Safety, immunogenicity, and broad-spectrum protective efficacy of a naturally attenuated GCRV-II strain against viral and bacterial challenges in grass carp.

Grass carp hemorrhagic disease (GCHD), caused by genotype II grass carp reovirus (GCRV-II), is a severe viral disease affecting Chinese aquaculture. In this study, a naturally attenuated GCRV-II strain, GCRV-GD2021, was isolated from healthy grass carp and evaluated as a live-attenuated vaccine candidate. The virus was confirmed as GCRV-II by immunofluorescence and S2 gene sequencing. Safety assessment demonstrated complete avirulence in grass carp, with no clinical signs or mortality observed. Serial in vivo blind passage confirmed that progressive viral load decline and no virulence reversion. Viral clearance kinetics showed peak replication at 7 days post-injection, followed by complete elimination by 60 days. Immunization with GCRV-GD2021 significantly upregulated both innate and adaptive immune genes in head kidney and elicited robust VP38-specific IgM and virus-neutralizing antibody responses. Challenge experiments revealed that the vaccine conferred potent protection against homologous GCRV-II challenge, with relative percent survival (RPS) exceeding 89%. Notably, it provided broad-spectrum cross-protection against heterologous GCRV-II isolates from distinct geographical regions, with RPS values ranging from 89.7% to 96.6%. Furthermore, immunization significantly enhanced resistance against secondary Aeromonas veronii infection. Collectively, these findings establish GCRV-GD2021 as a safe and highly immunogenic naturally attenuated vaccine candidate that confers potent homologous protection, broad heterologous cross-protection against diverse GCRV-II field isolates, and partial resistance to bacterial co-infection, offering a promising strategy for integrated disease management in grass carp aquaculture.