First isolation and identification of red-grouper nervous necrosis virus (RGNNV) from adult hybrid Hulong grouper (Epinephelus fuscoguttatus × Epinephelus lanceolatus) in China

With the sharp development of present aquaculture, outbreaks of viral diseases have become a major obstacle for the sustainable development of the aquaculture industry with massive economic losses. Viral encephalopathy and retinopathy (VER) represents one of the main viral threats to world aquaculture, which is caused by nervous necrosis virus (NNV). In the present study, a new RGNNV strain named RGNNV-HL was isolated from cultured adult hybrid Hulong grouper (Epinephelus fuscoguttatus(♀) × Epinephelus lanceolatus(♂)) in Guangdong province, China. Cell culture-based virus isolation, RT-PCR detection, tissue section, electron microscopy, and complete genome sequence were used for virus identification. Furthermore, an artificial infection experiment was conducted for virulence testing. Gross examination of affected fish showed no noticeable lesions, no parasites in the gills or on the body surfaces, and no pathogenic bacteria. Histopathological examination revealed severe vacuolations in the brain of diseased groupers. Inoculation of the tissue homogenates of diseased fish induced obvious cytopathic effects in SSN-1, GF-1, and GS cell cultures. Electron microscopic observation revealed the presence of small-sized icosahedral-shaped viral particles (~30 nm) with no viral envelope, similar to betanodaviruses. RT-PCR and sequencing analysis confirmed the presence of red-spotted grouper nervous necrosis virus (RGNNV) capsid protein sequences. The virus clustered with RGNNV genotype, genus Betanodavirus within the Nodaviridae family on an evolutionary tree constructed based on the complete genome sequence. Thus, this newly isolated virus from adult Hulong grouper is tentatively designated RGNNV-HL. The artificial infection experiment showed that groupers began to die at 4 and 5 days after intraperitoneal injection and bath immersion, respectively. The cumulative mortality rates by injection and bath were 90% and 83.33%, respectively. To the best of our acknowledge, this is the first report of the isolation and characterization of RGNNV from Hulong grouper. Findings from this study will provide a candidate material and useful epidemiology information for further study on the infection mechanism and prevention and control strategies for RGNNV.