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目的了解2009年山东省临沂市分离的肠道病毒71型(EV71)基因组5’非编码区(5’UTR)序列特征,探讨基因组中引起神经毒性作用的候选位点。方法从手足口病患者的粪便标本中分离EV71,运用一步法逆转录聚合酶链反应扩增8个病毒株的5’UTR区,运用DNAstar和MEGA 4软件进行序列分析。结果 8个病毒株5’UTR区核苷酸为741～745 nt,核苷酸序列同源性介于97.0%～99.9%,8个病毒株的5’UTR区与C4亚型的同源性最高。序列比对发现,在5’UTR区第265 nt及703 nt处出现了核苷酸的突变(G265A,G703T)。遗传进化分析显示,8个病毒株序列与C4亚型的亲缘关系最为密切。结论此8个病毒株为C4亚型,核苷酸突变(G265A,G703T)可能与EV71的致神经毒性作用有关。
Objective To understand the sequence of 5 ’untranslated region (5’UTR) of enterovirus 71 (EV71) genome isolated from Linyi City in Shandong Province in 2009 and to explore candidate sites for causing neurotoxicity in the genome. Methods EV71 was isolated from stool samples of HFMD patients. One-step RT-PCR was used to amplify the 5’UTR of the eight strains. Sequence analysis was performed using DNAstar and MEGA 4 software. Results The nucleotide sequences of the 5’UTR of the 8 strains were 741-745 nt, the homologies of nucleotide sequences were between 97.0% -99.9%. The homologies between the 5 ’UTR regions of 8 strains and the C4 subtype highest. Sequence alignment revealed nucleotide mutations (G265A, G703T) at the 265 nt and 703 nt of the 5’UTR. Genetic evolution analysis showed that the eight virus strains were most closely related to the C4 subtype. Conclusion These 8 strains are subtypes C4, and nucleotide mutations (G265A, G703T) may be related to the neurotoxicity induced by EV71.