Insertion of the P gene of spring viremia of carp virus into the snakehead rhabdovirus genome increases virulence.

Spring viremia of carp virus (SVCV) is highly lethal in common carp (Cyprinus carpio) and other cyprinid fish species. In this study, we evaluated the effect of the SVCV phosphoprotein (P protein) on the virulence of snakehead rhabdovirus (SHRV) by generating a recombinant SHRV expressing the SVCV P protein. The SVCV P gene was inserted between the N and P genes of the SHRV genome, resulting in the generation of rSHRV-A-Psvcv, which contains not only its own P gene but also the P gene of SVCV. In preliminary experiments, we determined the minimum titer of SHRV that was lethal in zebrafish (Danio rerio). We then conducted experiments using titers below this threshold to assess the in vivo virulence of rSHRV-A-Psvcv in zebrafish. Under these conditions, no mortality was observed in fish infected with rSHRV-A-eGFP, a modified SHRV containing the enhanced green fluorescent protein (eGFP) gene between the N and P genes, which was used as a control. However, fish infected with rSHRV-A-Psvcv exhibited mortality accompanied by hemorrhage and ophthalmia. In a minigenome assay, the SVCV P protein failed to induce reporter protein expression when co-expressed with the SHRV N and L proteins, suggesting that the SVCV P protein is unable to form a ribonucleoprotein complex with SHRV N and L proteins. Therefore, the enhanced virulence of rSHRV-A-Psvcv resulting from the addition of the SVCV P gene can probably be attributed to the intrinsic properties of the SVCV P protein itself. Epithelioma papulosum cyprini (EPC) cells infected with rSHRV-A-Psvcv exhibited significantly lower type I interferon (IFN) responses compared to those infected with rSHRV-A-eGFP, suggesting that the SVCV P protein expressed by the recombinant SHRV inhibits type I IFN responses. Thus, the immune-suppressive activity of the SVCV P protein might be one of the causes of the enhanced virulence of rSHRV-A-Psvcv.