大口黑鲈肝脾肿大病病原研究

摘要

为了查明2009年10月广东省佛山地区养殖大口黑鲈(Micropterus salmoides)中暴发的传染性疾病的病原,对病鱼的肝脏、脾脏和腹隔膜进行切片电镜观察,发现细胞质中有大量病毒颗粒,切面为六角形,直径约145～150 nm,病毒为二十面体对称结构、无囊膜、似虹彩病毒的病毒粒子.用除菌的病鱼组织滤液感染健康大口黑鲈,被感染鱼死亡率达90%以上.根据已知虹彩病毒主要衣壳蛋白(MCP)基因序列设计特异引物,提取人工感染发病鱼的肝脏、脾脏、肾脏组织的DNA进行PCR扩增,将扩增片段进行序列测定与分析,结果表明该序列与已报道的鳜(Siniperca chuatsi)传染性脾肾坏死病毒(Infectious Spleen and Kidney Necrosis Virus,ISKNV)MCP基因同源性为98%.电镜观察和MCP基因测序分析结果显示,该病毒的分类地位为虹彩病毒科(Iridoviridae)细胞肿大病毒属(Megalocytivirus).%During October, 2009, a serious infectious disease began to break out in cultured largemouth bass(Micropterus salmoides) of Foshan area in Guangdong province of China. Affected fish had following clinical signs including slowly swimming, floating at the surface of water, pale gill, swollen liver, spleen and kidney, pale liver and dark red spleen. Transmission electron microscopy of the focus liver, spleen and kidney revealed numerous cytoplasmic, icosahedral virion without envelope. The diameter of the virion was about 156-160 nm. Filtrate dilutions of liver, spleen and kidney were inoculated onto the monolayers of EPC, FHM and CIK cells respectively, but cytopathic effect(CPE) was not observed at the 8th days after infection. According to known MCP gene sequences of iridovirus, special primers were designed to amplify the MCP gene of the current virus. Liver,spleen and kidney DNA was extracted as PCR templates. PCR products were sequenced and the nucleic acid sequence of viral MCP gene is 98％ identical to those of ISKNV (Infectious spleen and kidney necrosis virus) and DGIV (Dwarf gourami iridovirus), and the amino acid sequence is 99％ to those of them. Transmission electron microscopy and MCP gene sequence of the virus indicated that the pathogen is a virus of genus Megalocytivirus,family Iridoviridae. Experimentally infection for healthy largemouth bass with filtrates of tissues by intramuscular injecting caused a more than 90％ mortality and the signs of the infected fish were similar to those of natural infection. In contrast, control fish exhibited no mortality and no clinical signs of infection. LMBV was a ranavirus isolated from diseased largemouth bass in USA. Naturally infected largemouth bass with LMBV appeared normal and lacked external lesions, but they floated at water's surface with an enlarge swim bladder. A similar ranavirus was isolated in China in 2008, and its predicted amino acid sequence was 98％ identical to corresponding sequence of LMBV, and naturally infected largemouth bass with the virus were all characterized by extensive ulceration in skin and muscle. Different from the symptoms of the above viruses, largemouth bass infected by the currently megalocytivirus exhibited swollen liver, spleen and kidney. All of the three viruses could cause death of largemouth bass, but the clinical signs of the infections were different. This study is the first report on largemouth bass naturally infected by megalocytivirus.