The Betanodaviruses caused nerve necrosis in several fishes. There are two RNAs in the Betanodaviruses. RNA1 encodes RNA dependent RNA polymerase and RNA2 encodes capsid protein. I analyzed the RNA2 sequences of the viruses isolated from two species of grouper, Epinephelus malabaricus (MGNNV) and E. lanceolatus (DGNNV). The similarity of two grouper viruses (GNNV) was 99%. Their similarities to DlEV and SJNNV were 87.8% and 78.6%, respectively. The capsid protein was successful expressed and assembled to virus-like particles. Deleting N- and C-termini revealed different impacts on VLP formation. Deletion of 35 or 52 residues at the N-terminus completely ruined the VLP assembly, presumably due to removal of positively charged residues for binding RNAs. When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained. The ability of VLP formation diminished when 4 to 11 C-terminal residues were deleted. The termini that can be deleted without seriously destructing the VLPs are 25 and 3 residues at N- and C-termini, respectively. Expression the ORF of RNA2 in E. coli formed virus-like particles, indistinguishable from native virus particles in appearance, whereas a mutant of Asp-75 expressed no VLPs. The emergence of a trimer band in mutant D75N as wild type suggested that the Asp-75 mutation could halt the packaging process in the trimer stage, not proceeding to assemble intact VLPs. The D54N mutant remained the ability of VLP formation but lost packaging high molecular weight of RNAs. Another Asp mutant at C-terminus, D335A, lost the ability of VLP assembly. The D335 may play an important role on the instability of VLP structure.

Three epitope-mapping procedures were used to identify B-cell epitopes on Betanodaviruses: neutralisation escape mutant sequence analysis, phage display, and pepscan. Betanodaviruses have emerged as major pathogens of marine fish. These viruses are the aetiological agents of a disease referred to as viral nervous necrosis (VNN), which affects many species of fish that are economically valuable to the aquaculture industry. The identification of betanodavirus B-cell epitopes will facilitate the rational development of vaccines to counter VNN. A panel of mouse monoclonal antibodies (MAbs) was produced using hybridoma methodology for use in each of the epitope mapping procedures. These antibodies were characterised in Western blotting, ELISA, and virus neutralisation tests. Rabbit polyclonal sera, and serum samples from nodavirus-infected fish were also used for pepscan analyses. Attempts to produce betanodavirus neutralisation escape mutants, using plaque assay or limiting dilution based methods, were not successful. Two phage libraries expressing random peptides of seven (Ph.D.7™) or twelve (Ph.D.12™) amino acids in length as fusions to the coat protein were used to identify the ligands recognised by MAbs directed against betanodavirus. Neither of these phage libraries yielded conclusive results. Phage clones containing tandem inserts were obtained after MAb selection from library Ph.D.7™. Extensive screening and nucleotide sequence analysis of MAb-selected clones from library Ph.D.12™) failed to yield a consensus sequence. Pepscan analyses were performed using the recently developed suspension array technology (SAT). This was used to map the recognition sites of MAbs and serum samples onto a panel of overlapping synthetic peptides (12mers) that mimicked the betanodavirus coat protein. The results of pepscan analyses required careful interpretation due to the binding of antibodies and serum samples to multiple peptides. However, three regions of the nodavirus coat protein were identified as containing B-cell epitopes: amino acids 1-50, 141-162, and 181-212. These results are discussed in relation to previous studies of immune responses to betanodaviruses, and to the future development of betanodavirus vaccines and diagnostic reagents.