A Z-DNA/PKZ-based immunoenhancer confers protective immunity against GCRV infection in grass carp

Grass carp reovirus (GCRV) is a devastating pathogen that causes hemorrhagic disease in grass carp, resulting in mortality rates of up to 80% in fingerlings and substantial economic losses to the aquaculture industry. The development of a broad-spectrum immunoenhancer to boost intrinsic host defense mechanisms represents an urgent and commercially attractive strategy. In the present study, we constructed a Z-DNA/PKZ-based immunoenhancer by cloning GC-rich Z-DNA sequences and grass carp protein kinase Z (PKZ) into a eukaryotic expression vector, and investigated its potential as an immunoprophylactic strategy against GCRV infection in grass carp. Fish immunized with the immunoenhancer showed a significant increase in serum IgM levels, reaching peak titers at 16 d post-injection. Dual-luciferase reporter assay and qRT-PCR analysis revealed that the immunoenhancer markedly activated the IFN I promoter and synergistically upregulated IFN I expression, with PKZ enhancing this effect. Furthermore, the immunoenhancer reduced cell viability in CIK cells. Tissue distribution analysis demonstrated that the immunoenhancer was detectable in the liver, kidney, intestine, spleen, and gills, with persistence up to approximately 20 d. Immunization also significantly elevated SOD, CAT, and MDA levels. Notably, the immunoenhancer conferred a relative percent survival of 70% following lethal GCRV challenge. In summary, this is the first study demonstrating that a Z-DNA/PKZ-based immunoenhancer effectively induces both humoral and innate immune responses and provides significant protection against GCRV infection in grass carp. These findings suggest that the Z-DNA/PKZ immunoenhancer represents a promising prophylactic candidate for the control of GCRV infection in aquaculture.