Protective immunity of orange-spotted grouper(Epinephelus coioids) against a nervous necrosis virus isolated from China,and determination of the complete sequences of the virus

On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus (OGNNV), China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV.The major capsid protein (MCP)gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction (RT-PCR)and ligated into the pET32a expression plasmid.The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa.Recombinant protein with a molecular mass of 57.4 kDa was expressed in E.coli BL21 (DE3).Vaccine was prepared from the recombinant protein expressed in recombinant cells.The juvenile orange-spotted groupers (8 cm in average length) were immunized by intraperitoneal injection.Group A was challenged with infected tissue filtrates 25 d post-vaccination.The mortality in the vaccined group (A1, 30%) was a little higher than the unvaccined group (B2, 27.8%).Group B was challenged after three vaccine injections.The mortality in the vaccined group (B1, 16.7%) was lower than the unvaccined group (B2, 27.8%), And the relative percentage survival (RPS) value of vaccined group, compared with the unvaccined group, was 40%.The anti-recombinant protein sera with a 1:100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper.Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish (Group C1, mortality of 18.18%), compared with the fish (Group C2, mortality of 40%) which received infected tissue filtrates treated with control serum.Results implied the potential use of the capsid protein in immunization against OGNNV.