WNV is susceptible to siRNA pretreatment. A. Scheme for generation of small hairpin siRNAs. B. Cap siRNA specifically inhibits WNV RNA accumulation. Huh7.5 cells were mock transfected or transfected with Cap siRNA or Cap Mut siRNA. Eighteen hours later cells were infected with WNV at MOI 0.1. Forty-eight hours later cells were collected and total RNA was recovered. WNV RNA was measured by quantitative real time RT-PCR. The results are an average of three independent experiments and the error bars indicate standard error of the mean. C. Capsid siRNA specifically inhibits WNV E protein expression. Huh7.5 cells were mock transfected or transfected with M2 siRNA, Cap siRNA, or Cap Mut siRNA as described above. Forty-eight hours after infection, cells were collected and processed for flow cytometry using anti-WNV envelope protein antibody E1. The results are one representative example of three independent experiments. D. Inhibitory activity of different WNV-specific siRNA. Huh7.5 cells transfected with the indicated siRNA, infected with WNV, and then analyzed for viral antigen as described in Materials and Methods. The fold inhibition was calculated after dividing the percentage of antigen positive cells from mock-transfected cells by the percentage of antigen positive cells from siRNA transfected cells. The results are one representative example of two independent experiments.