新疆2011-2013年肠道病毒71型VP1区基因特征分析

Genetic characteristics of VP1 region of the enterovirus 71 strains isolated in 2011-2013 from Xinjiang,China

目的了解新疆肠道病毒71型（EV71）分离株VP1区基因特征。方法选取2011-2013年新疆部分地州手足口病病例标本，临床标本或其病毒培养物经实时荧光PCR鉴定后，通过RT—PCR法进行VP1区编码基因扩增，并对扩增产物进行序列测定，所得序列用ChromasPro1．7．4，BioEdit7．1．11和MEGA5．1软件进行序列校正、拼接、整理和分析，并与EV71各型及亚型参考序列构建基于VPl序列的系统进化树。结果新疆EV71分离株之间核苷酸和氨基酸序列同源性在92．8％～100．0％和97．9％～100．0％，22株病毒株与安徽阜阳和山东临沂C4a基因参考株最为相近，核苷酸和氨基酸序列序列同源性在93．9％～98．6％和98．6％～99．6％。而与A、B基因型参考株差异较大，核苷酸和氨基酸序列序列同源性在82．1％～84．8％和95．9％～98．3％。直接用临床标本进行核酸提取、扩增和序列测定的阳性率为30．0％，低于用病毒培养物的阳性率（100．0％）。结论2011—2013年新疆EV71分离株均为C4a基因型，与安徽和山东分离的EV71毒株可能有相同的起源。直接用临床标本进行核酸提取、扩增和序列测定可用于肠道病毒的分型鉴定。

Objective To study the genetic characteristics of enterovirus 71 （EV71） isolated from hand-footmouse disease （HFMD） in 2011- 2013 from Xinjiang, China. Methods Real time RT-PCR was used for EV71 detection from the stool and throat swab specimens collected from HFMD patients in several districts of Xinjiang, China. Both original specimens and viral isolates from RD cell culture were used to do RT-PCR for the amplification and sequencing analysis of the VP1 gene of EV71. The nucleotide and deducted amino acid sequences of the VP1 gene were analyzed by ChromasPro 1.7.4, BioEdit 7.1.11 and MEGA 5.1 software. Phylogenetic tree was constructed with comparison of representative strains reported in China. Results The 22 strains isolated from Xinjiang shared 92.8 % -100.0 % and 97.9 %-100.0 % homology in nucleotide acid and amino acid sequences, respectively. All these strains shared the closest genetic relationship with the representative strain C4a which was isolated from Anhui and Shandong provinces, with 93.9%- 98.6% and 98.6%- 99.6% homology in nucleotide and amino acid sequences, respectively. However, these strains showed low genetic relationships with genotypes A and B EV71 strains （82.1% -84.8% and 95.9%-98.3% homology in nucleotide acid and amino acid sequences, respectively）. The 30.0% positive amplification in original speci mens was lower than that from the viral isolates of cell culture （100.0%）. Conclusions The 22 strains isolated in 2011--2013 from Xinjiang of China were grouped as subgenotype C4a. These strains are closely related to those from Anhui and Shandong provinces of China. The VP1 gene can be directly amplified and identified by sequencing using original specimen for rapid identification of enterovirus serotypes.