Ranaviruses are globally emerging pathogens causing mass mortality in both farmed and wild populations. In this study, we confirmed that a novel ranavirus was related to mass die‐offs of black‐spotted pond frogs (Rana nigromaculata) in Sichuan Province, China. Histological examination revealed marked degeneration or necrosis in parenchymatous organs, and intracytoplasmic inclusions can be observed in the liver and intestine cells. The virus was isolated from diseased tadpoles (tentatively named the virus as RNRV), and the ranavirus‐like particles could be observed in infected EPC cells by the electron microscope. The tadpoles challenged with the isolated virus displayed similar clinical symptoms and pathogenicity as those naturally infected. Physicochemical characteristics showed that it was sensitive to chloroform treatment, trypsin treatment, heating treatment, acidity and alkalinity. Biological characteristics showed that RNRV can induce cytopathic effect (CPE) in various fish cell lines and the optimum growth temperature is 25~28°C. In addition, the complete genome sequence of RNRV was determined (GenBank accession number MG791866) and analysed. The results showed that the genome of RNRV consists of 104,286 bp containing 55.2% GC content and 104 predicted ORFs were identified in the genome. Phylogenetic analysis of the complete genome revealed that RNRV should belong to the FV3‐like group in genus Ranavirus, and is more closely related to RGV and STIV. These studies confirmed that the RNRV was the causative agent of this natural epizootic event and genome analysis indicated that it belongs to the FV3‐like group. In addition, viral physicochemical and biological characteristics will provide a scientific basis for prevention and control.
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