Isolation and identification of a koi herpesvirus and phylogenetic analysis from Sichuan Province

In May 2015, an unknown disease caused severe economic losses in a common carp (Cyprinus carpio) culture pond in Sichuan Province. The mortality rate was 80%. Necropsy, bacteriologic test, histological examination, pathogenicity tests, virus isolation, PCＲ assay and phylogenetic analysis were performed to explore its causes. Principal symptoms included sunken eyes, hemorrhage at the base of the pectoral and pelvic fins, severe gill necrosis and swollen kidney. Bacteriologic test was negative. Histopathologically, the most important changes were observed in the gills and kidneys. The respiratory epithelial cells of gill lamellae was swelling and/ or dropping and congestion of the blood vessels in the gill filaments. The base of branchial epithelium also exhibit hyperplasia, hypertrophy and cellular layer increased. The obvious disorder in structure, swelling, and disintegration and necrosis were found in epithelial cells of renal tubulus. Positive results were obtained from all detected samples according to a standard PCR diagnosis of koi herpesvirus (KHV/ CyHV-3) Sph gene proposed by Office International des Epizooties (OIE) by using gills and kidney tissue DNA isolated from diseased C. carpio. After filtration treatment the kidney and liver homogenate was injected intraperitoneally into 20 healthy common carp and the injected carp showed similar clinical symptoms as the fish that was naturally infected. C. carpio died acutely in the trial group with a cumulative mortality rate 90%. Tissue suspension was inoculated to the common carp brain (CCB) monolayer cells and a typical cytopathic effect (CPE) was observed in CCB cells after three blind passages. Icosahedral viral particles were observed in cells under the electron microscope. The virus particles were spherical in shape measuring 180-200 nm in diameter. Based on Neighbor-Joining analyses of the TK gene sequences, phylogenetic tree was constructed and the result showed that the isolate belongs to Asian 1 genotype. This is the first report of KHV infection in cultured common carp associated with high mortality in Southwestern China. The result present in this study is important for virus evolution, classification, viral diagnosis and disease control.