Generation of Recombinant Snakehead Rhabdovirus (SHRV) Expressing Artificial MicroRNA Targeting Spring Viremia of Carp Virus (SVCV) P Gene and In Vivo Therapeutic Use Against SVCV Infection

Spring viremia of carp virus (SVCV) is a highly lethal virus in common carp ( Cyprinus carpio ) and other cyprinid fish species. The aim of the present study was to develop an in vivo therapeutic measure against SVCV using artificial microRNA (AmiRNA) targeting the SVCV P gene transcript. Three candidates of AmiRNAs (AmiR-P1, -P2, and -P3) were selected, and their ability to downregulate SVCV P gene transcript was analyzed by both synthesized AmiRNA mimics and AmiRNA-expressing vector system, in which AmiR-P3 showed the strongest inhibitory activity among the three candidates. To overcome in vivo limitation of miRNA mimics or plasmid-based miRNA expression systems, we rescued recombinant snakehead rhabdoviruses (SHRVs) expressing SVCV P gene–targeting AmiRNA (rSHRV-AmiR-P3) or control AmiRNA (rSHRV-AmiR-C) using reverse genetic technology. The successful expression of AmiR-P3 and AmiR-C in cells infected with the rescued viruses was verified by quantitative PCR. To evaluate the availability of rSHRV-AmiR-P3 for in vivo control of SVCV, zebrafish ( Danio rerio ) were (i) infected with either rSHRV-AmiR-C or rSHRV-AmiR-P3 followed by SVCV infection or (ii) infected with SVCV followed by either rSHRV-AmiR-C or rSHRV-AmiR-P3 infection. Fish infected with rSHRVs before and after SVCV infection showed significantly higher survival rates than fish infected with SVCV alone. There was no significant difference in survival rates between groups of fish infected with rSHRV-AmiR-C and rSHRV-AmiR-P3 before SVCV infection; however, fish infected with SVCV followed by infection with rSHRV-AmiR-P3 showed significantly higher survival rates than fish infected with rSHRV-AmiR-C. These results suggest that rSHRV-AmiR-P3 has therapeutic potential against SVCV in fish when administered after SVCV infection, and rSHRVs expressing artificial microRNAs targeting SVCV transcripts could be used as a tool to control SVCV infection in fish for a therapeutic purpose.