石斑鱼病毒性神经坏死病的病原分离和鉴定  

Isolation and identification of viral nervous necrosis virus from Epinephelus

作　　者：林楠 吴斌 李苗苗 王巧煌 林国清 樊海平[2] 林克冰[3] LIN Nan;WU Bin;LI Miaomiao;WANG Qiaohuang;LIN Guoqing;FAN Haiping;LIN Kebing(Fujian Provincial Fishery Technical Extention Center,Fuzhou,Fujian 350002,China;Freshwater Fisheries Research Instituteof Fujian,Fuzhou,Fujian 350002,China;Fisheries Research Institute of Fujian,Xiamen,Fujian 361000,China)

机构地区：[1]福建省水产技术推广总站,福建福州350002 [2]福建省淡水水产研究所,福建福州350002 [3]福建省水产研究所,福建厦门361000

出　　处：《福建农林大学学报（自然科学版）》2019年第4期474-479,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建省海洋与渔业结构调整专项资金项目(闽财指[2019]0062)

摘　　要：对福建省漳州市某水产养殖场发生的疑似病毒性神经坏死病的病鱼进行病原分离和鉴定.从出现疑似病毒性神经坏死病症状的石斑鱼苗上取眼部、脑部组织进行研磨,将匀浆液接种到条纹月鳢细胞系(SSN)细胞株SSN-1及其克隆细胞株E-11,两株细胞均出现明显的细胞病变,细胞肿胀变圆,呈空泡化并脱落,E-11细胞较SSN-1细胞更早出现细胞病变,且病变更明显.将病鱼眼部、脑部组织的研磨液、细胞培养上清进行RT-PCR检测,均检测出病毒性神经坏死病病毒(VNNV)的特异性条带,目的片段大小为427bp,序列比对结果提示该病原为赤点石斑鱼神经坏死病毒(RGNNV).电镜观察结果显示,细胞培养上清中存在直径约30nm的病毒颗粒,细胞质内大量病毒颗粒聚集成团,细胞器受损,细胞结构被破坏.将分离得到的VNNV进行温度、酸碱度敏感性试验,结果显示:50℃下处理30min后该病毒滴度明显下降,60℃处理30min后病毒失活;在pH为3、11的条件下病毒滴度均明显下降.To verify a suspected viral nervous necrosis from Epinephelus in Zhangzhou, eye and brain tissue were sampled and ground. The homogenate was inoculated to striped snakehead (SSN-1) cell and its cloned cell line E-11. Subsequently, both SSN-1 and E-11 showed obvious cytopathic effect, the cell swelled and then became vacuolate and detached.The cytopathic effect took place earlier and more severe for E-11 cells than SSN-1. Furthermore, specific fragment of viral nervous necrosis virus (VNNV) was detected from the homogenate and cell culture supernatant by RT-PCR, whose sequence was 427 bp. Under electron microscope, the virus particles were around 30 nm in diameter. A large number of virus particles aggregated into groups in the cytoplasm, resulting in damaged organelles and cell structure. Temperature and pH sensitivity test showed virus titer of the isolated VNNV decreased significantly after being under 50 ℃ for 30 min, and then became inactivated after being under 60 ℃ for 30 min;besides, its virus titer decreased obviously after being under pH 3 and pH 11.

关 键 词：石斑鱼 病毒性神经坏死病 分离 鉴定 

分 类 号：S943[农业科学—水产养殖]