To explore the importance of bat IRF7 in the IFN production pathway, a knockdown approach was used in our P. alecto PaKiT03 cells. Transfection of siRNA targeting bat IRF7 for 24 h resulted in a reduction of native IRF7 mRNA expression to approximately 20% compared to mock transfected cells. The statistical analysis of the knock down effects was calculated by comparing mRNA expression in the knock down samples to mock transfected cells. To exclude the possibility of off-target effects of siRNA transfection, the expression of a closely related gene, IRF3 was examined in transfected cells. As shown in Figure 4A, there was a decrease in IRF3 transcription in siIRF7 transfected cells due to possible toxic effects and/or off target effects of the siRNA smart pool but this change was not statistically significant. Having confirmed the knockdown effect of siIRF7, we then explored the downstream effect of reduced IRF7 on IFN-b production and viral replication. Two experiments were performed; firstly, 24 h after siIRF7 transfection, cells were stimulated with SeV for 6 h and IFN-b mRNA was detected by qPCR. Knockdown of IRF7 impaired the induction of IFN-b by SeV by 2.5 fold relative to Bay 11-7085 untransfected cells. Notably, bat cells maintained some IFN-b induction in siIRF7 cells, a result which likely reflects insufficient knockdown of IRF7 or IFN-b induction through alternative pathways. To examine the effect of IFN knockdown on the Clopidol replication of a bat-borne virus, PulV, a dsRNA reovirus originating from pteropid bats, was used to infect siIRF7-transfected bat cells. A dose of 10 moi was used to infect PaKiT03 cells one day after siIRF7 transfection. Cell supernatant containing virus was collected 24 h after infection and applied to a TCID50 test. Figure 4C shows that when bat IRF7 was knocked down, PulV replicated to a titer more than four-fold higher than in mock-transfected cells. These data demonstrate that bat IRF7 is functionally important in SeV induced IFN-b production and antiviral defense against PulV infection of bat cells.