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.