云南省双江县三带喙库蚊中乙型脑炎病毒分离及编码区序列  

Isolation and coding region sequence of Japanese encephalitis virus from Culex tritaeniorhynchus in Shuangjiang County,Yunnan Province

作　　者：顾杨杨 何于雯 阮方超 杨杰林 杨绍昌 冷珺 李富雨 王佳丽 王静林[3] GU Yangyang;HE Yuwen;RUAN Fangchao;YANG Jielin;YANG Shaochang;LENG Jun;LI Fuyu;WANG Jiali;WANG Jinglin(Dali University,Dali,Yunnan 671003,China;Yunnan Provincial Hospital of Infectious Diseases,Kunming,Yunnan 650301,China;Yunnan Provincial Key Laboratory of Tropical and Subtropical Animal Virus Diseases,Yunnan Academy of Animal Science,Kunming,Yunnan 650224,China;Kunming Medical University,Kunming,Yunnan 650500,China;Animal Husbandry and Veterinary Technology Extension Center,Agriculture and Rural Bureau of Shuangjiang Autonomous County,Lincang City,Yunnan Province,Lincang,Yunnan 677399,China)

机构地区：[1]大理大学,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,云南昆明650500 [5]云南省临沧市双江自治县农业农村局畜牧兽医技术推广中心,云南临沧677399

出　　处：《中国热带医学》2025年第1期22-27,共6页China Tropical Medicine

基　　金：国家自然科学基金项目(No.32260896,No.81960605);中央引导地方科技发展资金项目(No.202207AB110006);云南省科技厅基础研究重点基金(No.202201AS070062);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(No.CXTD202111)。

摘　　要：目的了解云南省临沧市双江县蚊虫携带乙型脑炎病毒(Japanese encephalitis virus,JEV)情况及其分子特征。方法2023年8月,在临沧市双江县牛圈采用诱蚊灯采集蚊虫标本,经蚊种鉴定后每25只为一组,采用BHK-21细胞和C6/36细胞进行病毒分离,阳性分离物分别采用黄病毒属引物进行鉴定;然后采用针对GⅠ型JEV全长15对引物进行RT-PCR扩增,测序、拼接,采用MEGA X、DNAstar、GeneDoc、SOPMA和SWISS-MODEL等生物信息学软件进行序列分析。结果共采集到三带喙库蚊1300只,分52组进行病毒分离,获得1株阳性分离株(SJM23-22),接种C6/36和BHK-21细胞后均出现细胞病变,黄病毒属引物扩增为阳性。对该株病毒全序列测定拼接后,获得一条10840个核苷酸长序列,编码3432个氨基酸;基于全基因序列和E基因序列构建的遗传进化分析结果显示,新分离SJM23-22与柬埔寨GⅠa型毒株(C081)的亲缘关系最近,编码区核苷酸同源性为98.5%,氨基酸同源性为99.8%,而与其他基因型编码区核苷酸同源性均在90%以下,氨基酸同源性均在98%以下;位点分析结果显示与减毒活疫苗株SA14-14-2在E基因上共有22个氨基酸差异位点,其中在8个与神经毒力相关氨基酸关键位点上,存在7个位点差异;重要抗原表位分析结果显示,双江分离株与减毒活疫苗株在结构域Ⅲ中的3个重要抗原表位完全一致;二级结构及三级结构预测结果显示,该毒株以无规则卷曲为主要结构特征。结论在双江县三带喙库蚊中分离1株GⅠa型JEV,与抗原性表位相关的关键氨基酸位点未发生明显改变。本研究丰富了云南省双江县三带喙库蚊携带病毒情况,为云南省虫媒疫情的防控提供了参考。Objective To investigate the status and molecular characteristics of Japanese encephalitis virus(JEV)carried by mosquitoes in Shuangjiang County,Lincang City,Yunnan Province.Methods Mosquito specimens were collected from cattle pens using mosquito traps in Shuangjiang County,Lincang City in August 2023.After mosquito species identification,BHK-21 cells and C6/36 cells were used in one group of 25 mosquitoes each.Positive isolates were identified by flavivirus primers.Subsequently,the full-length GⅠ-type JEV was amplified using 15 pairs of primers with RT-PCR,sequenced,and spliced,and sequence analysis was performed using bioinformatics software such as MEGA X,DNAstar,GeneDoc,SOPMA,and SWISS-MODEL.Results A total of 1300 Culex tritaeniorhynchus were collected and divided into 52 groups for virus isolation,leading to the identification of one positive isolate(SJM23-22).After inoculation with C6/36 and BHK-21 cells,cytopathic effects(CPE)were detected,and flavivirus primers were amplified as positive.After the complete sequence of the virus was determined and spliced,a 10840 nucleotide long sequence was obtained,encoding 3432 amino acids.Phylogenetic analysis based on the whole gene sequence and E gene sequence showed that:The newly isolated SJM23-22 was most closely related to the GⅠa strain(C081)in Cambodia,with 98.5%nucleotide homology and 99.8%amino acid homology,while the homology with other genotypes was below 90%for nucleotides and below 98%for amino acids.The results of site analysis revealed 22 amino acid difference sites on the E gene compared to the live attenuated vaccine strain SA14-14-2,with 7 differences at 8 neurovirulence-related key amino acid sites.The results of important epitopes analysis indicated an exact match in three important epitopes in domainⅢbetween the Shuangjiang isolates and the live attenuated vaccine strains.The results of secondary structure and tertiary structure prediction showed that the strain was characterized by random curling.Conclusions One strain of GⅠa-type JEV w

关 键 词：GⅠa乙型脑炎病毒 全基因组 系统进化分析 E蛋白结构预测 

分 类 号：R384.1[医药卫生—医学寄生虫学]