Mutations in the glycoprotein of viral haemorrhagic septicaemia virus thataffect virulence for fish and the pH threshold for membrane fusion

To study the molecular basis of virulence of viral haemorrhagic septicaemia virus (VHSV), we used a cross-reactive neutralizing MAb to select MAb-resi stant (MAR) mutants with reduced pathogenicity for fish. From sequence dete rmination of the G gene of MAR mutants, attenuated laboratory variant and a virulent field strains, we identified two distant regions of the glycoprote in associated with virulence: region I (aa 135-161), homologous to the puta tive fusion peptide of both rabies virus (RV) and vesicular stomatitis viru s (VSV), and region II (surrounding aa 431-433), homologous to RV and VSV d omains controlling the conformational changes necessary for the fusion proc ess to take place. Simultaneous mutations in both regions resulted in the m ost attenuated phenotype and we obtained genetic evidence that regions I an d II may be structurally linked. As the MAR mutants had mutations in or nea r domains involved in fusion, the fusion properties of VHSV and its variant s were analysed. This work allowed us to postulate that the fusion domain o f VHSV is probably constituted of two distinct regions of the protein conne cted through a disulfide bridge between cysteines 110 and 152. Finally, we obtained evidence suggesting that the pH threshold for fusion is a determin ant for virulence: restriction of fusion to a more acidic pH was associated with attenuation for the variant tr25 which had a shift of the threshold f or maximal fusion from pH 6.30 (for the parental strain) to pH 6.00; conver sely, two field strains which had maximal fusion at pH 6.60 were the most v irulent.