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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 molecular characterization and gene characterization of four healthy children in Yunnan Province at the border crossing. Methods The primers 292/222 of universal coding region of VP1 of human enterovirus were amplified by reverse transcription - polymerase chain reaction. The products were purified and sequenced. The obtained sequences were genotyped by BLAST program and analyzed homology with other virus strains at home and abroad, and then the phylogenetic tree was constructed for genotyping and its evolutionary origin and epidemiological characteristics were analyzed. Results The four viruses in this study were novel enterovirus EV96. The nucleotide homology of the four strains was 82.7% ~ 95.7%, which was highly homologous to the previous EV96 nucleotide in Yunnan Province. The 28 EV96 VP1 sequences were divided into 3 genotypes by phylogenetic analysis. The genetic distances between genotype 1 and genotype 2 and geno-type 3 were 0.193 and 0.205, respectively. The genotype 2 and genotype 3 The genetic distance was 0.220, indicating that all the genotypes were distant from each other. Conclusion All Yunnan strains, including the 4 strains in this study, are relatively clustered in geno-type 1 with different branches, indicating that EV96 has different transmission chains in Yunnan province. The continuous use of molecular biology to monitor novel enterovirus in Yunnan Province can provide valuable information on viral genetics.