Abstract: Objective　To isolate the Japanese encephalitis virus carried by Culex tritaeniorhynchus in Dongchuan District of Yunnan Province and analyze its molecular characteristics, so as to provide insights into the prevention and control of Japanese encephalitis in Yunnan Province. Methods　Mosquito specimens were collected using mosquito⁃trapping lamps from pig farms in Batang Village and Xiaoxin Village, Dongchuan District, Kunming City, Yunnan Province in July 2016, and the mosquito species was identified according to the mosquito morphology. Then, 60 to 100 mosquitoes of each species served as a group and were ground. Baby hamster kidney⁃21 (BHK⁃21) cells and Aedes albopictus clone C6/36 cells were used for virus isolation, and positive isolates were identified using flavivirus primers. The positive isolates were amplified using reverse transcription polymerase chain reaction (RT⁃PCR) assay with 15 pairs of specific primers covering the full length of the genotypeⅠJapanese encephalitis virus, and DNA sequence assembly was performed using the software SeqMan in the DNASTAR package. The obtained sequences were aligned with the complete sequences of 38 Japanese encephalitis virus downloaded from the GenBank with the software MegAlign, and the nucleotide and amino acid homology analyses of the obtained sequences were performed. The difference in amino acid sites was analyzed with the software GeneDoc, and phylogenetic trees were created based on the sequences of the coding region and E protein of the isolated Japanese encephalitis virus with the software Mega X. In addition, the secondary and tertiary structures of the E protein of the Japanese encephalitis virus were predicted using the online tool SOPMA and the software Swiss⁃Model. Results　A total of 5 820 mosquitoes were collected and 3 843 Cx. tritaeniorhynchus (66.03%) were identified according to the mosquito morphology. A positive virus isolate, termed YNDC55⁃33, was isolated from Cx. tritaeniorhynchoides following batches of virus isolation from mosquito specimens, and cytopathic effect was observed following inoculation into BHK⁃21 and C6/36 cells. The YNDC55⁃33 virus isolate was successfully amplified with the flavivirus primes, and a long sequence containing 300 nucleotides was obtained. Following sequence alignment using the BLAST tool, the sequence of the YNDC55⁃33 virus isolate had high homology with that of the genotypeⅠJapanese encephalitis virus. A long sequence with 10 845 nucleotides in length, which encoded 3 432 amino acids, was obtained by splicing the full sequence of the YNDC55⁃33 virus isolate. Phylogenetic analysis based on the whole⁃genome sequence and E gene sequence of the YNDC55⁃33 virus isolate showed that the new YNDC55⁃33 virus isolate was most closely related to the genotypeⅠGuizhou isolate (GenBank accession number: HM366552), with nucleotide homology of 98.5% and amino acid homology of 99.4%, and the YNDC55⁃33 virus isolate shared 97.96% ± 0.33% nucleotide homology and 99.35% ± 0.08% amino acid homology with other genotypeⅠJapanese encephalitis virus isolates, and < 90% nucleotide homology and < 98% amino acid homology with other genotypes of Japanese encephalitis virus. The YNDC55⁃33 virus isolate and the live attenuated virus vaccine candidate SA14⁃14⁃2 isolate differed at 16 amino acid sites on E gene, and 7 out of 8 key amino acid sites related to neurovirulence. The secondary and tertiary structures of the E protein of the YNDC55⁃33 virus isolate were predicted to be characterized by random coils. Conclusions　A genotype I Japanese encephalitis virus was isolated from Cx. tritaeniorhynchus in Dongchuan District, Kunming City. This virus isolate and the live attenuated virus vaccine candidate SA14⁃14⁃2 isolate does not differ at antigenic epitopes⁃related key amino acid sites, and the major protein structure of the virus isolate is random coils. This study adds new data for the epidemiological distribution of Japanese encephalitis virus in Yunnan Province, which may provide insights into the prevention and control of Japanese encephalitis in the province.

Key words: Japanese encephalitis virus, Culex tritaeniorhynchus, Whole?genome sequencing, Phylogenetic analysis, Protein structure

摘要： 目的　分离云南省昆明市东川区三带喙库蚊携带的乙型脑炎病毒，分析其分子特征，为云南省乙型脑炎防治提供科学依据。方法　2016年7月，在云南省昆明市东川区坝塘村、小新村养猪场采用诱蚊灯采集蚊虫标本，根据蚊虫形态进行蚊种鉴定。每种蚊虫均按60～100只为一组进行研磨后，采用乳仓鼠肾BHK⁃21细胞和C6/36白蚊伊蚊细胞进行病毒分离，阳性分离物采用黄病毒属引物进行鉴定。采用15对覆盖Ⅰ型基因型乙型脑炎病毒全长特异性引物对阳性分离物进行逆转录聚合酶链反应（reverse transcription polymerase chain reaction，RT⁃PCR）扩增，采用DNASTAR软件包中的SeqMan软件进行序列拼接，采用MegAlign软件对所获取序列及从GenBank 中下载的38条乙型脑炎病毒全序列进行比对、核苷酸和氨基酸同源性分析，采用GeneDoc软件进行氨基酸差异位点分析，采用Mega X软件构建所分离乙型脑炎病毒编码区及E蛋白序列的系统进化树，分别采用SOPMA在线工具和Swiss⁃Model软件进行E蛋白二级和三级结构预测。结果　共采集蚊虫5 820只，经形态学鉴定，共有三带喙库蚊3 843只（66.03%）。将蚊虫标本分批次进行病毒分离，从三带喙库蚊中分离到1株阳性病毒分离物，编号为YNDC55⁃33。将其接种至BHK⁃21和C6/36细胞后均出现细胞病变，黄病毒属引物扩增均为阳性，测序获得含300个核苷酸的长序列。经BLAST比对，YNDC55⁃33株病毒该段序列与Ⅰ型基因型乙型脑炎病毒同源性较高。对该株病毒全序列进行拼接后，获得一条长度为10 845个核苷酸的长序列，编码3 432个氨基酸。基于全基因序列和E基因序列构建的系统进化树分析结果显示，新分离的YNDC55⁃33株与贵州Ⅰ型基因型毒株（GenBank登录号：HM366552）亲缘关系最近，核苷酸同源性为98.5%、氨基酸同源性为99.4%；与Ⅰ型基因型其他乙型脑炎病毒毒株核苷酸同源性为97.96% ± 0.33%、氨基酸同源性为99.35% ± 0.08%；与其他基因型核苷酸同源性均< 90%、氨基酸同源性均< 98%。氨基酸位点分析结果显示，YNDC55⁃33株与减毒活疫苗株 SA14⁃14⁃2在E基因上共有16个氨基酸差异位点；在8个与神经毒力相关氨基酸关键位点上，两者有7个位点存在差异。YNDC55⁃33株E蛋白二级结构及三级结构预测结果均显示，该毒株以无规则卷曲为主要结构特征。结论　在昆明市东川区三带喙库蚊体内分离得到1株氨基酸型乙型脑炎病毒，该病毒与减毒活疫苗株 SA14⁃14⁃2在抗原性表位相关关键氨基酸位点上无明显差异，其主要蛋白结构为无规则卷曲。本研究丰富了云南省乙型脑炎病毒分布资料，可望为云南省流行性乙型脑炎防控提供参考。

关键词: 乙型脑炎病毒, 三带喙库蚊, 全基因组测序, 系统进化分析, 蛋白质结构