广西壮族自治区2001年急性弛缓性麻痹病例中2株C_(4b)亚型进化分支人肠道病毒71型VP_1编码区基因特征分析

Genetic Analysis of the VP 1 Region of Human Enterovirus Type 71 Subgenotype C 4b Detected from Acute Flaccid Paralysis Cases in Guangxi Zhuang Autonomous Region during 2001

目的分析广西壮族自治区2001年急性弛缓性麻痹(Acute Flaccid Paralysis,AFP)病例中分离到的两株人肠道病毒71型(Human Enterovirus Type71,HEV71)VP1编码区的基因特征。方法 对2001年AFP病例中的非脊髓灰质炎肠道病毒(Non-Polio Enterovirus,NPEV)分离培养物,进行HEV71特异性逆转录-聚合酶链反应(Reverse Transcript-Polymerase Chain Reaction,RT-PCR)检测,对鉴定为HEV71的病毒分离物,随机选取其中2株进行VP1编码区核苷酸序列测定和分析。结果在25份NPEV分离物中,有5份鉴定为HEV71核苷酸阳性,选取GX(Guangxi,广西)01-64和GX01-71株进行进一步分析。VP1编码区核苷酸序列分析结果显示,它们与基因C型4b亚型(Subgenotype C4b)进化分支参考株的相似性最高,核苷酸序列相似性为95.2%,在遗传进化树中与C4b亚型病毒株同属一个分支。与1997～2008年国内HEV71流行株的氨基酸序列比对中发现,GX01-64和GX01-71株的第22位氨基酸、多肽SP(Synthetic Peptide,合成多肽)31～33以及CD4+T(Thymus,胸腺)细胞表位上的氨基酸发生了点突变。结论广西壮族自治区最早有HEV71感染的病例可以追溯到2001年,GX01-64、GX01-71株与国内1998～2003年所分离的C4b进化分支HEV71的亲缘关系很近,提示可能存在共同的祖先,并且它们的某些可能与抗原以及细胞表位有关的氨基酸序列发生了点突变。

Objective To analyze the genetic characteristics of the VP1 region of human enterovirus71 （HEV71） isolates from acute flaccid paralysis cases in Guangxi zhuang autonomous region in 2001. Methods The HEV71 strains were detected by reverse transcript-polymerase chain reaction （RT-PCR） from non-polio enterovirus （NPEV）isolates from acute flaccid paralysis cases in 2001. Two HEV71 strains were selected randomly for sequencing analysis. Results Five of the 25 NPEV isolates were tested as positive for HEV71 ribonucleic acid （RNA）. The VP1 nucleotide sequences of the strains GX （Guangxi） 01-64 and GX01-71, which were selected for sequencing, showed the highest homology of 95.2%with the subgenotype C4b reference strains. The nucleotide similarities were 93.2%-97.9 % between the two strains and the highly similar strains in China and abroad from 1997 to 2008. The phylogenetic analysis indicated that the two strains clustered as subgenotype C4b. The amino acid sequence alignment of the two strains and circulating strains in China during 1997-2008 revealed that the amino acid residues of the two strains had point mutations at position 22, polypeptide SP （Synthetic Peptide）31-33 and CD4＋T cell epitope. Conclusion HEV71 infection has existed in Guangxi since 2001. The relationship was close between the two HEVT1 strains studied and the subgenotype C4b isolates from China during 1998- 2003, indicating genetic linkages among those strains. Their amino acid sequences that may be associated with antigens and cell epitopes exhibit point mutations.