However, QRT-PCR demonstrated that DF-1 cells express almost non-detectable levels of TLR7, suggesting that other dsRNA-sensing molecules, such as other TLRs or RIG-like helicases, may recognise PB1-2257. siRNAs were transfected into DF-1 cells to create cell populations knocked down for melanoma-differentiation-associated gene 5 and TLR3. siRNAs to silence chicken TLR3 and chicken Mda5 have been described previously and reduce gene expression levels by.75%. QRT-PCR results confirmed that siRNAs and did not themselves induce significant increases in type I IFN. A strong induction of IFN-b in control DF-1 cells was observed 24 h after stimulation with PB1-2257. This response was reduced by 90% in TLR3 knock down DF-1 cells. In contrast, cells with reduced levels of Mda5 did not show a significant reduction in IFN-b expression. While results in figure 2 and figure 3 show VU0405601 isPB1-2257 is more immunostimulatory than PB1-2257, this effect was achieved by altering the PB1-2257 sequence, which is critical for silencing. To create a multi-functional antiviral with both immunostimulatory and silencing properties, we attached 59-UGUGU-39 to either the 59 or the 39 ends of c-PB1-2257 without disrupting the silencing sequence itself. This modification resulted in a 3-fold enhancement of the immunostimulatory profile for the PB1-2257 variant tagged with the Danshensu sodium salt UGUGU-39 motif at the 59 end relative to the untagged molecule. In contrast, tagging at the 39 end appeared to have little enhancing effect. We tested siRNA-mediated inhibition of influenza A/Vietnam/ 1203/2004 in vitro. Since DF-1 cells are poor at supporting H5N1 virus replication, the immortalised chicken macrophage cell line, HD-11, was used for this experiment. Firstly, we investigated whether 59-UGUGU-39-tagged PB1-2257 showed enhanced immunostimulatory properties in HD-11 cells. Similar to results observed for DF-1 cells, HD-11 cells transfected with uPB1-2257 showed increased levels of IFN-b at 8 h compared to cells transfected with PB1-2257 or PB1-2257u. When infected with H5N1 virus, HD-11 cells transfected with uPB1-2257 showed significantly reduced levels of virus compared to cells transfected with PB1-2257 or PB1- 2257u.