Construction of influenza virus siRNA expression vectors and their inhibitory effects on multiplication of influenza virus.

Three plasmid constructs were prepared that express small interfering RNAs (siRNAs) targeted to sequences encoding the ribonucleoprotein member, nucleoprotein (NP) and/or PA, of influenza virus genome. The antiviral properties of siRNAs against the H5N1 strain of influenza virus were studied by evaluating their capacity to silence expression of target genes as well as their effect on influenza virus-induced apoptosis in Madin-Darby canine kidney cells, chicken embryo fibroblast cells, and embryonated chicken eggs in a transient replication model. The results demonstrated that all three siRNAs expressing plasmids efficiently transcribed the short hairpin RNAs and inhibited expression of the NP or PA proteins measured by northern blot and western blot analyses, respectively, in the transfected cells. We also found that the integrated siRNA expression plasmid pEGFP/NP+PA, which we constructed for the first time to synchronously target NP and PA segments of the influenza virus genome, could more efficiently inhibit synthesis of influenza virus detected by cytopathogenic effects, hemagglutinin, and plaque-forming unit assays in the transfected cells. Furthermore, the integrated siRNA expression plasmid pEGFP/NP+PA could remarkably interrupt the cellular apoptotic course caused by influenza virus, which protected infected cells from apoptotic damage. In contrast, a control siRNA expression plasmid, pEGFP/HK, could neither inhibit the protein expression and production of influenza virus nor interrupt the cell apoptotic course mediated by influenza virus. These results demonstrate that RNA interference (RNAi) can be used to inhibit protein expression and replication of influenza virus and that RNAi treatment holds potential as a new approach to prevent avian influenza.