大口黑鲈蛙虹彩病毒的分离鉴定与病理学研究  

Isolation,Identification and Histopathology of Largemouth Bass Ranavirus(Iridoviridae)from Micropterus salmoides

作　　者：李佳宇 绳秀珍[1] 唐小千[1] 邢婧[1] 迟恒[1] 战文斌[1] Li Jiayu;Sheng Xiuzhen;Tang Xiaoqian;Xing Jing;Chi Heng;Zhan Wenbin(Laboratory of Pathology and Immunology of Aquatic Animals,Key Laboratory of Mariculture,Ministry of Education,Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国海洋大学水产动物病害与免疫学实验室,海水养殖教育部重点实验室,山东青岛266003

出　　处：《中国海洋大学学报(自然科学版)》2025年第3期184-192,共9页Periodical of Ocean University of China

基　　金：国家重点研究发展计划项目(2023YFD2400701);国家自然科学基金项目(32273163,31872599)资助。

摘　　要：为了丰富大口黑鲈病毒(Largemouth bass virus,LMBV)的流行病学资料,深入理解病毒的特性及致病机理,本文从山东省烟台市某养殖场采集了患病的大口黑鲈(Micropterus salmoides),采用细胞培养、PCR扩增及系统进化分析等方法进行病原的分离鉴定。肉眼可见患病大口黑鲈多处出现红肿出血的症状,显微镜检查未发现寄生虫和细菌感染。将病鱼组织匀浆液接种鲤上皮瘤细胞(Epithelioma papulosum cyprini,EPC),出现明显的细胞病变;对EPC细胞进行透射电镜观察,在细胞质内发现直径约为150 nm的成熟病毒粒子,病毒粒子呈现虹彩病毒典型的正六边形结构。根据NCBI公布的蛙病毒的主要衣壳蛋白(Major capsid protein,MCP)基因序列设计引物,经PCR扩增和测序后得到一段长度为1392 bp的基因序列。对获得的MCP序列进行系统进化分析,发现该病毒株与蛙病毒属的其他13种病毒聚成一支,与Santee-Cooper蛙病毒(SCRV)、鳜鱼虹彩病毒(MRV)、大口黑鲈溃疡综合征病毒(LBUSV)等7株病毒的MCP序列同源性高达100%。向健康大口黑鲈腹腔注射200μL的105 TCID 50/mL的病毒液,7 d的累计死亡率可达100%。研究结果表明,我们分离到的病毒为虹彩病毒科蛙病毒属成员,将该病毒株命名为大口黑鲈蛙虹彩病毒LMBV-CN2022株。对病鱼组织进行病理学观察,石蜡切片苏木精-伊红(Hematoxylin-Eosin,H&E)染色结果显示肝、脾、肾组织中出现大量铁血黄素沉积,透射电镜结果显示脾和体肾中存在病毒粒子。In order to enrich the epidemiological data of largemouth bass virus(LMBV)and further understand the characteristics and pathogenic mechanism of the virus,in this study,the diseased largemouth bass(Micropterus salmoides)with signs of redness,swelling and hemorrhages on body surface was collected from a fish farm in Yantai,Shandong Province,China.The virus isolation and identification were performed by cell culture,histopathological analysis,PCR detection and phylogenetic analysis.No parasitic or bacterial infection was found in the diseased fish.Epithelioma papulosum cyprini(EPC)cells were inoculated with the homogenate of diseased fish tissues,and the cytopathic effect including rounding up and detachment of cultured cells appeared.The enveloped virions with a diameter of about 150 nm were observed in the cytoplasm of EPC cells under transmission electron microscope(TEM),which were hexagonal in shape like iridovirus.According to the main capsid protein(MCP)gene sequence of ranavirus retrieved from NCBI,the specific primers were designed and a gene sequence with a length of 1392 bp was obtained after PCR amplification.By phylogenetic analysis,this virus strain clustered together with other 13 ranaviruses.Among them,the similarity of the isolated virus with 7 virus strains including Santee-Cooper ranavirus(SCRV),Mandarinfish ranavirus(MRV)and largemouth bass ulcerative syndrome virus(LBUSV)was up to 100%.In vivo infection of healthy largemouth bass demonstrated that the mortality rate reached to 100%on day 7 after intraperitoneal injection of 200μL 105 TCID 50/mL virus.These results collectively revealed that the isolated virus belonged to the genus Ranavirus of family Iridoviridae,and accordingly we named it largemouth bass ranavirus LMBV-CN2022 strain.The histopathological observation showed that a large amount of hemosiderin was deposited in liver,spleen and kidney tissues.In addition,we found the virus particles in spleen and kidney under TEM.

关 键 词：大口黑鲈 蛙病毒 分离鉴定 组织病理 

分 类 号：S941.41[农业科学—水产养殖]