湖北襄阳2016-2017年埃可病毒11型流行株基因特征分析

Genetic characteristics of Echovirus 11 circulating in Xiangyang,Hubei Province between 2016 and 2017

目的对2016—2017年中国湖北襄阳地区分离的埃可病毒11型(Echovirus 11,Echo11)基于VP1基因进行分子进化特征分析。方法采集2016—2017年中国湖北襄阳地区手足口病患儿肛拭子或咽拭子标本,应用real-time RT-PCR技术对肠道病毒阳性样本进行筛选,对肛拭子或咽拭子样本进行病毒分离。采用常规RT-PCR方法对在人横纹肌瘤细胞上分离的Echo11病毒襄阳流行株VP1区和3株代表株全长基因扩增并进行序列分析,使用DNAStar7.0软件包中的MegAlign、MEGA6.0中的Data和Phylogeny软件对核苷酸和氨基酸序列同源性、突变位点进行分析和构建系统进化树等。使用SimPlot软件进行重组分析,对3株代表株全长序列和GenBank中下载的核苷酸序列进行BootScan扫描,生成相似性图。结果本研究从3494份手足口病患儿样本中共分离出Echo11病毒11株,占比0.31%。11株Echo11流行株的VP1基因同源性比较高。核酸序列和氨基酸序列同源性分别为98.4%~100.0%和98.3%~100.0%。11株Echo11襄阳流行株与Echo11原型株(Echo11/Gregory,X80059)核酸序列和氨基酸序列同源性分别为73.9%~74.8%和87.7%~88.7%。襄阳地区流行的Echo11毒株均属于D进化分支,与中国大陆部分地区2013年以来的流行株亲缘关系相近。襄阳流行株和2010年Echo1河南流行株和2015年的Echo6湖北流行株在多个区域有较高的相似度,说明襄阳流行株基因组的多个区域可能发生了重组。结论襄阳地区流行的Echo11毒株均属于D进化分支,2016—2017年襄阳地区流行的Echo11是重组株。

Objective To analyze the molecular characteristics of Echovirus 11(Echo11)strains isolated in Xiangyang,Hubei Province from 2016 to 2017 based on the sequences of VP1 gene.Methods Rectal and throat swab specimens were collected from children with hand,foot and mouth disease(HFMD)in Xiangyang from 2016 to 2017.Echo11 strains were detected by real-time reverse transcriptase PCR(RT-PCR)and isolated after cultured in human rhabdosarcoma(RD)cells.The VP1 regions of Echo11 strains isolated from RD cells and the whole genomes of three representative Echo11 strains were amplified by conventional RT-PCR and the sequences were analyzed.DNAStar7.0(MegAlign)and MEGA6.0(Data)were used to analyze the homology and mutation sites in nucleotide and amino acid sequences.Neighbor-joining method was used to construct phylogenetic trees.Recombination analysis was performed with SimPlot software(BootScanning).Results A total of 11 Echo11 strains were isolated from 3494 HFMD cases,accounting for 0.31%.They were highly homologous in the VP1 gene.These strains shared 98.4%-100.0%homology in nucleotide sequences and 98.3%-100.0%homology in amino acid sequences.The homology between the 11 Echo11 strains and the prototype strain(Echo11/Gregory,X80059)was 73.9%-74.8%in nucleotide sequences and 87.7%-88.7%in amino acid sequences.All of the Echo11 strains circulating in Xiangyang were classified into lineage D,having a similarity to the strains circulating in some regions of China's Mainland since 2013.In multiple regions of the genome,the Echo11 strains isolated in Xiangyang were highly similar to the Henan Echo1 strains in 2010 and the Hubei Echo6 strains in 2015,suggesting there was recombination within the genome of Echo11 strains in Xiangyang.Conclusions The Echo11 strains circulating in Xiangyang from 2016 to 2017 belonged to lineage D and were recombinant strains.