云南省昆明市东川区三带喙库蚊携带乙型脑炎病毒分子特征  

Molecular characteristics of Japanese encephalitis virus carried by Culex tritaeniorhynchus in Dongchuan District,Kunming City,Yunnan Province

作　　者：顾杨杨 何于雯 陈易居 杨振兴[3] 李楠[3] 吕顺燕 朱燕涛 阮方超 王佳丽 王静林[3] GU Yangyang;HE Yuwen;CHEN Yiju;YANG Zhenxing;LI Nan;LU Shunyan;ZHU Yantao;RUAN Fangchao;WANGJiali;WANG Jinglin(School of Public Health,Dali University,Dali,Yunnan 671003,China;Yunnan Provincial Hospital of Infectious Diseases,Kunming,Yunnan 650301,China;Yunnan Academy of Animal Husbandry and Veterinary Science,Yunnan Provincial Key Laboratory of Tropical and Subtropical Animal Viral Diseases,Kunming,Yunnan 650224,China;School of Public Health,Kunming Medical University,China;Dongchuan District Center for Animal Disease Control and Prevention,Kunming City,Yunnan Province,China)

机构地区：[1]大理大学公共卫生学院,云南大理671003 [2]云南省传染病医院,云南昆明650301 [3]云南省畜牧兽医科学院、云南省热带亚热带动物病毒病重点实验室,云南昆明650224 [4]昆明医科大学公共卫生学院 [5]云南省昆明市东川区动物疫病预防控制中心

出　　处：《中国血吸虫病防治杂志》2024年第4期361-369,共9页Chinese Journal of Schistosomiasis Control

基　　金：云南省科技厅基础研究重点基金(202201AS070062);国家自然科学基金(32260896,81960605);中央引导地方科技发展资金项目(202207AB110006);昆明医科大学艾滋病共感染传染性疾病诊疗科技创新团队(CXTD202111);云南省科技厅外国专家引智项目(202305AO350020)。

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5820只,经形态学鉴定,共有三带喙库蚊3843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10845个核苷酸的长序列,编码3432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�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 SwissModel.Results A total of 5820 mosquitoes were collected and 3843 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 obs

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 

分 类 号：R373.3[医药卫生—病原生物学]