Combined amino acid mutations in position 67 and 438 in the glycoprotein of infectious hematopoietic necrosis virus can further reduce the pathogenicity

The outbreak of infectious hematopoietic necrosis (IHN), driven by the severe acute IHN virus (IHNV), poses a significant risk to rainbow trout farming in China. As such, the development of effective vaccines and an understanding of their mechanisms are essential. Our previous research demonstrated that mutations at sites 67 and 438 respectively in the G protein of IHNV could reduce its pathogenicity and enhance the survival rate (SR) of rainbow trout post-infection. In this study, we mutated at both amino acid positions 67 and 438 simultaneously in the G protein of IHNV. The resulting recombinant IHNVs (rIHNVs) exhibited similar growth patterns to the parental strain. Additionally, rIHNV-G_67–438 induced reduced apoptosis and lower activity of caspase-3 and caspase-9 compared to other groups in vitro. Rainbow trout vaccinated with rIHNV-G_67–438 showed significant protection against IHNV, with a relative percent survival (RPS) of approximately 88.9 %, outperforming other groups. Furthermore, vaccination with rIHNV-G_67–438 triggered a notable increase in specific IgM antibodies. Notably, an antiviral immune response was rapidly activated early in the vaccination process, evidenced by the upregulation of IL1β, IFN1, IL8, TNF-α, MX1, and TLR3. The viral genomic load in fish vaccinated with rIHNV-G_67–438 was significantly lower compared to the other groups. These findings indicate that this vaccine enhances immune responses while reducing viral virulence, making it a promising candidate for the prevention of IHN outbreaks. Additionally, the study provides new insights into the virulence mechanisms of rhabdoviruses and the development of attenuated vaccines.