Inhibition of viral hemorrhagic septicemia virus replication using a short hairpin RNA targeting the G gene.

RNA interference (RNAi), a mechanism for post-transcriptional silencing of homologous genes by double-stranded RNA (dsRNA), has emerged as an antiviral strategy in animals. In this study, the epithelioma papulosum cyprini (EPC) cell line, in combination with a fugu-U6-promoter-driven shRNA construct designed against G gene, was used to investigate whether short hairpin RNA (shRNA) could inhibit viral hemorrhagic septicemia virus (VHSV) proliferation by sequence-specific RNAi. The results showed that transfection with a shRNA-producing construct (shRNA-VG594) resulted in a sequence-specific knockdown of G gene mRNA in EPC cells. There were no significant differences in IFN-induced Mx1 gene expression among cells transfected with each shRNA vector including shRNA-VG594, -VG594sc (two nucleotides mismatch) and -EGFP (non-specific control), suggesting that knockdown of G gene expression was not due to an IFN response but instead by sequence-specific RNAi. Transfection of EPC cells with shRNA-VG594 conferred resistance to VHSV, and this anti-VHSV effect was not observed when using a two-nucleotide-mismatched shRNA-VG594sc or a shRNA targeting EGFP. Furthermore, shRNA-VG594 expressed in EPC cells did not confer protection against infectious hematopoietic necrosis virus (IHNV), suggesting sequence-specific RNAi-dependent suppression of viral replication.