We investigate the practical performance of the applied

Double-stranded RNA may activate the type I interferon pathway as an immune mechanism directed to impact on viral replication. Downstream outcomes of type I IFN production and binding to its receptor include the shutdown of RNA and protein synthesis in virally-infected cells, apoptosis of virally-infected cells and the induction of IFNstimulated genes that coordinate downstream antiviral measures. Stimulation of innate immune responses by RNAi molecules would therefore appear to not be a random process,CU-CPT22 and several studies have identified factors that confer immunostimulatory properties to RNA, including nucleoside sequence, short hairpin RNA promoters and polymerases used to synthesise short interfering RNAs. Whilst so called off-target effects of RNAi are unwanted in many instances, anti-viral therapeutics may profit greatly from multiple response pathways directed towards prevention of viral replication. In this study we have explored the application of isRNAi in chickens with the aim of developing isRNAi antivirals that combats H5N1 by silencing viral genes whilst simultaneously triggering antiviral host immune responses to eradicate the virus. We identify a nucleoside motif that strongly induces type W-13 IFN in chicken cells, and explore the strategy of attaching this motif to siRNAs designed against H5N1. By combining a silencing RNA with a nucleoside immuno-enhancers, we have created a new RNAi molecule that complement and synergise in a double-action antiviral. siRNAs designed against three different conserved regions of the influenza A genome were selected based on their silencing ability. To test the ability of these siRNAs to induce cytokine production in chicken cells, these three siRNAs were synthesized using T7 RNA polymerase and transfected into the immortalized chicken fibroblast cell line, DF-1. Since DF-1 cells have previously been shown to produce high levels of IFN- b in response to the dsRNA mimetic polyI:C, they provide a model cell line for assessing the type I IFN response to siRNA. M- 592, PA-2087 and PB2-2240 induced IFN-b to differing levels, effectively giving low, moderate and high induction of IFN-b, respectively, relative to induction by polyI:C, with maximum levels observed after 24 h.