Identification of potential virulence sites of IHNV G protein and its impact on pathogenicity and immunogenicity of IHNV in rainbow trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) is the major pathogen responsible for acute hemorrhagic disease in salmonids and causes significant economic losses worldwide. The viral glycoprotein (G protein) contains key virulence determinants that regulate pathogenicity and immunogenicity. It is therefore an important target for vaccine development. In this study, G protein sequences of J-genotype IHNV were compared with those of other genotypes to identify amino acid residues potentially associated with differences in virulence. A reverse genetics system was used to rescue recombinant viruses carrying specific mutations. Five recombinant viruses were generated and designated rIHNV-G67, rIHNV-G107, rIHNV-G124, rIHNV-G221, and rIHNV-G256. To determine the roles of these putative virulence-related sites, pathogenicity and immunogenicity assays were conducted. Mutations at amino acid positions 107 and 124 of the G protein markedly reduced viral virulence. The cumulative survival rates of rainbow trout infected with rIHNV-G107 and rIHNV-G124 were 74% and 35%, respectively. These rates were substantially higher than those of fish infected with the parental strain rIHNV HLJ-09 and wild-type IHNV HLJ-09. Innate immune-related genes, including IL-1β and TLR3, were significantly upregulated in rainbow trout infected with rIHNV-G107 and rIHNV-G124 compared with those infected with wtIHNV HLJ-09. Serological analysis showed that fish immunized with rIHNV-G107 produced specific serum IgM antibodies earlier than those infected with wtIHNV HLJ-09. This finding suggests that the G107 mutation enhances antigen presentation and accelerates immune activation. Moreover, rainbow trout immunized with rIHNV-G107 exhibited strong protection against IHNV challenge. The relative percent survival (RPS) reached approximately 87.50%, which was higher than that observed in fish immunized with the other recombinant strains. Overall, these results demonstrate that amino acid residues 107 and 124 of the G protein play critical roles in IHNV virulence and immunogenicity. Among the recombinant viruses, rIHNV-G107 showed the most favorable immunogenic profile and represents a promising candidate for the development of an attenuated IHNV vaccine.