Isolation, Identification and Histopathology of Largemouth Bass Ranavirus (Iridoviridae) from Micropterus salmoides

In order to enrich the epidemiological data of largemouth bass virus (LMBV) and further understand the characteristics and pathogenic mechanism of the virus, in this study, the diseased largemouth bass (Micropterus salmoides) with signs of redness, swelling and hemorrhages on body surface was collected from a fish farm in Yantai, Shandong Province, China. The virus isolation and identification were performed by cell culture, histopathological analysis, PCR detection and phylogenetic analysis. No parasitic or bacterial infection was found in the diseased fish. Epithelioma papulosum cyprini (EPC) cells were inoculated with the homogenate of diseased fish tissues, and the cytopathic effect including rounding up and detachment of cultured cells appeared. The enveloped virions with a diameter of about 150 nm were observed in the cytoplasm of EPC cells under transmission electron microscope (TEM), which were hexagonal in shape like iridovirus. According to the main capsid protein (MCP) gene sequence of ranavirus retrieved from NCBI, the specific primers were designed and a gene sequence with a length of 1392 bp was obtained after PCR amplification. By phylogenetic analysis, this virus strain clustered together with other 13 ranaviruses. Among them, the similarity of the isolated virus with 7 virus strains including Santee-Cooper ranavirus (SCRV), Mandarinfish ranavirus (MRV) and largemouth bass ulcerative syndrome virus (LBUSV) was up to 100%. In vivo infection of healthy largemouth bass demonstrated that the mortality rate reached to 100% on day 7 after intraperitoneal injection of 200 μL 10⁵ TCID₅₀/mL virus. These results collectively revealed that the isolated virus belonged to the genus Ranavirus of family Iridoviridae, and accordingly we named it largemouth bass ranavirus LMBV-CN2022 strain. The histopathological observation showed that a large amount of hemosiderin was deposited in liver, spleen and kidney tissues. In addition, we found the virus particles in spleen and kidney under TEM.