Chimeric snakehead rhabdoviruses and zebrafish as a model system for investigating the role of fish rhabdoviral glycoproteins in in vivo virulence

Fish rhabdoviruses pose significant threats to aquaculture due to their pathogenicity and economic impact. This study explores the role of viral glycoprotein (G) and non-virion (NV) genes in determining virulence by generating a panel of chimeric snakehead rhabdoviruses (SHRVs) that express heterologous G or NV genes from pathogenic fish rhabdoviruses. Zebrafish (Danio rerio), which shows susceptibility to multiple rhabdoviruses and low mortality upon SHRV infection, was employed as an in vivo model to evaluate virulence. All chimeric SHRVs replicated efficiently in ZF-4 cells, indicating functional compatibility of the heterologous glycoproteins with the SHRV backbone. However, in the in vivo virulence analysis, G gene substitutions alone, even from highly virulent parental viruses (e.g., VHSV Ia, SVCV), did not significantly enhance mortality, suggesting that the G protein is insufficient by itself to confer high virulence in zebrafish. In contrast, a chimeric SHRV harboring the NV gene from VHSV Ia demonstrated elevated mortality and viral replication in vivo compared to wild-type SHRV, indicating the NV gene's role as a virulence determinant. These findings underscore the complexity of rhabdoviral virulence mechanisms and support the utility of the zebrafish–SHRV system for dissecting gene-specific contributions to pathogenicity in aquatic hosts.