宁波市手足口病优势毒株肠道病毒A组71型VP_1编码区基因进化分析

Evolution of the Hand,Foot and Mouth Disease Dominant Viral Strain A71 VP_1 Coding Region in Ningbo

目的研究浙江省宁波市手足口病（Hand,Foot and Mouth Disease;HFMD）优势毒株肠道病毒A组71型（Enterovirus Group A Type 71,EVA71）VP1编码区基因分子进化特征。方法收集宁波市2003~2010年HFMD患者标本,2003~2007年标本经过病毒分离后利用逆转录-聚合酶链反应（Reverse Transcription-Polymerase Chain Reaction,RT-PCR）对EV、EVA71和柯萨奇病毒A组16型（Coxsackie virus Group A Type 16,CVA16）进行检测;2008年标本利用RT-PCR直接进行检测;2009~2010年标本利用实时荧光（Real-time）RT-PCR直接进行检测,选择32例EVA71阳性标本进行VP1编码区基因扩增,扩增产物经过测序后利用序列分析软件（Lasergene）进行分析。结果宁波市2003~2010年共检测HFMD标本1335份,其中EV阳性1159份,总检出率为86.82%;EVA71阳性645份,占确证病例数的55.65%;CVA16阳性205份,占确证病例数的17.69%;未分型标本309份,占确证病例数的26.66%;未发现EVA71和CVA16同时阳性标本。32株代表株VP1编码区核苷酸序列的长度均为891碱基对（base pair,bp）,编码297个氨基酸,核苷酸和氨基酸的同源性分别为95.2%~100%和99.3%~100%,与C4a基因亚型代表株的核苷酸和氨基酸的同源性最高,分别为95.4%~99.2%和98%~100%。氨基酸进化位点分析表明,来源于不同病例类型的毒株在氨基酸的组成上无明显差异。在基因进化树上,32株流行株全部处于C4a基因亚型分支上,但位于不同的进化簇中。结论 EVA71是引起宁波市2008~2010年HFMD的优势毒株,也是导致HFMD重症病例和死亡病例的主要病原。来源于不同病例类型的毒株在氨基酸的组成上无明显差异。32株代表株均属于C4a基因亚型,与同期我国其他地区流行株共同进化。

Objective To determine the evolution of the VP1 coding region of hand, foot and mouth dis- ease （HFMD） enterovirus group A type 71 （EVA71） in Ningbo. Methods Specimens were collected from HFMD patients during 2003-2010. In 2008, EV, EVA71 and coxsackie virus group A type 16 （CVA16） were detected by reverse transcription-polymerase chain reaction （RT-PCR）. Real-time RT- PCR was conducted during 2009-2010. The VP1 coding region of EVA71 from 32 samples was cloned and sequenced. Sequence results were analysed by Lasergene software. Results A total of 1335 samples was collected. The positive rates of EV,EVA71, and CVA16 were 86.82% ,55.65% ,17.69% , respectively. Nueleotide and amino acids homology rates of VP1 gene among these 32 strains were 95.2%- 100% and 99.3%-100% , respectively. Strains shared the highest similarity with the C4a subgenotype with nucleo- tide and amino acid identity of 95.4%- 99.2% and 98%- 100%. The VP1 amino acid constituents of EVAT1 isolated from different cases showed no significant differences. Phylogenetie analysis revealed that the 32 Ningbo strains clustered within the C4a evolutionary branch of the C4 subgenotype. Conclusions EVA71 was the predominant patho- gen of HFMD in Ningbo during 2008-2010. EVAT1 was the main pathogen causing severe HFMD cases and deaths. The VP1 amino acid constituents of EVA71 shows no significant difference between severe HFMD cases （ including death ca- ses） and mild cases. The strains belonged to the C4a evolution branch of C4 subgenotype and coevolved with the common strains from other provinces in China's Mainland.