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目的对2008年分离自广西壮族自治区柳州市手足口病死亡儿童的1株肠道病毒71型(Enterovirus Type71,EV71),进行了全基因组的序列测定,并对其分子变异和进化特点进行了分析。方法采用逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR),分段扩增覆盖病毒全长序列的9个重叠片段(未包括多聚腺苷酸尾),并进行序列测定和分析。结果 EV71广西株基因组全长7405个核苷酸(nt),5’端和为3’端分别为742nt和84nt的非编码区,5’和3’非编码区间为一个6579nt长的开放阅读框,编码一个含2193个氨基酸的多聚蛋白,编码区内未见核苷酸的插入或缺失。在整个编码区,广西株与浙江株FJ158601的核苷酸及氨基酸序列同源性最高,为98.81%;在VP1编码区,与浙江株FJ158601、安徽阜阳株FJ439769核苷酸及氨基酸序列同源性最高,均为99.10%;在VP4编码区,与安徽阜阳株FJ439769的核苷酸及氨基酸序列同源性最高,为99.51%。结论首次完成广西EV71全基因组核苷酸序列的测定,经核苷酸序列同源性比对和进化树分析,证实EV71广西株属于C基因型中的C4基因亚型,与2008年国内各地区所爆发的EV71的亚型相吻合。遗传进化分析也发现,广西株与国内2008年各地区所分离的EV71毒株亲缘关系很近,提示可能源于同一种系。
OBJECTIVE: To determine the whole genome sequence of Enterovirus Type 71 (EV71) isolated from children with HFMD in Liuzhou City, Guangxi Zhuang Autonomous Region in 2008 and analyze its molecular variation and evolutionary characteristics . Methods Nine overlapping fragments (excluding poly A tail) covering the full-length sequence of the virus were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced Determination and analysis. Results The genome of EV71 Guangxi strain was 7405 nucleotides in length (nt), with non-coding regions of 742 nt and 84 nt at the 5 ’end and 3’ end, respectively. The 5 ’and 3’ non-coding region was a 6579nt long open reading frame , Encoding a 2193 amino acid polyprotein, coding region no nucleotide insertions or deletions. In the entire coding region, the nucleotide and amino acid sequences of Guangxi strain and FJ158601 strain were the highest, accounting for 98.81%. In the coding region of VP1, the nucleotide and amino acid sequence homologies with Zhejiang strain FJ158601 and Anhui Fuyang strain FJ439769 The highest was 99.10%. In VP4 coding region, the nucleotide and amino acid sequence of FJ439769 shared the highest homology of 99.51% with Anhui Fuyang strain FJ439769. Conclusion The complete nucleotide sequence of EV71 in Guangxi was firstly determined. The alignment of nucleotide sequences and phylogenetic tree showed that the EV71 Guangxi strain belonged to C subtype of C4 genotype. The subtypes of EV71 erupted coincide. Genetic analysis also found that Guangxi strain was closely related to the EV71 strain isolated in various regions of China in 2008, suggesting that it may be derived from the same strain.