title: shRNAs targeting either the glycoprotein or polymerase genes inhibit Viral haemorrhagic septicaemia virus replication in zebrafish ZF4 cells.
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.
authors.affiliation: CSIRO-Australian Animal Health Laboratory, PO Bag 24, Geelong, Victoria 3220, Australia; Deakin University, School of Medicine, Centre for Molecular Medical Research, Waurn Ponds, Victoria 3216, Australia.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Clarke
authors.affiliation: CSIRO-Australian Animal Health Laboratory, PO Bag 24, Geelong, Victoria 3220, Australia.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: McColl
authors.affiliation: Deakin University, School of Medicine, Centre for Molecular Medical Research, Waurn Ponds, Victoria 3216, Australia.
authors.forename: Alister C
authors.initials: AC
authors.lastname: Ward
authors.affiliation: CSIRO-Australian Animal Health Laboratory, PO Bag 24, Geelong, Victoria 3220, Australia. Electronic address: Timothy.Doran@csiro.au.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Doran
mesh_terms: D000818:Animals; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D004261:DNA Replication / Q000187:drug effects; D018274:Electroporation; D005393:Fish Diseases / Q000517:prevention & control; D031941:Hemorrhagic Septicemia, Viral / Q000517:prevention & control; D007372:Interferons / Q000235:genetics; D029141:Novirhabdovirus / Q000187:drug effects / Q000201:enzymology / Q000472:pathogenicity / Q000502:physiology*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics*; D014779:Virus Replication / Q000187:drug effects*; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: C096089:G protein, viral hemorrhagic septicemia virus; D034741:RNA, Small Interfering; D014759:Viral Envelope Proteins; D029961:Zebrafish Proteins; C543713:interferon phi1, zebrafish; D007372:Interferons; D012324:RNA-Dependent RNA Polymerase