In vitro and in vivo characterization of molecular determinants of virulence in reassortant betanodavirus

Abstract

We have previously reported that betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole exhibited a modified SJNNV capsid amino acid sequence, with two amino acid changes at positions 247 and 270. In the current study, we have investigated the possible role of both residues as putative virulence determinants. Three recombinant viruses harbouring site-specific mutations in the capsid protein sequence, rSs160.03247 [S247A], rSs160.03270 [S270N], and rSs160.03247+270 [S247A/S270N], have been generated using a reverse genetics system. These recombinant viruses were studied in cell culture and in vivo in the natural fish host. The three mutant viruses were shown to be infectious and able to replicate in E-11 cells, reaching final titers similar to the wild-type virus, although with a somewhat slower kinetics of replication. When the effect of the amino acid substitutions on virus pathogenicity was evaluated in Senegalese sole, typical clinical signs of betanodavirus infection were observed in all groups. However, fish mortality induced by all three mutant viruses was clearly affected. Roughly 40% of the fish survived in these 3 groups in contrast to the wild-type virus which killed 100% of the fish. These data demonstrate that residues 247 and 270 play a major role in the betanodavirus virulence although when both mutated amino acids 247 and 270 are present, corresponding recombinant virus was not further attenuated.