2株Cluster 3鹅源坦布苏病毒的分离鉴定及其致病性研究  

Isolation,Identification and Pathogenicity of Two Strains of Cluster 3 Goose Tembusu Virus

作　　者：陈作鑫 陈宇欣 潘彦林 黄允真 李林林 董嘉文 向勇 徐志宏 孙敏华 张俊勤 黄淑坚 廖明 CHEN Zuoxin;CHEN Yuxin;PAN Yanlin;HUANG Yunzhen;LI Linlin;DONG Jiawen;XIANG Yong;XU Zhihong;SUN Minhua;ZHANG Junqin;HUANG Shujian;LIAO Ming(Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases,Ministry of Agriculture and Rural Affairs,Key Laboratory of Livestock Disease Prevention of Guangdong Province,Guangdong Provincial Observation and Research Station for Animal Disease,Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;College of Life Science and Engineering,Foshan University,Foshan 528225,China;College of Animal Science&Technology,Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China;National Key Laboratory of Agricultural Microbiology,College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]广东省农业科学院动物卫生研究所,农业农村部禽流感等家禽重大疾病防控重点实验室,广东省畜禽疫病防治研究重点实验室,广东省动物疫病野外科学观测研究站,广州510640 [2]佛山大学生命科学与工程学院,佛山528225 [3]仲恺农业工程学院动物科技学院,广州510225 [4]华中农业大学动物医学院,农业微生物学国家重点实验室,武汉430070

出　　处：《中国畜牧兽医》2025年第4期1750-1762,共13页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(32102691);广东省自然科学基金(2024A1515012732);广州市科技计划项目(2024A04J6754);广东省畜禽疫病防治研究重点实验室项目(2023B1212060040);广东省动物疫病野外科学观测研究站项目(2021B1212050021)。

摘　　要：【目的】明确Cluster 3鹅源坦布苏病毒(Tembusu virus, TMUV)基因组变异情况及其对鹅的致病性,为Cluster 3 TMUV的防控提供参考。【方法】利用BHK-21细胞对感染TMUV的鹅肝脏组织样品进行病毒分离,通过RT-PCR、间接免疫荧光试验(IFA)、透射电镜观察进行鉴定,并测定分离株的生长曲线。对分离株完成全基因组扩增后,使用ModelFinder、MrBayes等软件对其进行遗传进化分析,并对分离株的E蛋白进行氨基酸突变位点分析;测定分离株病毒滴度后,攻毒30日龄鹅,观察鹅各组织器官临床剖检病变及组织病理变化,使用实时荧光定量PCR检测鹅各组织脏器中的病毒载量。【结果】RT-PCR成功鉴定得到2份TMUV核酸阳性病料,接种至BHK-21细胞后,60 h即可观察到明显病变。将3代病毒液IFA检测可观察到明显红色荧光,透射电镜可观察到直径约50 nm、有囊膜的病毒粒子。从发病鹅肝脏组织成功分离得到2株TMUV,分别命名为JM3与JM1205。病毒一步生长曲线结果显示,JM3和JM1205株分别在培养60和48 h时病毒滴度最高。全基因扩增结果显示,JM3和JM1205株基因组全长均为10 994 bp。遗传进化树显示,JM3和JM1205株均为Cluster 3 TMUV成员,与Cluster 3 TMUV鸡源分离株CTLN遗传距离最近。氨基酸突变位点分析结果显示,与GenBank中最早上传的TMUV毒株MM1775株相比,JM3和JM1205株的E蛋白存在多个氨基酸位点突变,其中V157A突变可能与TMUV毒力增强相关。攻毒后1 d后鹅开始出现排绿色稀粪症状,攻毒后7 d开始出现神经症状。JM3组在攻毒后14 d仍持续排毒,JM1205组排毒持续至攻毒后11 d。攻毒后6 d,鹅出现体重增长减缓、下降的情况,至10 d开始恢复缓慢上升。剖检发现攻毒组鹅出现不同程度的脾脏肿大、胰脏坏死、肝脏发白、大脑充血;此外JM3株攻毒组鹅出现卵巢出血、心包积液;JM1205株攻毒组鹅出现心脏出血。攻毒后各时间点脾脏病毒载量均最高【Objective】This study was aimed to understand the genomic variation of Cluster 3 goose Tembusu virus(TMUV)and its pathogenicity to geese.【Method】BHK-21 cells were used to isolate the liver tissue samples of geese infected with TMUV,and identified by RT-PCR,indirect immunofluorescence assay(IFA),transmission electron microscope observation,and the growth curve of isolated viruses were detected.After the whole genome amplification of TMUV isolated strains,the genetic evolution was analyzed by ModelFinder,MrBayes and other softwares.The amino acid mutation sites of E protein of TMUV isolated strains were analyzed.After detecting the viral titers of TMUV isolates,the isolates were used to challenge 30-day-old geese,respectively.Then the clinical dissection of each tissue and organ,and the histopathological changes were observed.The viral load in each tissue and organ was detected by Real-time PCR.【Result】Two TMUV nucleic acid positive samples were successfully identified by RT-PCR.After inoculation into BHK-21 cells,significant lesions were observed within 60 h.Significant red fluorescence could be observed by IFA detection of the third-generation virus solution,and virus particles with a diameter of about 50 nm and a capsule could be observed by transmission electron microscopy.Two strains of TMUV were successfully isolated from diseased goose liver tissue and named JM3 and JM1205,respectively.The one-step growth curve of the virus showed that JM3 and JM1205 strains had the highest virus titers after 60 and 48 h of cultivation,respectively.The whole genome amplification results showed that the full-length genomes of JM3 and JM1205 strains were both 10994 bp.The genetic evolutionary tree showed that both JM3 and JM1205 strains were members of Cluster 3 TMUV,and had the closest genetic distance to the CTLN isolate of Cluster 3 TMUV chicken source.The analysis of amino acid mutation sites showed that compared with the earliest uploaded TMUV strain MM1775 in GenBank,JM3 and JM1205 strains had multiple amino

关 键 词：坦布苏病毒(TMUV) 分支3 分离鉴定 鹅 致病性 

分 类 号：S852.65[农业科学—基础兽医学]