Isolation and characterization of Indian betanodavirus strain from infected farm-reared Asian seabass Lates calcarifer (Bloch, 1790) juveniles

We isolated the infectious viral agent from infected Asian seabass, Lates calcarifer (Bloch, 1790) using SSN-1 cells. The isolated virus was cultivated in indigenous marine fish cell lines developed from ornamental sea anemone fish Amphiprion sebae and from Asian seabass. Reverse transcriptase PCR using betanodavirus-specific primers gave positive results for the RNA from virus-infected cells (hereinafter referred to as L. calcarifer nervous necrosis virus Indian strain - LCNNV-In01). Serum neutralization test indicated partial homogeneity between Atlantic halibut nervous necrosis virus (AHNNV 692/9/98) and LCNNV-In01. Transmission electron micrographs of the virus-infected cell culture showed large numbers of 25-30-nm particles in cellular vacuoles with features similar to betanodaviruses. Pathogenicity study in seabass fingerlings using cell culture grown virus revealed nervous necrosis in retinal cells following a 21-day challenge trial. The nodavirus was reisolated from the infected fish using clownfish and seabass cell lines. Sequence analysis of the coat protein gene showed that the virus belonged to RGNNV group (GenBank Acc. No. FR669249). The isolation of the nodavirus from seabass in India and its potential to grow in non-homologous cell lines indicate the serious threat of the Indian strain of betanodavirus (LCNNV-In01) to cultured and ornamental marine fish that have their hatchery production successfully standardized in India.