Identification and characterization of a new strain of nervous necrosis virus isolated from pearl gentian grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) in China

Viral nervous necrosis (VNN), caused by nervous necrosis virus (NNV), is a worldwide disease that severely imperils many kinds of marine and freshwater fish. In this study, a NNV strain CNPgg2018 isolated from pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) juveniles was identified and characterized by histopathology examination, transmission electron microscopic (TEM), reverse transcription-polymerase chain reaction (RT-PCR), virus culture, phylogenetic analysis and experimental infection. The results showed that the diseased fish exhibited symptoms with loss of appetite, darkened body and abnormal swimming behavior. Histopathological examination revealed widespread diffuse vacuolation degeneration appeared in the brain, retina and peripheral nerve tissues. TEM observation showed that numerous virions with a diameter of about 25 nm scattered around the retinal vacuolation. Significant cytopathic effects (CPE) were observed in striped snakehead (SSN-1) and grouper brain (GB) cell lines, not in Chinook salmon embryo (CHSE-214), epithelioma papillosum cyprini (EPC) and fat-head minnow (FHM) cell lines after inoculation of pathological tissue homogenates. RT-PCR and phylogenetic tree analysis indicated that CNPgg2018 was a member of NNV with highly homology to RGNNV genotype. Besides, both monomer and tripolymer forms of capsid protein were detected through structural polypeptide analysis and three-dimensional model prediction. Infection experiment demonstrated that CNPgg2018 was virulent to pearl gentian grouper juvenile with a LD50 of 1.0 × 106.1 TCID50/fish. In conclusion, we investigated a natural disease in pearl gentian grouper and confirmed that the pathogen was NNV. The results of this study provide new insights into the current research on the biology, ecology and host range of NNV.