Isolation, identification and phylogenetic analysis of a ranavirus isolated from Rana nigromaculata

Objective  To explore the etiology of a serious infectious disease occured in Rana nigromaculata tadpoles in a farm of Sichuan Province.

Method  Pathological examination and virus isolation were carried out for the diseased tadpoles of R. nigromaculata. The isolated pathogen was identified by artificial infection test, transmission electron microscopy, PCR detection and phylogenetic analysis.

Result  External clinical signs included hemorrhage on body surface and swollen abdomen with yellow ascites. Based on histopathological observation, we found that liver, spleen, kidney, pancreas and other organs had damages with obvious degeneration and necrosis focus, and basophilic inclusions appeared in the cytoplasm of some pathological cells. The tissue homogenates of diseased tadpoles were inoculated into epithelioma papulosum cyprini (EPC) cells under 25 ℃ and caused typical cytopathic effect (CPE) after four days with the TCID₅₀ of 10⁸ mL^–1. In the artificial infection test of virus fluid, the tadpoles showed the symptoms similar to those observed in naturally infective tadpoles and the mortality reached 80%, which suggested that the isolated virus was the determined pathogen of this disease. Transmission electron microscopic observation showed that the virus was regular hexagon with capsule, the diagonal diameter was (135±8) nm, and virus particles arrayed in crystalline or freely in the cytoplasm. PCR examination of the MCP gene showed positive results for samples of diseased tadpoles, aquaculture water source and isolated virus. Phylogenetic analysis based on MCP gene sequences indicated that the sequence of isolated virus had above 99% similarity to ranavirus, and the isolated virus belonged to the FV3-like virus group.

Conclusion  This study confirmed that ranavirus was the causative agent of this outbreak, and the virus is named as Rana nigromaculata ranavirus (RNRV).