This synergistic effect was recently achieved in a mammalian system by introducing a microRNA-like non-pairing uridine-bulge into siRNA passenger strands, resulting in immunostimulatory siRNAs that enhanced protection of human immune cells from Semliki Forest virus. We have harnessed the immunostimulatory properties of the 59- UGUGU-39 motif,SDZ 220-581 hydrochloride and illustrated how the differential positioning of the 59-UGUGU-39 motif at the 59 end of PB1-2257 can have a major impact on immunostimulation and subsequent antiviral protection. In chicken cells infected with H5N1, the effect of 59 tagging lead to a 2-log enhancement of anti-viral activity. Interestingly, a 21 bp siRNA duplex consisting entirely of poly- UG has recently been shown to induce type I IFNs in chicken splenocytes more effectively than siRNAs lacking poly-UG repeats. This result further demonstrates the immunostimulatory properties of siRNAs featuring poly-UG motifs such as 59- UGUGU-39 and supports our strategy to enhance PB1-2257 Fialuridine using this motif. A major finding from this study is that immunostimulation of chicken cells by T7-siRNAs involves TLR3, a result that is perhaps surprising given that immune recognition of short dsRNA has been assigned to TLR7 and TLR8 in other species. However, it is well established that TLR3, while long-associated with the recognition of long dsRNA, can be activated by RNA duplexes as short as 21 bp. An example of this activation is the design of siRNAs against vascular endothelial growth factor-A as a treatment for age-related macular degeneration. It was subsequently demonstrated that numerous dsRNAs inhibited angiogenesis in a sequence-independent manner by activating TLR3 and the downstream production of IFN-c and IL-12. Our results suggest a hitherto unreported role for chicken TLR3 in the recognition of short dsRNA. This is of particular interest given that the apparent lack of RIG-I in the chicken genome would suggest other receptors are responsible for viral dsRNA surveillance. Having already established the role for chicken TLR3 in the recognition of long dsRNA, our results suggest that TLR3 plays a dual role in recognition of both long and short dsRNA.