RNAi to control SVC& CyHV-3 infection of cyprinid fish

Viral infection causes lethal diseases to cyprinid fish with sever economic consequences. For example, two viruses; one RNA virus spring viraemia of carp (SVC) and the DNA virus cyprinid herpes virus-3 (CyHV-3) both are notifiable diseases and causing severe economic losses for both consumption and ornamental cyprinid fish. Control of viral infection is difficult and complex because antiviral drugs are not currently available to treat any viral diseases of cultured fish and due also to absence of commercially available effective vaccines. Therefore, new effective approaches are urgently needed to treat and control viral infection in cyprinid. RNA interference (RNAi) is a conserved mechanism in the posttranscriptional gene silencing (PTGS) phenomenon observed in a variety of species such as plants, fungi and animals. Recent experiments have shown that the application of RNAi can inhibit viral infection by targeting viral or host genes. With the advent of RNAi technology and the refinement of techniques to silence gene, drugs based on small interfering RNA (siRNA) will surely make great advances in the prevention and treatment of viral diseases in fish. This project aims to use RNAi technology to inhibit SVC and CyHV-3 replication in cell culture system. Important gene(s) in each virus will be targeted with a specific siRNA and the silencing effect of each siRNA on the target gene will be assessed by quantitative real-time PCR. Results of this project will provide significant contributions for prevention and control of these viruses.

As there is no current treatment strategy for fish viruses; RNA mediated interference (RNAi) technology was used to control two important fish viruses that causing high mortality rate in common carp and koi carp populations, namely, the Spring Viraemia of Carp Virus (SVCV) and the Cyprinid Herpes Virus -3 (CyHV-3). Small interfering RNAs (siRNAs) were tested to inhibit, in vitro, replication of these viruses in cell lines. Important genes in each virus were targeted with specific siRNA and the knock down effect of each siRNA on the target gene was assessed by quantitative real-time PCR (qPCR). Synthetic siRNA molecules were designed to target SVCV- nucleoprotein and SVCV- phosphoprotein transcripts to inhibit SVCV replication in Epithelioma Papulosum Cyprinid (EPC) cell line. Inhibition of nucleoprotein and phosphoprotein gene expression by siRNA reduced SVCV replication. However, use of tandem siRNAs that target phosphoprotein and nucleoprotein worked best at reducing SVCV replication. Similarly, siRNAs were tested to inhibit, in vitro, viral replication of CyHV-3 in Common Carp Brain (CCB) cell line. The siRNAs were designed to target either thymidine kinase (TK) or DNA polymerase (DP) genes involved in CyHV-3-DNA replication. Treatment with siRNA targeting either TK or DP genes reduced the release of the virus particles from infected CCB cells. However, siRNA targeting DP was most effective at reducing viral release as measured by qPCR. Our work should be followed up in vivo to investigate the possibility of applying RNAi to inhibit the replication of SVCV and CyHV.3 in fish.