In vitro inhibition of CSFV replication by multiple siRNA expression

Classical swine fever (CSF) is a highly contagious viral disease of pigs which causes major economic losses worldwide. No specific drug is currently available for the effective treatment of CSFV infection; however, RNA interference (RNAi) has been applied successfully to inhibit the replication of human and other animal viruses. In this study, three effective siRNAs targeting NS3 of CSFV were selected. siNS3-2 targeting NS3 gene was chosen for further experimentation, while siN1 and siN2 targeting N(pro) gene, and siNS5B targeting NS5B gene describe previously. Single, double and quadruple anti-CSFV siRNA expression plasmids, with loxp sites at each end of the selectable marker genes, were constructed and analyzed using the same promoters or four different promoters, targeting N(pro), NS3 and NS5B genes of CSFV. Results indicate that single or multiple siRNA expression plasmids can efficiently inhibit CSFV replication and that inhibition was markedly stronger when multiple siRNAs were expressed targeting different genes of CSFV. Since RNAi applied to anti-CSFV research, this study provides anti-CSFV methods by single and multiple siRNA expression which can target most viral isolates of different subtypes and prevent viral escape. It also provides a basis for development of CSFV-resistant transgenic pigs.