一株虹鳟源传染性胰腺坏死病病毒的分离与鉴定

为明确引起四川石棉某养殖场饲养的虹鳟患病死亡的病原体,实验对自然发病虹鳟进行大体病变观察并对其病原体进行分离,通过人工感染实验及多重RT-PCR鉴定确定病原体WZ160509,并对病原体的主要结构蛋白VP2进行扩增分析,同时对病变组织进行组织病理学观察。结果显示,患病鱼主要临床症状表现为体表发黑,腹部膨大,挤压腹部可见肛门喷射淡黄色黏液便;剖检可见肝脏、肾脏苍白;肠道内无食物,内积黄色黏液。将患病虹鳟组织匀浆液无菌接种虹鳟鱼生殖腺细胞系(rainbow trout gonad cell line,RTG-2)细胞,盲传3代均出现典型的细胞病变。人工感染实验显示死亡率高达90%,并出现与自然患病鱼相同的症状。多重RT-PCR检测发现,自然发病鱼、人工感染鱼以及病变RTG-2细胞均为传染性胰腺坏死病病毒(infectious pancreatic necrosis virus,IPNV)阳性,其主要结构蛋白VP2基因与美国分离株基因组1型聚为一支,且同源性分析表明,WZ160509-VP2与IPNV-VP2(AY026345)的同源性最高,序列一致性为95.8%。组织病理学观察显示,患病鱼胰腺细胞空泡变性,坏死;肝细胞空泡变性,坏死;肾小球轻度炎症,毛细血管通透性增加,肾小囊腔内有红色絮状蛋白类物质渗出,肾小管上皮细胞空泡变性。研究表明,从该养殖场患病虹鳟中分离到的病毒为IPNV。

Isolation and identification of infectious pancreatic necrosis virus from rainbow trout (Oncorhynchus mykiss)

This study investigated the etiology of Oncorhynchus mykiss that suffered fulminant death in Shimian of Sichuan. General observation, virus isolation, pathogenicity tests, multiple RT-PCR assay and histological examination were performed to explore its causes, and to indentify the pathogen WZ160509 in which the major structural protein VP2 was amplified and analyzed. Principal symptoms included darkened body, abdomen distension, yellow mucoid fluid fecal casts from anus. Internally, no food was found in digestive tract and enteritis, pale liver and kidney were observed. The filtrated homogenate was also inoculated into the O. mykiss gonad cell line cells, and the typical cytopathic effect was formed after three blind passages. The result of artificial infection test showed that IPNV could cause 90% death rate, and the injected trouts displayed similar clinical symptoms as the fish that was naturally infected. Multiple RT-PCR assay was performed to detect the presence of the virus from the fish naturally infected, the fish artificially infected and the infected cells, and all the results showed IPNV-positive, and the homology analysis, in which the test sample mixed the main structural protein VP2 gene and genomic U.S. isolates 1 type, showed the highest homology between WZ160509-VP2 and IPNV-VP2 (AY026345) and the sequence identity was 95.8%. Histopathologic analysis, pancreas and liver showed degeneration and necrosis. A mild inflammation was observed in glomerular. Besides, increased capillary permeability and red floccule proteins were also observed in capsular space. Renal tubule showed vacuolar degeneration and proteinuria. All the results show the isolated virus is IPNV.