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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 understand the gene signature of VP1 coding region of human Enterovirus Type 71 (HEV71) in acute flaccid paralysis (AFP) cases in Jilin Province. Methods The original stool samples from AFP cases in children aged <15 years from 2006 to 2007 in Jilin Province were isolated by using human Rhabdomyosarcoma (RD) cells. The positive samples were confirmed by Enterovirus (EV) combined serum and HEV71 Serotypes were identified and identified as HEV71 strains. VP1 coding region-specific primers were used to amplify full-length VP1 encoding by reverse transcription-polymerase chain reaction (RT-PCR) The nucleotide sequence and the nucleotide sequence were determined. The nucleotide sequence and amino acid sequence were analyzed by Bioedit software. The phylogenetic tree was constructed by MEGA software and the genetic relationship was analyzed. Results Four HEV71 strains were isolated from 176 patients with AFP. The serotypes were determined by HEV71 specific serum neutralization test and further confirmed by molecular biology method. The nucleotide homology of VP1 coding region of four HEV71 strains ranged from 96.6% to 100% and the amino acid homology was from 98.3% to 100%. Compared with the HFMD in 2009-2010, The homology of nucleotide sequence of HEV71 isolated from patients was 96.2% ~ 98.8% and the amino acid homology was 98.3% ~ 99.3%. The C4a evolutionary branch representative strains isolated from HFMD patients in Fuyang, Anhui Province in 2008 were EU703813 -Fuyang-08/2-C4 showed nucleotide homology of 96.5% -98.8% and amino acid homology of 98.3% -99.3% .Compared with C4b evolutionary clade isolated from HFMD patients in Shenzhen, Guangdong Province in 1998, AF302996-SHZH98 (Shenzhen, Shenzhen) -C4 had nucleotide homology of 92.1% -93.1% and amino acid homology of 96.2% -97.3%. The constructed phylogenetic tree showed that all four viruses were located in C4a evolutionary branch. Conclusion HEV71 isolated from AFP cases in Jilin province in 2006 and 2007 belonged to the C4a evolutionary branch of C4 subtype. By comparing with HEV71 strain isolated from HFMD patients in Jilin Province in recent years and HEV71 in GenBank In comparison, no HEV71 isolated from AFP cases and HEV71 isolated from HFMD cases showed significant difference in nucleotide level of VP1 coding region.