Figure 3. ISG and IFN-β induction in primary myeloid cells in response to WNV infection.

Primary myeloid cells from WT, Irf3^−/−×Irf7^−/− DKO, Irf3^−/−×Irf5^−/−×Irf7^−/− TKO, and Ifnar^−/− mice were infected with WNV-NY. A and B. Bone marrow-derived mDC (A) and macrophages (B) were infected at an MOI of 0.001 (mDC) or 0.01 (macrophages), and viral replication was measured by focus-forming assay. Data represent the mean ± SEM of two independent experiments performed in triplicate. The dotted line represents the limit of detection of the assay. C and D: WT and TKO mDCs (C) and macrophages (D) were infected at an MOI of 1 or mock-infected (M). At 12, 24, or 48 hours after infection, cells were lysed, separated by SDS-PAGE and analyzed by western blot to detect expression of specific ISGs or viral proteins. One representative experiment of three is shown. E and F: mDCs (E) and macrophages (F) were infected with WNV at an MOI of 0.1, RNA was isolated at 24 hours after infection, and relative expression of the indicated target genes was measured by qRT-PCR. Gene expression was normalized to Gapdh and is displayed as the fold increase compared to uninfected cells on a log₂ scale. Data represent the average of three independent experiments and are expressed as the mean ± SEM. A–B and E–F: DKO and Ifnar^−/− groups were compared to TKO by two-way ANOVA; asterisks indicate differences that are statistically significant (****, P<0.0001; ***, P<0.001; **, P<0.01).