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目的对2011年吉林省肠道病毒71型(enterovirus type 71,EV71)分离株全基因组进行序列分析。方法将手足口病伴发重症脑炎患者的粪便样品接种至Vero细胞,分离EV71;采用RT-PCR法分8段扩增全基因组序列,进行双酶切及序列测定;应用Mega5软件构建EV71系统进化树;通过DNAMAN7和DNASTAR7软件进行EV71分离株核苷酸和氨基酸序列的同源性比较;经RDP4软件对EV71分离株进行同源重组分析。结果粪便样品接种至Vero细胞2 d后,细胞出现圆缩、透亮、聚堆的病变现象;各PCR扩增产物经双酶切鉴定,均可见与预期大小一致的目的条带;经种系进化树分析显示,EV71分离株属C4a亚型,命名为EV71-JiLin-11-China;其与A、B和C亚型间核苷酸序列的平均同源性分别为22.44%、17.72%和11.96%,与A、B和C亚型间氨基酸序列的平均同源性分别为96.0%、97.2%和97.3%;全基因同源重组分析显示,EV71分离株的165～1 946和7 289～7 366核苷酸区有重组现象,分别源于分离自湖北的C4a-EV71-Hubei-09-China及马来西亚的B4-SB2864-SAR-00和C1-S18062-SAR-98。结论 2011年吉林地区流行的EV71为C4a基因型,为EV71基因特征及流行病学的研究提供了实验依据。
Objective To analyze the whole genome of enterovirus type 71 (EV71) strain in Jilin province in 2011. Methods Faeces samples of hand, foot and mouth disease patients with severe encephalitis were inoculated into Vero cells and EV71 was isolated. The whole genome sequence was amplified by RT-PCR and double-digested and sequenced. Mega5 software was used to construct EV71 system The nucleotide and amino acid sequences of EV71 isolates were compared by DNAMAN7 and DNASTAR7 software. The homology analysis of EV71 isolates by RDP4 software was carried out. Results The stool samples were seeded into Vero cells for 2 days, and the cells showed round, translucent and polyheaded lesions. The PCR products were identified by double enzyme digestion, and all the bands were consistent with the expected size. The germline evolution The tree analysis showed that the EV71 isolates belong to the C4a subtype named as EV71-JiLin-11-China, and the average homology with the nucleotide sequences of subtypes A, B and C were 22.44%, 17.72% and 11.96 %, And the average homologies of the amino acid sequences with the subtypes A, B and C were 96.0%, 97.2% and 97.3%, respectively. Genome-wide homologous recombination analysis showed that the average homologies between 165-1 946 and 7 289-7 The 366 nucleotide region has a recombination originating from C4a-EV71-Hubei-09-China isolated in Hubei and B4-SB2864-SAR-00 and C1-S18062-SAR-98 in Malaysia, respectively. Conclusion The genotypes of EV71 in Jilin Province in 2011 were C4a genotypes, which provided experimental evidence for the characterization and epidemiology of EV71 gene.