Diagnosis and ORF gene sequencing analysis of the nervous necrosis virus (NNV) isolated from cultured pearl gentian grouper, Epinephelus lanceolatus × Epinephelus fuscoguttatus, in China

Pearl gentian grouper, Epinephelus lanceolatus × Epinephelus fuscoguttatus, is a hybrid species with increasing market requirement in China. A disease outbreak occurred in the fish juvenile stages in Shandong Province, P.R. China. The gross signs were characterized by mass mortality, exhibiting dark skin coloration, reduce of feeding and having abnormal swimming behavior. Based on histopathological study, a large number of cellular vacuoles were observed in the diseased fish tissues of brain and retina. In addition, RT-PCR confirmed nervous necrosis virus (NNV) infection, and it was considered as the causative agent of the outbreak. This is, so far, the first report to detect NNV infection in Pearl gentian grouper. Furthermore, the open reading frames (ORF) of RNA dependent-RNA polymerase gene and coat protein gene of NNV also were sequenced after purification and cloning. Nucleotide sequence and the phylogenetic tree analysis of RNA1 and RNA2 showed extra high similarity (98.9%, 99.3% respectively) to red-spotted grouper nervous necrosis virus (RGNNV). These indicated that the NNV belong to RGNNV genotype, which is the only one genotype in P.R. China. Based on molecular study, the Cp genome included the ORF of 1017(6-1022) bases which encode protein of 338 amino-acids and the RdRp genome included the ORF of 2949 (49-2997) bases which encode protein of 982 amino-acids. These results would be helpful to understand NNV infection and provide information for fish health management.