A naturally occurring substitution in the E2 protein of Salmonid alphavirus subtype 3 changes viral fitness

•A naturally occurring mutation in the SAV E2 protein was studied through homology modelling and reverse genetics.•The mutation has a role in cell culture adaptation.•No difference in virulence or tissue tropism was observed in Atlantic salmon as a result of the mutation.•The mutation was associated with changes in persistence and shedding in vivo.

Phylogenetic analyses of the Salmonid alphavirus subtype 3 (SAV3) epizootic have suggested that a substitution from proline to serine in the receptor binding protein E2 position 206 has occurred after the introduction of virus from a wild reservoir to farmed salmonid fish in Norway. We modelled the 3D structure of P62, the uncleaved E3-E2 precursor, of SAVH20/03 based on its sequence homology to the Chikungunya virus (CHIKV), and studied in vitro and in vivo effects of the mutation using reverse genetics. E2206 is located on the surface of the B-domain of E2, which is associated with receptor attachment in alphaviruses. Recombinant virus expressing the E2206S codon replicated slower and produced significantly less genomic copies than virus expressing the ancestral E2206P codon in vitro in Bluegill Fry (BF2) cells. The E2206S mutant was out-competed by the E2206P mutant after 5 passages in an in vitro competition assay, confirming that the substitution negatively affects the efficacy of virus multiplication in cell culture. Both mutants were highly infectious to Atlantic salmon (Salmo salar), produced similar viral RNA loads in gills, heart, kidney and brain, and induced similar histopathologic changes in these organs. The E2206S mutant produced a less persistent infection in salmon and was shed more rapidly to water than the E2206P mutant. Reduced generation time through more rapid shedding could therefore explain why a serine in this position became dominant in the viral population after SAV3 was introduced to farmed salmon from the wild reservoir.