title: Inhibition of viral hemorrhagic septicemia virus replication using a short hairpin RNA targeting the G gene.
abstract: 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.
authors.affiliation: Department of Aquatic Life Medicine, Pukyong National University, Nam-gu 599-1, Busan 608-737, South Korea.
authors.forename: Min Sun
authors.initials: MS
authors.lastname: Kim
authors.forename: Ki Hong
authors.initials: KH
authors.lastname: Kim
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D019677:Artificial Gene Fusion; D001688:Biological Products / Q000494:pharmacology*; D002460:Cell Line; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D029142:Infectious hematopoietic necrosis virus; D029141:Novirhabdovirus / Q000235:genetics / Q000254:growth & development*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D001688:Biological Products; C096089:G protein, viral hemorrhagic septicemia virus; D034741:RNA, Small Interfering; D014759:Viral Envelope Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins