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目的对我国新分离乙脑病毒02-76株进行全基因序列测定和分析,了解乙脑病毒基因组结构及毒力特性。方法设计乙脑病毒全基因组扩增引物,RT-PCR扩增片段,PCR产物直接测序,拼接后获得全基因序列。通过ClustalX(1.8)、DNASTAR、GENEDOC(3.2)等生物学软件进行核苷酸序列及氨基酸序列分析和病毒的系统进化分析。结果新分离乙脑病毒02-76株全基因组全长10977个核苷酸,从96位到10391位,共10296个核苷酸编码一个开放阅读框,编码3432个氨基酸。与我国1949年分离的Beijing-1株相比较共存在248个核苷酸差异,16个氨基酸差异。与GenBank中选择的29株乙脑病毒全基因序列比较发现,其核苷酸总体差异率为0.6%~15.1%,氨基酸总体差异率为0.2%~4.6%。通过PrM/C区段、E区段、3′NTR区段及全基因序列进行系统进化分析均显示该毒株属于基因3型乙脑病毒。结论新分离的乙脑病毒02-76株属于基因3型,与中国分离株SA-14进化关系最接近。
Objective To determine and analyze the genomic structure and virulence of Japanese encephalitis virus (JEV) by sequencing the whole genome of newly isolated JE virus 02-76 in China. Methods Genomic DNA amplification primers of JEV were designed and amplified by RT-PCR. The PCR products were directly sequenced and the whole gene sequence was obtained after splicing. Nucleotide sequence and amino acid sequence analysis and phylogenetic analysis of the virus were performed by biological softwares such as ClustalX (1.8), DNASTAR and GENEDOC (3.2). Results The whole genome of newly isolated JE virus 02-76 strain was 10977 nucleotides in length, ranging from 96 to 10391. A total of 10296 nucleotides encoded an open reading frame encoding 3432 amino acids. Compared with the Beijing-1 strain isolated in 1949 in our country, there are 248 nucleotide differences and 16 amino acid differences. Compared with 29 JEV selected from GenBank, the overall nucleotide difference was 0.6% -15.1%, and the overall amino acid difference was 0.2% -4.6%. Phylogenetic analysis of PrM / C, E, 3'NTR and whole genome showed that this strain belonged to genotype 3 Encephalitis virus. Conclusion The new isolate of JE virus 02-76 belongs to genotype 3, which is closest to the evolution of SA-14 in China.