Characterization of immunogenic peptide derived from grouper betanodavirus

Abstract

Betanodavirus is one of the major pathogens causing viral nervous necrosis in fish, leading to mass mortality in infected larvae of farmed fish. Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) has a broad range of hosts, including grouper species. Currently, there is still no effective strategy addresses this viral infection. Vaccines are recognized as effective preventive and therapeutic measures for infectious diseases. This study employs reverse vaccinology to identify immunogenic peptides against grouper betanodavirus. The nucleotide sequence of the grouper nervous necrosis virus strain was obtained from NCBI. Using the Immune Epitope Database and NetCTL 1.2 server, B- and T-cell epitopes were predicted and analyzed for antigenicity, immunogenicity, physico-chemical properties, and surface accessibility. As a result, epitope BCE-1 and TCE-3 were selected and synthesized for grouper immunization. Physico chemical analysis revealed that the BCE-1 epitope exhibited a 30-hour half-life and has a large accessible surface area on the virus particle, indicating strong antigen exposure. Immunization with the BCE-1 epitope produced significantly higher antigenspecific antibodies titre compared to the TCE-3 epitope and control groups. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze fish serum from the BCE-1 epitope treatment group, identifying a total of 1440 proteins. Further analysis identified 655 proteins consistently present in at least two technical and two biological replicates. A comparative proteome analysis, using the transformed and normalized protein expression data, followed by one-way ANOVA (q ≤ 0.05) and fold change (FC ≥ 2), revealed 182 differentially expressed proteins (DEPs). Upregulated and downregulated DEPs in peptide treatment groups when compared to peptide with adjuvant treatment group were further identified by differential expression (DE) analysis. KEGG pathway enrichment analysis (q-value ≤ 0.05) highlighted associations of upregulated DEPs with immune response pathways, including phagosome, apoptosis, and protein processing in endoplasmic reticulum, while neuroactive ligand-receptor interaction and ferroptosis pathways were enriched with downregulated DEPs. Upregulated DEPs actin and tubulin in phagosome pathway link serum responses to viral entry, trafficking, and assembly, reflecting their roles in host defense and viral exploitation. The study highlights DEPs' roles in grouper immunity post-immunization therefore providing insights into immune protein expression for managing betanodavirus infections.