In vitro inhibition of CSFV replication by multiple siRNA expression

Abstract

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.

Evaluation of single and dual siRNAs targeting rabies virus glycoprotein and nucleoprotein genes for inhibition of virus multiplication in vitro

“…The reduction in RV titer in the infected cell culture supernatant was 87.4 %, which was significantly similar to the effect produced by individual siRNAs, indicating no cumulative effect. The approach of using multigene siRNAs or multi-siRNAs/miRNAs targeting a single gene has been reported for many viruses, with a variable degree of inhibition of virus replication [11,[33][34][35].…”

The role of axon guidance factor semaphorin 6B in the invasion and metastasis of gastric cancer

“…Ampicillin-resistant colonies were cultured on solid luria broth medium at 37 C overnight on a rocking bed, and the sequence of the inserted 76 bp siRNA was verified by electrophoresis of the recombinant semaphorin 6B siRNA plasmid. 19 Next, SGC-7901 cells were seeded in sixwell plates (1 Â 10 5 /well in 100 ml) and transfected with 100 nM pSilencer TM plasmid, containing either semaphorin 6B siRNA or control siRNA when confluence reached 90%. Transfection was performed using XtremeGene siRNA transfection reagent (Roche, Indianapolis, IN, USA), according to the manufacturer's instructions.…”

Construction of multiple shRNA vectors targeting PEDV and TGEV and production of transgenic SCNT porcine embryos in vitro

“…(2) Given the transfection efficiency, one vector more effectively expresses multiple shRNAs than can be achieved by cotransfection with multiple shRNAs vectors. (3) Multiple shRNAs can prevent viral escape because that would require acquisition of mutations in all shRNA-target sites [21,22,28] . (4) Previous studies involving the expression of multiple shRNAs employing an expression strategy in which three or four shRNAs were expressed from the same promoters found that this caused frequent recombination at repeat sequences of the expression cassette, resulting in deletion of one or two of the cassettes [2] .…”