ABSTRACT
Viral haemorrhagic septicaemia virus (VHSV) represents an important disease of finfish. To explore the potential of shRNAs to combat this disease nucleotide sequences of either the VHSV glycoprotein (G) or polymerase (L) gene were targeted. To test their function, shRNAs were expressed in zebrafish epithelial ZF-4 cells utilizing the zebrafish U6-2 promoter. Five of the six shRNA molecules successfully reduced VHSV replication by between 2 and 4 logs in titre relative to an irrelevant control shRNA at all MOIs and also reduced viral CPE at the highest MOI. To ensure that observed reductions in viral titre were dependent on shRNA silencing, potential non-specific antiviral responses were assessed. Only the ineffective shRNA, which formed an improper hairpin when analysed in silico, induced an antiviral response as measured by induction of interferon (ifnphi1) and Mx (MxA) genes. These results represent an important preliminary step in the generation of transgenic zebrafish resistant to VHSV.
Subject(s)
Novirhabdovirus/physiology , RNA Interference , RNA, Small Interfering/genetics , RNA-Dependent RNA Polymerase/genetics , Viral Envelope Proteins/genetics , Virus Replication/drug effects , Animals , Cell Line , Cytopathogenic Effect, Viral , DNA Replication/drug effects , Electroporation , Fish Diseases/prevention & control , Hemorrhagic Septicemia, Viral/prevention & control , Interferons/genetics , Novirhabdovirus/drug effects , Novirhabdovirus/enzymology , Novirhabdovirus/pathogenicity , Promoter Regions, Genetic , Transfection , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
In a landmark finding published in Science, Hyde et al. have demonstrated that a hairpin RNA structure adjacent to the 5' cap of alphavirus genomic RNA confers the ability of these viruses to evade restriction by the interferon-induced host protein IFIT1.
Subject(s)
Alphavirus Infections/immunology , Alphavirus/pathogenicity , Host-Pathogen Interactions/immunology , RNA Caps/chemistry , RNA Caps/immunology , RNA, Viral/chemistry , RNA, Viral/immunology , AnimalsABSTRACT
RNA interference (RNAi) is a powerful, sequence specific, and long-lasting method of gene knockdown, and can be elicited by the expression of short-hairpin RNA (shRNA) molecules driven via polymerase III type 3 promoters from a DNA vector or transgene. To further develop RNAi as a tool in zebrafish, we have characterized the zebrafish U6 and H1 snRNA promoters and compared the efficiency of each of the promoters to express an shRNA and silence a reporter gene, relative to previously characterized U6 promoters from pufferfish, chicken, and mouse. Our results show that the zebrafish polymerase III promoters were capable of effective gene silencing in the zebrafish ZF4 cell line, but were ineffective in mammalian Vero cells. In contrast, mouse and chicken promoters were active in Vero but not ZF4 cells, highlighting the importance of homologous promoters to achieve effective silencing.
