为了解2007 ― 2008 年北京地区流行的肠道病毒71 型( EV71) 是否存在基因序列变异及其与病毒毒力的关系, 我们选择2007 年分离的3 株EV71( 其中1 株分离自重症手足口病患儿的咽拭子标本, 其余2 株分离自普通手足口病患儿咽拭子标本) 和2008 年分离的5 株EV71( 其中3 株分离自重症手足口病患儿的咽拭子或鼻拭子标本, 2 株分离自普通手足口病患儿的疱疹液标本) , 提取基因组RNA, 经反转录-聚合酶链反应
( RT-PCR) 扩增得到VP4 基因片段, 并进行核苷酸序列测定, 使用生物信息软件与GenBank 中的EV71 VP4 基因进行序列及病毒型别分析。结果表明, 所测得的8 株EV71 VP4 基因全长均为207 bp, 编码69 个氨基酸, 理论相对分子质量( Mr) 为7 ×10³。8 株EV71 病毒VP4 基因的核苷酸同源性在94% ～100% , 与GenBank 中其他EV71 病毒株VP4 的核苷酸同源性为82% ～100% , 与阜阳、深圳和台湾等地区流行的EV71 VP4 的核苷酸同源性比其他地区高。除了与印度报道的VP4 编码的氨基酸在第7 和54 位不同外( 印度株: 7 位蛋氨酸, 54位苏氨酸; 其余株7 位苏氨酸,54 位丙氨酸) , 这8 株EV71 VP4 编码的氨基酸序列之间以及与其他EV71 VP4编码的氨基酸同源性均为100%。8 株EV71 病毒VP4 与文献报道的3 株重症感染病毒株VP4 ( BrCr、MS 和NCKU9822) 核苷酸有较大差别, 而8 株病毒株中从重症感染( BJ97、BJ110B、BJ110Y 和BJ4243) 与轻症感染( BJ25、BJ47、BJ65 和BJ67) 分离到的毒株之间VP4 基因序列未见明显改变, 只有几个核苷酸存在差别。VP4 核苷酸序列的进化树分析表明, 这8 株EV71 均属于C4 亚型, 显示2007 ― 2008 年北京地区流行的EV71的VP4 基因相当保守, 分离自伴有神经系统感染的重症手足口病和普通手足口病患儿的EV71 的VP4 基因之间在核苷酸水平未出现同样的变异。结果提示, 近2 年来北京地区所流行的EV71 属C4 亚型。

To understand the relationship between variation of enterovirus 71 ( EV 71) and its virulence, the sequence of the VP4 gene of EV71 virus was analyzed. Eight strains of EV71 isolated from clinical samples collected from infants and children with hand, foot and mouth disease in the Children’s Hospital Affiliated to Capital Institute of Pediatrics in Beijing during 2007 to 2008 were used for comparison. Full length VP4 genes from these eight EV71 strains were amplified by reverse-transcriptase PCR ( RT-PCR) and sequenced after the amplicons were cloned into pUCm-T. The sequences were compared with VP4 genes from EV71 in GenBank. Sequence analysis and type identification were performed by bioinformatics ( DNAStar) . The full length of VP4 gene has 207 bp coding for a protein of 69 amino acids with estimated molecular weight of 7 ×10³ . The nucleotide homology of VP4 genes among the eight strains were 94% ～100% and homology of the deduced amino acids were 100% . The nucleotide sequence homology between these eight strains isolated in Beijing and those isolated in Fuyang, Shenzhen and Taiwan was higher than those isolated elsewhere. The homology of deduced amino acid sequences encoded by VP4 between these eight isolates in Beijing and those in GenBank was 100% except one strain isolated from India. The amino acids at the 7th and 54th position of VP4 of the strain from India were different from others. There were many differences between the nucleotide sequences of VP4 from the eight Beijing strains and those from severe cases ( BrCr, MS and NCKU9822) , as reported in the literature, whereas only a few nucleotide differences were observed between severe
cases ( BJ97, BJ110B, BJ110Y and BJ4243) and mild cases ( BJ25, BJ47, BJ65 and BJ67) among these eight Beijing strains. Phylogenetic analysis of the VP4 sequences from these eight strains indicates that the EV71 viruses that circulated in Beijing during 2007 to 2008 should be classified as subtype C4. The VP4 genes from the isolates in Beijing from2007 and 2008 were highly conserved. There was no consistent divergence in the sequences of VP4 between strains isolated fromsevere cases and those frommild cases, suggesting that the virulence of the EV71 virus does not seem to be related to the sequence of VP4.