四川地区一株传染性造血器官坏死病毒的分离鉴定及系统发育分析

2014年5月,四川省都江堰市某虹鳟养殖场暴发一种传染性疾病,幼鱼和鱼苗死亡率分别高达40％和80％.为探究此次疾病病因和流行规律,将病料进行解剖及细菌学检查、病理组织观察、人工感染实验、病毒分离、多重RT-PCR鉴定和系统发育分析.结果显示,病鱼主要临床症状表现为腹部膨大,体表发黑,肛门拖淡黄色黏液便,解剖见鳔壁、腹膜出血,胃胀气膨大和明显肠炎;细菌学检查为阴性;组织病理学上,头肾、肾脏和脾脏造血组织广泛性变性、坏死,肠黏膜下层嗜酸性颗粒细胞浸润与坏死,肝细胞变性、坏死形成局灶性的坏死灶,并在一些肝细胞胞浆内见嗜酸性包涵体.将病鱼的肝脏、脾组织研磨过滤灭菌后,腹腔注射20尾健康虹鳟,注射组均表现为急性死亡(累积死亡率=75％),并出现与自然发病鱼相同的症状.取病鱼组织匀浆滤菌液接种到鲤上皮瘤细胞(epithelioma papulosum cyprini,EPC),盲传3代出现典型的细胞病变效应(cytopathic effects,CPE).针对IHNV、IPNV与VHSV的多重RT-PCR检测显示自然发病鱼、人工感染病鱼和病变细胞均为IHNV阳性,扩增序列与IHNV核蛋白(nucleoprotein,N)基因同源性为99.9％.对分离株的糖蛋白基因“Mid-G”区域进行系统发育分析,结果显示该分离株与亚洲分离株聚为一支,属于JRt基因型.本研究首次报道我国西南地区养殖虹鳟中IHNV感染引起的疾病.

Isolation and identification of an infectious hematopoietic necrosis virus isolate and phylogenetic analyses from Sichuan Province

In May 2014, a severe infectious disease occurred among the farmed rainbow trout(Oncorhynchus mykiss)in Dujiangyan, Sichuan Province, associated with 40% and 80% mortality in juvenile fish and fingerlings, respectively. Bacteriologic test, histological examination, pathogenicity tests, virus isolation, multiple RT-PCR assay and phylogenetic analysis were performed to explore its causes and the epidemic law. Principal symptoms included darkening of the body, distension of abdomen, yellow mucoid fluid fecal casts from anus. Internally, hemorrhage occurred in the swim bladder and peritoneum, gastric distention and enteritis were observed. Bacteriologic test was negative. Histopathologically, anterior kidney, kidney and spleen showed degeneration and necrosis in the haematopoeitic tissue. Necrosis of the eosinophilic granular cells(EGCs)in the intestinal wall was also observed. Hepatocytes also showed degeneration and necrosis with some intracytoplasmic inclusions emerged in the hepatocyte. After filtration treatment, the spleen and kidney homogenate was injected intraperitoneally into 20 healthy trouts, and the injected trouts displayed similar clinical symptoms as the fish that was naturally infected. Trouts died acutely in the trial group with a cumulative mortality rate 75%. The filtrated homogenate was also inoculated into the epithelioma papulosum cyprini(EPC)cells, and the typical cytopathic effect was formed after three blind passages. Multiple RT-PCR reaction of tissue homogenates from the fish naturally infected, the fish artificially infected and the infected cells were performed, and the results showed all IHNV-positive. The sequenced amplification products shared 99. 9% identity with that of IHNV nucleoprotein gene(GenBank access number: KJ421216). Phylogenetic analysis based on the "mid-G" sequences showed that the isolate was classified into Asian isolates, belonging to the genogroup JRt. This is the first report of IHNV infection in cultured rainbow trout associated with mass mortality in Southwestern China.