主免疫系统的选择性压力引起的，这有利于EV71疫苗的应用。 Abstract:Objective To understand the genetic characteristics and evolutionary relationships of the VP1 gene of enterovirus group A 71 (EV71) in Weifang, Shandong province, from 2014 to 2019, and further analyze the selective pressure on the VP1 region codon. Methods Positive samples from the patients with hand foot and mouth disease (HFMD) caused by EV71 were randomly selected in Weifang from 2014 to 2019 and total viral RNA was extracted after human rhabdomyosarcoma (RD) cell isolation and culture. Sequence determination was performed after amplification of the VP1 region gene by reverse transcription-polymerase chain reaction (RT-PCR), then nucleotide and amino acid homologies with reference strains of each EV71 genotype were analyzed. The VP1 gene sequences of EV71 strains detected after 2008 in China were obtained from GenBank for the construction of a phylogenetic tree with the VP1 gene sequences of local EV71 strains. The natural selection pressure on VP1 codon of C4a subtype EV71 were analyzed by single-likelihood ancestor counting (SLAC) and fixed effects likelihood (FEL) method respectively on Datamonkey website service. Results A total of 41 local EV71 strains were obtained, all of which had the highest homology with C4a genotype, with 94.9%−98.2% nucleotide homology and 98.3%−99.6% amino acid homology. The results of the phylogenetic tree showed that all the local EV71 strains belonged to C4a genotype, and formed two distinct clades, with clusters of strains and sporadic strains, and had close relationship with the strains in multiple areas. Totally 136 codons and 190 codons were identified as negative selected pressure sites by SLAC and FEL respectively, and no codon was under positive selection pressure. Conclusion The EV71 strains circulating in Weifang from 2014 to 2019 belonged to sub-genotype C4a and multiple viral transmission chains existed. The strong negative selection pressure in the VP1 region indicated the stability of the antigen in the VP1 region, suggesting that the variation in the VP1 protein was not caused by selective pressure from the host immune system, which is favorable for the application of EV71 vaccine. -
Key words:
- Enterovirus A 71 /
- VP1 /
- Phylogeny /
- Selective pressure
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图 1 基于VP1区构建的189株肠道病毒 A 组 71 型毒株的最大似然系统进化树
注：本地分离株采用★标记；不同基因亚型及本地分离株形成的2个分支用不同颜色表示，其中：●为A基因亚型； ●为E、D及G基因亚型； ●为F基因亚型； ●为B基因亚型； ●为C1、C2、C3及C5基因亚型； ●为C4b基因亚型； ●为C4a基因亚型； ●为分支1； ●为分支2
Figure 1. Phylogenetic tree of 189 EV71 strains based on VP1 gene constructed by using Maximum Likelihood method
表 1 41株本地毒株与肠道病毒 A 组 71 型各基因型代表株的核苷酸及氨基酸序列同源性
Table 1. Homology of nucleotide and amino acid between 41 local strains and reference EV71 strains with different genotypes
参考株 基因型 核苷酸同源性（%） 氨基酸同源性（%） U22521 A 80.3～82.4 93.6～95.2 U22522 B 83.1～85.2 96.2～97.6 AF135945 C1 87.8～90.1 97.6～98.6 AF135941 C2 87.4～89.3 97.5～98.5 AB115494 C3 86.9～89.4 97.9～98.9 JQ514785 C4a 94.9～98.2 98.3～99.6 AF302996 C4b 89.8～92.1 96.9～97.9 AM490141 C5 85.4～87.7 96.2～97.3 AY179600 D 78.7～80.4 93.9～94.6 JN255590 E 81.9～83.0 94.9～96.2 HG421068 F 81.5～82.9 95.2～96.2 KF906417 G 81.8～83.0 96.6～97.9 
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