肠道病毒71型北京分离株全基因组序列分析

目的 了解2008年北京地区肠道病毒71型(EV71)分离株的全基因序列特点.方法 收集北京地区手足口病患儿的咽拭子样本12份,对其中1份样品08YM-3,经Vero细胞分离培养,提取病毒RNA.利用RT-PCR和5'、3'RACE扩增EV71全长基因.对PCR产物克隆和测序.利用DNAStar软件包的MegAlign进行核苷酸序列分析,构建系统进化树.结果 分离的EV71病毒株经过扩增后克隆得到3个涵盖EV71全基因组的阳性质粒,测序后拼接为EV71全基因组,命名为BJ08株.BJ08的5'非编码区(UTR)、P1、P2、P3、3'非编码区(UTR)和全基因组的核苷酸与C4亚型的参考序列同源性均最高,分别为95.6%～96.7%、88.3%～96.1%、78.1%～94.0%、90.8%～94.6%、85.9%～94.1%和90.9%～93.9%.BJ08株与其他亚型各区段的核酸同源性均低于90%.在全基因序列与VP1区构建的系统进化树中,BJ08株均与C4亚型在同一分支.BJ08株与C4亚型参考株VP1区的6个亚型相关氨基酸序列一致,VP1抗原性表位(92～107aa)无变异.结论 北京BJ08 EV71病毒分离株为C4业型.

Genetic characterization of enterovirus 71 complete genome isolated in Beijing, 2008

Objective To investigate the characterization of the complete genome of EV71 in Beijing, 2008 and to provide basis for selecting appropriate virus strain to develop vaccine. Methods 12 throat swab samples were collected from children with hand-foot-mouth disease (HFMD). One sample named 08YM-3 was cultured and isolated in vero cells. Viral RNA was extracted and carried out by RT-PCR and 5' , 3' rapid amplification of eDNA ends (RACE) to obtain the sequence from 08YM-3. PCR products were cloned and analyzed. Nucleotide identity between sequences was calculated and sequence alignments were made to generate phylogenetic trees using MegAlign in DNAStar. Results 3 clones were constructed that covered EV71 complete genome. Data from sequences analysis showed that this viral strain named BJ08 shared 95.6%-96.7%, 88.3%-96.1%,78.1%-94.0%,90.8%-94.6%, 85.9%-94.1% and 90.9%-93.9% in 5' UTR, PI, P2, P3, 3' UTR region and complete genome with C4 subtype, respectively. B J08 showed low nucleotides identity (<90%) with other subtypes. Phylogenetic trees established from alignment of the complete genome and VPI region indicated that B J08 belonged to C4 subtype. BJ08 and C4 subtype strains shared the same amino acids in 6 sites in VP1 region, which were associated with EV71 subtype. There was no mutation in VP1 antigen epitope (92-107aa). Conclusion This BJ08 strain belonged to C4 subtype. Further study on EV71 complete genome would have great significance for vaccine research.