The ability of distinct sequences to modulate the immunostimulatory properties of siRNAs in mammalian cells was illustrated recently where the induction of pro-inflammatory cytokines was directly related the presence of a 5 bp motif, 59-UGUGU-39. We conducted a bioinformatics search of RNAi sequences targeting influenza A and found that PB1-2257, a 19 bp siRNA targeting the PB1 segment of influenza A contains a motif from base pairs 3–7, 59-UCUGU-39, remarkably similar to 59-UGUGU- 39. Previous studies have shown PB1-2257 to be an effective silencer of influenza A subtype H1N1 replication. We created a modified version of PB1-2257 PB1-2257) where a single substitution at position 4 was made, hence creating a 59-UGUGU-39 motif. An irrelevant siRNA was also synthesized as a negative control by random shuffling of the PB1-2257 sequence. isPB1-2257 induced IFN-a,Febrifugine dihydrochloride and IL-1b mRNA to a greater extent than PB1-2257 and Scramble after transfection into DF-1 cells. Interestingly, PB1-2257, isPB1-2257 and Scramble induced IL-1b, but not the related cytokine, IL-18. It has been reported that mode of synthesis can impact the immunostimulatory properties of siRNAs. To test this parameter in our study, chemically-synthesized siRNAs and siRNAs synthesized using DDRI-18 RNA polymerase were transfected into DF-1 cells at identical concentrations and the induced changes in IFN-b mRNA levels were measured. By 8 h post-transfection, the T7-isPB1-2257 had induced a large increase in IFN- b. However, this increase was not observed for the c-siRNA variant, nor either form of PB1-2257 or Scramble. At 24 h, induction of IFN- b was 10-fold higher for cisPB1- 2257 compared to T7-isPB1-2257. Scramble induced moderate IFN-b expression at 24 h as a T7-siRNA, but not as a c-siRNA. These results demonstrate that for both T7-siRNAs and c-siRNAs, there is a sequence-dependent induction of IFN-b. This induction is more rapid with the additional input of T7 synthesis. We next investigated the receptor responsible for inducing type I IFN in response to immunostimulatory siRNAs in chicken cells. Immunostimulatory siRNAs act via Toll-like receptor 7 in mammals.