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引用本文:许峰,鲁建飞,魏永伟,苗亮,陈炯.一株大口黑鲈(Micropterus salmoides)虹彩病毒(Iridoviridae)的分离及鉴定.海洋与湖沼,2020,51(1):156-162.
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一株大口黑鲈(Micropterus salmoides)虹彩病毒(Iridoviridae)的分离及鉴定
许峰1, 鲁建飞1, 魏永伟1, 苗亮1, 陈炯1,2
1.农产品质量安全危害因子与风险防控国家重点实验室 宁波大学 宁波 315211;2.应用海洋生物技术教育部重点实验室 宁波大学海洋学院 宁波 315832
摘要:
2018年7月,浙江省某大口黑鲈(Micropterus salmoides)养殖场暴发疑似病毒引起的疾病。现场采样发现,病鱼体长约15-20cm,鱼体于水面下暗游,反应迟钝,体表有出血点或溃疡症状。本研究通过采用鲤鱼上皮瘤细胞(epithelioma papulosum cyprinid,EPC)培养、超薄切片透射电镜观察、病毒主要衣壳蛋白(major capsid protein,MCP)克隆与测序分析等方法,从患病大口黑鲈中分离得到一株病毒,鉴定其属于虹彩病毒科蛙病毒属,命名为大口黑鲈虹彩病毒宁波分离株(LMBIV-NB001)。EPC经患病鱼组织匀浆液接种后出现细胞圆缩、死亡、脱落等典型的细胞病变症状。将感染后的EPC细胞制作超薄切片,通过电镜观察发现,EPC细胞质中存在大量直径约120nm具囊膜的正六边形成熟病毒粒子,形态与虹彩病毒相似。根据虹彩病毒MCP基因保守区域序列设计特异性引物对病鱼组织样本进行PCR扩增,获得了1029bp的目的基因片段。将该扩增片段连入pMD19-T simple质粒后测序,经BLAST比对分析显示,其与GenBank中已报道的鳜鱼蛙病毒NH-1609、大口黑鲈溃疡综合征病毒BG/TH/CU3、EPC060608-08的MCP基因同源性最高,相似度均达到99.13%。构建系统进化树分析表明,本研究分离的LMBIV-NB001株与NC_038508、GU256635、MG941005等虹彩病毒科蛙病毒属毒株聚成一簇。本论文研究结果为不同地区蛙病毒属成员的起源和分化等相关研究等提供了基础材料。
关键词:  大口黑鲈  虹彩病毒科  MCP基因  鲤鱼上皮瘤细胞(EPC)  分离鉴定
DOI:10.11693/hyhz20190800163
分类号:S941.41
基金项目:国家自然科学基金项目,31772876号;浙江省自然科学基金项目,LZ18C190001号;宁波市科技创新团队项目,2015C110018号。
CHARACTERIZATION OF AN IRIDOVIRUS ISOLATE FROM LARGEMOUTH BASS MICROPTERUS SALMOIDES
XU Feng1, LU Jian-Fei1, WEI Yong-Wei1, MIAO Liang1, CHEN Jiong1,2
1.State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, China;2.Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Meishan Campus, Ningbo University, Ningbo 315832, China
Abstract:
A suspected virus-induced disease broke out in July 2018 in a large-mouth bass farm in Zhejiang Province, South China. The diseased fish showed slow reaction and swam underneath the water surface. The body length of diseased fish samples were 15-20cm with typical clinical signs such as haemorrhages on the skin and ulcer symptoms. Epithelioma papulosum cyprinid (EPC) culture, ultrathin section of transmission electron microscope, and major capsid protein (MCP) cloning and sequencing were used, and a virus from diseased largemouth bass was isolated, identified, and named as Ningbo isolate of largemouth bass iridovirus-NB001 (LMBIV-NB001). The tissue homogenate of diseased fish could cause typical cytopathic effect such as cell shrinkage, death, and exfoliation in EPC cells. Electron microscopy observation showed that there were a large number of virus particles about 120 nm in diameter in infected EPC cells, which is similar to iridescent virus. The conserved region of major capsid protein (MCP) gene of iridovirus was amplified by PCR, and a 1029bp specific gene fragment was obtained. Sequence alignment analysis of the DNA fragment showed that LMBIV-NB001 shared high identity (up to 99.13%) with the published MCP gene sequence of iridovirus. Phylogenetic analysis showed that LMBIV-NB001 strain had the highest homology with Mandarin fish ranavirus strain NH-1609, Santee Cooper ranavirus strain BG/TH/CU3, and largemouth bass ulcerative syndrome virus strain EPC060608-08, belonging to the genus Ranavirus of the Family Iridoviridae.
Key words:  largemouth bass Micropterus salmoides  Iridoviridae  major capsid protein (MCP) gene  epithelioma papulosum cyprinid, EPC  isolation and identification
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