吉林省急性弛缓性麻痹病例中人肠道病毒71型VP_1编码区基因特征分析

Genetic Characteristic Study on VP_1 Coding Region of Human Enterovirus Type 71 Isolated from Acute Flaccid Paralysis Cases in Jilin Province

目的了解吉林省急性弛缓性麻痹(Acute Flaccid Paralysis,AFP)病例中,人肠道病毒71型(Human Enterovirus Type71,HEV71)VP1编码区的基因特征。方法 原始粪便标本来自吉林省2006～2007年<15岁儿童AFP病例,采用人横纹肌肉瘤(Human Rhabdom yosarcoma,RD)细胞进行病毒分离,阳性标本用肠道病毒(Enterovirus,EV)组合血清和HEV71特异性血清进行血清型鉴定,鉴定结果为HEV71的毒株,利用VP1编码区特异性引物,通过逆转录-聚合酶链反应(Reverse Transcription-Polym erase Chain Reaction,RT-PCR)扩增全长VP1编码区片段,并进行核苷酸序列测定,序列结果利用Bioedit软件进行核苷酸和氨基酸同源性分析,用MEGA软件建立基因进化树并进行基因亲缘关系分析。结果 176例AFP病人中共分离到4株HEV71,血清型由HEV71特异性血清中和试验确定,并通过分子生物学方法 进一步证实。4株HEV71的VP1编码区核苷酸同源性为96.6%～100%,氨基酸同源性为98.3%～100%;与2009～2010年吉林省手足口病(Hand,Footand Mouth Disease,HFMD)患者中分离到的HEV71毒株核苷酸同源性为96.2%～98.8%,氨基酸同源性为98.3%～99.3%;与2008年分离于安徽省阜阳HFMD患者中的C4a进化分支代表株EU703813-Fuyang-08/2-C4的核苷酸同源性为96.5%～98.8%,氨基酸同源性为98.3%～99.3%;与1998年广东省深圳HFMD患者中分离到的C4b进化分支代表株AF302996-SHZH98(Shenzhen,深圳)-C4的核苷酸同源性为92.1%～93.1%,氨基酸同源性为96.2%～97.3%。构建的基因进化树显示,4株病毒均位于C4a进化分支。结论吉林省2006年和2007年AFP病例中分离到的HEV71属于C4基因亚型的C4a进化分支;通过与近年来吉林省HFMD患者中分离到的HEV71毒株以及基因数据库(GenBank)中的HEV71相比较,未见AFP病例中分离到的HEV71与HFMD病例中分离到的HEV71在VP1编码区核苷酸水平上有明显差别。

Objective To study the genetic characteristic of VP1 coding region of human enterovirus type 71 （HEV7a） isolated from clinical specimens of patients under 15 years old with acute flaccid paralysis （AFP） in Jilin province during 2006-2007. Methods Stool specimens were collected from AFP patients, then virus isolation was performed by inoculating in human rhabdomyosarcoma （RD） cell. Positive isolates were identified using neutralization test with enterovirus serum set and specific HEV71 serum. The identified HEV71 viruses were amplified by reverse transcription-polymerase chain reaction （RT-PCR） method, and the PCR products were sequenced and the homogeneity was analyzed by Bioedit sequence alignment editor and the phylogenetic dendrogram was constructed by the Mega software. Results Four HEV71 were isolated from 176 AFP cases , by inoculating RD cells, and were identified as HEV71 by neutralization test and confirmed by molecular typing methods. The homology of the nucleotide sequences in VP1 coding region between the four Jilin HEV71 strains was 96.6%-100%, and which of the amino acid sequences was 98.3 %-100%; The nucleotide homogeneity with C4a HEV71 strains isolated from hand, foot and mouth disease （HFMD） cases in Jilin province was 96.2%-98.8%, which of the amino acid sequences was 98.3%-99.3 %. The nucleotide homogeneity with C4a representative strain EU703813-Fuyang-08/2-C4 isolated from HFMD patients in Fuyang was 96.5 %-98.8 %, which of amino acid was 98.3 %-99.3%; The nucleotide homogeneity with the C4b representative strain AF302996- SHZH98-C4 isolated from HFMD patients in Shenzhen city of Guangdong province was 92.1%-93.1 %, and which of amino acid is 96.2%-97.3%. Phylogenetic analysis revealed that the four viruses were on the same evolution lineage with C4a linage. Conclusion The four HEV71 isolated from the AFP cases in Jilin province belong to C4a linage of 44 genotype. Comparing with HEV71 isolated from HFMD cases in Jilin province and different sources in recent years that available from GenBank, there was no significant difference in VP1 coding region gene.