Development of the reverse genetics system for viral hemorrhagic septicemia virus genotype IVa and its application in antiviral compound screening

Viral hemorrhagic septicemia virus (VHSV) is a major pathogen affecting freshwater and marine fish species, posing a significant threat to global aquaculture. Reverse genetics systems are essential for studying viral replication, and host interactions, as well as developing vaccines and therapeutics. In this study, we developed a reverse genetics platform for VHSVLB2018 strain, a genetically distinct VHSV genotype IVa strain which exhibits low genomic identity with other Asian isolates, using a dual RNA polymerase I/II transcription vector. We successfully rescued recombinant VHSV in mammalian (B7GG) and fish (FHM and EPC) cell lines, and engineered recombinant VHSV strains expressing EGFP (rVHSV-EGFP) and cherry (rVHSV-Cherry) fluorescent proteins. Phenotypic analysis revealed that unmodified recombinant VHSV (rVHSV) exhibited growth kinetics and virulence similar to the wild-type VHSV, while fluorescent protein-expressing variants showed attenuated replication and virulence, with the rVHSV-EGFP strain displaying the greatest attenuation. Utilizing the rVHSV-EGFP strain, we conducted antiviral compound screening and identified three promising inhibitors-xanthohumol, octyl gallate, and rottlerin that effectively inhibit VHSV replication. Time-of-addition assays further revealed that xanthohumol and rottlerin targeted the viral replication stage, while octyl gallate interfered with viral internalization. This reverse genetics system provides a versatile platform for studying VHSV pathogenesis, developing live-attenuated vaccines, and screening antiviral compounds, enhancing our understanding of this pathogen and offering new tools for aquaculture disease management.