云南省边境地区健康儿童中肠道病毒96型的鉴定及型分析

Identification and genotyping analysis of human enterovirus 96 isolated from healthy children in the border area of Yunnan province with Myanmar

目的对云南省4例边境口岸健康儿童的阳性病毒株进行分子生物学鉴定及基因特征分析。方法用扩增人类肠道病毒VP1部分编码区通用引物292/222进行逆转录—聚合酶链反应,反应产物纯化后进行序列测定。所得序列通过BLAST程序进行基因定型,并与国内外其他病毒株进行同源性分析,进而构建系统发生树进行基因分型,分析其进化来源及流行特征。结果本次研究的4株病毒为新型肠道病毒EV96型;4株间核苷酸同源性为82.7%～95.7%,且与云南省既往EV96核苷酸同源性较高;GenBank中获得的28株EV96型VP1区序列通过系统发生学分析可分为3个基因型,genotype 1和genotype 2、geno-type 3之间的遗传距离分别是0.193、0.205,genotype 2和genotype 3之间的遗传距离是0.220,表明各个基因型代表株之间的亲缘关系均较远。结论包括本次研究的4株毒株在内的所有云南株相对聚集在geno-type 1中,但出现不同的分支,说明EV96在云南省的流行存在不同的传播链。利用分子生物学方法持续对云南省新型肠道病毒进行监测,可提供有价值的病毒遗传学信息。

Objective To identify the enterovirus strains isolated from stool specimens of 4 healthy children in the border areas of Yunnan province with Myanmar.Methods RT-PCR was done using 292/222 primers described in Oberste′s methods and the amplified VP1 region products were sequenced for genotyping analysis.Phylogenetic classification was performed together with other available reference enterovirus strains in GenBank.Results Four enterovirus strains were isolated and identified as human enterovirus 96（HEV96） by RT-PCR and sequencing analysis.Four strains share nucleotide homology between 82.7% and 95.7%,and they also showed very high similarities to previous strains isolated in Yunnan.Phylogenetic analysis of these 4 strains together with all currently available strains from GenBank divided HEV96 into 3 genotypes,and the genetic distances were 0.193,0.205 and 0.220 between genotype I and II,I and III,II and III,respectively with our 4 strains grouped as genotype I.Conclusions Four strains of enterovirus isolated in the border area of Yunan province were classified into genotype I in the family of EV96.Phylogenetic analysis indicated that these 4 strains were further sub-grouped into different branches,suggesting that there might be different transmission chains of HEV96 co-circulating in Yunnan province.Efficient identification of human enterovirus will be helpful in the surveillance and control of HEV96 infection.