shRNAs targeting either the glycoprotein or polymerase genes inhibit Viral haemorrhagic septicaemia virus replication in zebrafish ZF4 cells

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 MOls 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 (ifnphil) and Mx (MxA) genes. These results represent an important preliminary step in the generation of transgenic zebrafish resistant to VHSV. (C) 2017 Elsevier B.V. All rights reserved.