To ensure that siRNA-mediated inhibition of H5N1 virus growth was not due to toxicity, the viability of HD-11 cells transfected with siRNAs was measured using Alamar Blue. At the time of H5N1 infection, cells transfected with siRNAs showed viability levels comparable to untreated cells, suggesting that antiviral properties of siRNAs are not related to compromised cell viability. Highly pathogenic avian influenza H5N1 virus remains a significant pandemic threat. The development of novel antivirals to assist in protection against H5N1 virus will present healthcare industries with a new approach in dealing with an outbreak of viral infection. This study is the first to demonstrate the effective inhibition of highly pathogenic avian influenza by directed immunostimulatory siRNAs. Furthermore, we have identified several factors critical to the 2-Methylserotonin maleate immunostimulatory properties of siRNAs in chicken cells. Firstly, the importance of sequence was demonstrated by comparing the immunostimulatory properties of PB1-2257 and isPB1-2257 in DF-1 cells, where a single nucleoside substitution had a large impact on the kinetics and extent of IFN production. In addition, we showed that siRNAs synthesized enzymatically had different immunostimulatory properties to chemically-synthesized sequence-matched variants, demonstrating how the mode of siRNA synthesis influences immunostimulation in the chicken. This difference in immunostimulation between siRNA variants was caused partially by cellular recognition of 59- triphosphate groups present in T7-siRNAs. Interestingly, LM11A-31 dihydrochloride a recent report suggested that DF-1 cells do not produce type I IFN in response to 59-triphosphate siRNAs due to an absence of retinoic acid-inducible gene I protein in chickens, and that a decreased type I IFN response in chickens in response to viral RNA epitopes may explain the increased susceptibility of chickens to H5N1 virus. However, our findings would suggest that DF- 1 cells can produce type I IFN in response to phosphorylated siRNAs and that this process is critically sequence-dependent. This study provides further evidence that the immunostimulatory properties of siRNAs, for several years considered an unwanted side effect of siRNA research, can be utilised to improve the performance of antiviral siRNAs.