Combined amino acid mutations in the glycoprotein of infectious hematopoietic necrosis virus attenuate virulence and confer strong immunogenicity in rainbow trout.

Infectious hematopoietic necrosis virus (IHNV) is a major threat to the salmonid aquaculture industry, causing mortality rates exceeding 90% in juvenile salmonids. Our previous study showed that single-site mutations at amino acid residues 67, 107, 221 or 256 of the IHNV G protein reduced IHNV virulence, and immunization of rainbow trout with attenuated mutants improved survival after challenge with wild-type (wt) IHNV-HLJ09. To investigate the effects of combining these G-protein substitutions on IHNV virulence and protective efficacy, we employed a reverse genetics system to construct four recombinant viruses (rIHNVs) carrying combined mutations in the G protein, designated rIHNV-G67-107, rIHNV-G67-256, rIHNV-G67-107-221, and rIHNV-G67-107-256. Pathogenicity assays showed that survival rates in fish inoculated with triple mutants rIHNV-G67-107-221 and rIHNV-G67-107-256 reached 86.67%, exceeding those of double mutants and the wtIHNV-HLJ09 group (13.33%). In vivo viral copy numbers were significantly lower in fish infected with the rIHNVs, with rIHNV-G67-107-221 showing the lowest viral load. Notably, vaccination with rIHNV-G67-107-221 provided significant protection against challenge with wtIHNV-HLJ09, achieving an 88.75% relative percent survival (RPS). Moreover, compared with wtIHNV-HLJ09, rIHNV-G67-107-221 induced higher expression levels of TLR3, IFN-I, and IL-1β, together with reduced IL-10 expression, and elicited earlier and higher levels of IHNV-specific IgM. In conclusion, these results suggest that combined mutations at sites 67, 107, and 221 of IHNV G protein effectively attenuate virulence and enhance immunogenicity. These findings indicate that rIHNV-G67-107-221 offer a favorable balance between attenuation and immunogenicity, supporting its further evaluation as a promising live-attenuated vaccine candidate against IHNV.