We attempted to generate siRNAs with two active strands, which can simultaneously knock down the expression of mRNA and viral genomic RNA. In this study, short hairpin RNAs (shRNAs) against N and F genes were used. Expression of F and N mRNA transcripts as well as genomic RNA was determined with relative real-time RT-PCR. The RSV load in infected cell culture supernatant was determined by absolute quantitative real-time PCR. We found that (i) in the presence of shRNA-N, a greater reduction in viral genomic RNA was found; (ii) the level of expression at MOI 0.01 was reduced more than MOI 0.1; (iii) reduction in N transcript was greater than F; and (iv) finally, in combination pre-treatment with two shRNAs, the reduction was not significant as compared to single shRNA transfection. shRNAs also inhibited the production of RSV progeny as shown by viral load in infected HEp-2 cells. (i) Virus load reduction was greater at MOI 0.01 than 0.1 and (ii) significant load reduction was not seen with combination shRNA pre-treatment. The antiviral potency was also confirmed by plaque assay and western blot analysis. Our results provided further evidence that RNAi could be a powerful treatment option against respiratory viruses.
Keywords: Dual-targeting siRNA; Fusion; Genomic RNA; Nucleoprotein; RSV; mRNA.