宁波市肠道病毒71型分离株全基因序列对比分析

Sequence analysis of enterovirus 71 strains isolated from patients with different clinical symptoms in Ningbo city

目的探讨浙江宁波地区2010年分离的肠道病毒71型(EV71)中神经毒性相关位点。方法采集宁波地区2010年手足口病不同症状患者粪便标本,进行荧光聚合酶链反应检测,阳性标本病毒分离;分8个片段对EV71全基因序列和VP1区进行逆转录聚合酶链反应(RT-PCR)、克隆、测序、拼接、同源性比较,用DNAstar推导其内含子编码的氨基酸序列并比对分析。结果 6株分离株基因全长均为7 406 bp,核苷酸同源性为97.9%～99.4%,其中3株死亡病例标本内部核糖体进入位点(IRES)中的124、272及408位,碱基分别为T、G、A,区别于其他3株手足口轻症患者分离株的A、A和G;3株死亡病例标本位于主要衣壳蛋白VP1区的第715位氨基酸和3C蛋白酶的第1 704位及1 706位的氨基酸分别为甲硫氨酸(met)、精氨酸(arg)及异亮氨酸(ile),区别于3株轻症病例的亮氨酸(leu)、赖氨酸(lys)及缬氨酸(val)。结论宁波地区2010年分离的EV71 IRES、VP1与3C位点变异与EV71神经毒性相关。

Objective To determine the neurovirulence-related sites of enterovirus 71 （ EV71 ） isolated in Ningbo area in 2010. Methods Fluorescent PCR was used to detect the excrements of patients with different clinical symptoms. EV71 was isolated and checked from the positive species, and the genome determined with reverse transcription-PCR （RT-PCR） was cloned,spliced,and sequenced with eight superimposed fragments. The homology was analyzed and the amino acid sequences were deduced and analyzed with DNAstar. Results All of the sequences of six strains were 7 406 bp in length and the homology of nucleotide were 97.9% - 99.4%. The nucleotide sequence of the three severe cases at the position of 124th ,272th and 408th were T, G, and A, respectively, distinguishing from the other three of A, A, and G. According to the deduced amino acid sequences, the three severe cases in the position of 715th, 1704th and 1706th were met,arg and ile, respectively, distinguishing from the other three of leu, lys, and val. Conclusion The variations in IRES ,VP1 ,and 3C may be associated with the neurovirulence of EVT1 isolated from patients in Ningbo area in 2010.