AbstractIn this study, the susceptibility of turbot juveniles to two betanodavirus strains was assessed, a RGNNV/SJNNV reassortant (Ss160.03) and a SJNNV strain. The reassortant isolate exhibits a slightly modified SJNNV CP, with two amino acid substitutions in the C‐terminal domain (positions 247 and 270). To analyse the role of these residues as virulence and host determinants in turbot, three recombinant strains (rSs160.03247, rSs160.03270, rSs160.03247+270) harbouring site‐specific mutations in the CP sequence were also tested in experimental trials. Moderate mortalities (up to 50%) were recorded at 18 °C in the fish challenged with the Ss160.03 strain, whereas low mortalities (17%) were observed in the group challenged with the SJNNV strain. A slight decrease (around 10%) was observed in the mortalities caused by the mutants rSs160.03247 and rSs160.03270, whilst the mutation of both positions reduced mortality by more than half of that observed in fish challenged with the wild strain. These results are confirmed by the replication in brain tissues, because whereas the wild strain was detected from 5 to 30 dpi and reached the highest viral load, the recombinant virus harbouring both mutations was not detected in the brain until 20 dpi and with a moderate viral load.