摘要
目的通过对2016-2017年襄阳市手足口病(hand, foot and mouth disease, HFMD)样品的分离与鉴定,了解主要病原之一的柯萨奇病毒A2型(Coxsackievirus, CV-A2)的分子生物学特征。方法收集2016年9月-2017年12月襄阳市HFMD患儿肛拭子样品,用人类横纹肌肉瘤细胞(RD细胞)培养,分离病毒。RT-PCR扩增CV-A2 VP1基因,Megalign软件分析VP1基因同源性,MEGA6软件构建系统发育树。比较3种疾病(急性迟缓性麻痹、手足口病和急性呼吸道感染)CV-A2分离株VP1氨基酸序列,分析可能的致病位点。扩增CV-A2代表株基因组全长序列,用SimPlot软件分析可能的重组事件。结果 2016-2017年CV-A2襄阳株之间VP1核苷酸及氨基酸同源性分别为96.4%~99.8%,98.0%~100.0%;襄阳株与CV-A2原型株(Fleetwood株)之间的核苷酸及氨基酸同源性分别为80.8%~81.9%,95.3%~95.9%。与襄阳株同源性最高的为2017年江西株(GenBank:MG926784),核苷酸及氨基酸同源性分别为96.7%~98.8%,98.0%~98.6%。襄阳市HFMD主要病原之一的CV-A2 VP1基因系统发育树显示,襄阳株与国内主要流行株同属于基因型D。3种疾病分离株的VP1氨基酸比对发现,急性迟缓性麻痹分离株和HFMD分离株在第21、60、82和215位存在差异;而HFMD分离株与呼吸道感染分离株之间只在167位存在差异,由谷氨酸变成天冬氨酸。CV-A2重组分析提示,襄阳株在P1区域与Fleetwood株同属一分支,在P2区域与CV-A5 Swartz株亲缘性最高,但在P3区域与CV-A16 G-10株有较高的相似度。结论虽然CV-A2襄阳株与其他流行株在VP1区序列上亲缘性较高,但其VP1氨基酸突变或与其他肠道病毒的型间重组可能导致其致病特性与流行病学特点发生变化。
Objective To understand the molecular characteristics of coxsackievirus A2(CV-A2) isolates by identifying the samples of hand, foot and mouth disease(HFMD) from 2016 to 2017 in Xiangyang City, Hubei Province. Metho-ds Rectal swabs were collected from September, 2016 to December, 2017 from children with HFMD and cultured in RD cells. The full-length VP1 genes of CV-A2 isolates were amplified by RT-PCR. The VP1 gene homology was analyzed by Megalign software. The phylogenetic tree was constructed by MEGA6 software. The isolated CV-A2 VP1 amino acid sequence was analyzed for possible pathogenic sites from three diseases including acute flaccid paralysis, HFMD and acute respiratory infection(RTI), and the possible recombination events were analyzed using the complete nucleotide sequence of a representative isolate by SimPlot software. Results The identities for nucleotide and amino acid sequence of the VP1 were 96.5% to 99.5% and 98.0% to 100.0% respectively, among CV-A2 Xiangyang isolates, and these were 80.8-81.9% and 95.3%-95.9% respectively, in comparison with Fleetwood prototype strain. The strain with highest identity to Xiangyang isolates was the 2017-Jiangxi strain(GenBank: MG926784), and the identities for nucleotide and amino acid sequence were 96.7%-98.8% and 98.3%-98.6% respectively. The phylogenetic tree of the VP1 gene of CV-A2 strains, one of the main pathogens of HFMD in Xiangyang, showed that the Xiangyang strain belongs to the predominant strain(with genotype D)in China.The amino acid sequences of the VP1 were aligned for the isolates from three different diseases. It was found that differences in amino acid residues existed at positions 21, 60, 82 and 215 between strains with acute flaccid paralysis and strains with HFMD, while there was only a single difference at 167, from glutamate to aspartic acid between the HFMD isolates and the respiratory infection isolates. The CV-A2 recombination analysis indicated that the Xiangyang strains were in the same branch in the P1 region as the Fleetwood strain, while it had the highest similarities with the Coxsackievirus A5 Swartz strain in the P2 region, and with the Coxsackievirus A16 G-10 in the P3 region. Conclusion The CV-A2 Xiangyang isolate is highly related to other epidemic strains in the VP1 gene, but in a final analysis of the amino acid sequence for the VP1 gene and recombination in the complete genome, it suggests that mutations and inter-serotype recombination of CV-A2 may result in changes in pathogenicity.
作者
罗志宇
王泽鋆
孟胜利
禹玉婷
麦健仪
李萌
申硕
LUO Zhi-yu;WANG Ze-jun;MENG Sheng-li;YU Yu-ting;MAI Jian-yi;LI Meng;SHEN Shuo(Laboratory I of Viral Vaccine, Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, Hubei Province,China)
出处
《微生物学免疫学进展》
2019年第4期14-21,共8页
Progress In Microbiology and Immunology
基金
2017湖北省技术创新专项(重大项目)(2017ACA078)
关键词
手足口病
柯萨奇病毒A2型
VP1基因特征
重组分析
Hand,Foot and Mouth Disease(HFMD)
Coxsackievirus A2
VP1 gene characteristics
Recombination
