云斑尖塘鳢肿大细胞病毒属虹彩病毒的分离与鉴定

IDENTIFICATION AND CHARACTERIZATION ANALYSIS ON A MEGALOCYTIVIRUS ISOLATED FROM DISEASED MARBLED SLEEPY GOBY, OXYELEOTRIS MARMORATUS

2009年10月,广东顺德地区一云斑尖塘鳢养殖场暴发不明病因疾病,发病尖塘鳢体长15—18 cm不等,死亡率约85%,濒死尖塘鳢从池塘底层游至水面,呈现游动失衡状态直至死亡。死亡尖塘鳢腹部膨大,剖检可见肝脏、脾脏、肾脏肿大,有出血斑点,从内脏器官肝脏、脾脏和肾脏未分离到致病菌。病鱼内脏组织研磨过滤除菌后,腹腔注射20尾尖塘鳢,7d后开始出现死亡,10d后全部死亡,对照组无死亡。自然发病鱼和人工感染鱼的病理切片显示肝脏、脾脏和肾脏出现大量肿大细胞,超薄切片经电子显微镜观察,肝脏、脾脏和肾脏观察到大量病毒颗粒。电镜下病毒呈六边形,直径约135 nm,形态与虹彩病毒相似。针对虹彩病毒主衣壳蛋白(Major capsid protein,MCP)序列设计引物,提取自然发病鱼和人工感染鱼的DNA作为模板,均能扩增出预期大小的特异性产物。利用NCBI的Blast搜索,结果显示扩增序列与肿大细胞病毒属的传染性脾肾坏死病毒(ISKNV)、闪电丽鱼虹彩病毒(DGIV)和条石鲷虹彩病毒(RBIV)MCP核苷酸序列同源性分别为98.8%、98.1%和94.7%。利用MCP序列构建的系统发育树显示,导致云斑尖塘鳢发病死亡的病毒为肿大细胞病毒属虹彩病毒,暂命名云斑尖塘鳢虹彩病毒(MSGIV)。

An unknown disease with highly mortality of 85% occurred in marbled sleepy goby,Oxyeleotris marmoratus,cultured in Shunde area,Guangdong Province,in Oct.,2009.The diseased gobies were mainly 15—18 cm in body length and showed abdominal distension,lethargy and unusual swimming behavior such as floating on the pond surface until died.The postmortem examination revealed that liver,spleen and kidney were obviously swollen with petechial haemorrhages.No pathogenic bacterium was isolated from organs including liver,spleen and kidney.After filtration treatment,the tissue suspension was injected intraperitoneally with 20 gobies.The affected gobies began died without any obvious signs at 7 days post-infection（p.i.）,and all gobies died until 10 days p.i.,whereas there were no deaths in the control group.The section samples of liver,spleen and kidney including diseased and artificial affected gobies exhibited similar histopathological changes,showing many enlargement and necrosis cells.Under electron microscope（EM）,many icosahedral viral particles were observed in the cytoplasm from liver,spleen and kidney.The hexagonal virus particles measured an average 135 nm from side to side,and the shape was highly similar to that of iridovirus.With the primers designed according to the sequence of major capsid protein（MCP） from the GenBank database,specific products with predicted size were obtained from all diseased and artificial affected gobies.Comparative analysis of nucleotide sequences was performed with the GenBank databases using BLAST database network service.The results showed that the putative gene products from infected gobies shared high identity with homologous 98.8%,98.1% and 94.7% of infectious spleen and kidney necrosis virus（ISKNV）,dwarf gourami iridovirus（DGIV） and Red sea bream iridovirus（RBIV） respectively.Based on neighbor-joining analyses of the MCP gene sequences,phylogenetic tree was constructed and the result indicated that the pathogen in present study was a species within the genus Megalocytivirus,which was named tentatively marbled sleepy goby iridovirus（MSGIV）.