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目的采用逆转录-聚合酶链反应-序列分析方法,对脑炎与非脑炎手足口病患儿粪便和脑脊液样本中肠道病毒71型(EV71)部分VP1区进行核苷酸和氨基酸序列比对,探讨核苷酸或氨基酸变异与不同临床类型的关系。方法以临床诊断和基因诊断方法确定手足口病和EV71感染,用自行设计的2对引物,在EV71 VP1区扩增核苷酸,连接T载体后进行序列分析。所得序列连接后与EV71各亚型和安徽阜阳流行株核苷酸序列,通过DNAMAN和BI-OEDIT软件比对,分析其基因特征,并构建系统发生树。结果 6个不同临床类型手足口病病人核苷酸序列与A型、B型、C型的同源性分别为82.2%～82.6%、82.9%～84.1%和89.1%～95.1%,其中与C4亚型同源性最高,为94.8%～95.1%,与安徽阜阳地区流行株的同源性在98.0%～98.4%之间;与安徽阜阳株氨基酸序列比对,同源性在99.3%～99.6%之间,且均在563位由I(异亮氨酸)变为T(苏氨酸),仅有1例脑炎病人zxuan_VP1_2438-3260(脑脊液)在584位A(丙氨酸)变为G(甘氨酸),1例HFMD病人A06_B816064.VP1_2438-3227(粪便)在736位由W(色氨酸)变为C(半胱氨酸),余未出现特征性改变。结论神经源性肺水肿、脑炎与非脑炎手足口病患儿分离株EV71 VP1区核苷酸序列和推导的氨基酸序列无特征性差异。
Objective To detect the nucleotide and amino acid sequence of VP1 region of enterovirus 71 (EV71) in feces and cerebrospinal fluid samples of children with encephalitis and non-encephalitis hand-foot-mouth disease by reverse transcription-polymerase chain reaction-sequence analysis Right, to explore the relationship between nucleotide or amino acid variation and different clinical types. Methods The clinical diagnosis and gene diagnosis were used to identify hand, foot and mouth disease and EV71 infection. Two pairs of primers designed by ourselves were used to amplify nucleotides in the EV71 VP1 region. The T vector was ligated and sequenced. The obtained sequences were ligated with the nucleotide sequences of EV71 subtypes and epidemic strains of Fuyang in Anhui Province. The DNAMAN and BI-OEDIT software were used to analyze the gene characteristics and phylogenetic tree. Results The nucleotide sequences of 6 different clinical types of HFMD patients were 82.2% -82.6%, 82.9% -84.1% and 89.1% -95.1%, respectively. The homology between them and C4 The homology of subtype was the highest (94.8% -95.1%), the homology was 98.0% -98.4% with the epidemic strain in Fuyang area of Anhui province, the amino acid sequence of Fuyang strain was 99.3% -99.6 % And both changed from I (isoleucine) to T (threonine) at 563, and only 1 patient with encephalitis became (serine) zzuan_VP1_2438-3260 (CSF) at A = 584 G (glycine), and 1 case of HFMD A06_B816064.VP1_2438-3227 (stool) changed from W (tryptophan) to C (cysteine) at 736, with no characteristic changes. Conclusion The nucleotide and deduced amino acid sequences of EV71 VP1 isolated from children with neurogenic pulmonary edema, encephalitis and non-encephalitis HFMD have no characteristic difference.