Type I IFN signaling mediates gene induction by IRF-5 and MAVS.

<p><b>A.</b> Heatmap displaying 22 genes induced upon WNV infection in <i>Ifnar^−/−</i> mDC (greater than 1.5 fold upregulated compared to mock-infected cells, <i>P</i><0.05). <b>B.</b> Expression of selected IFN-independent genes was validated by qRT-PCR using the same RNA samples analyzed by microarray. Gene expression was normalized to <i>Gapdh</i> and is displayed as the fold increase compared to mock-infected cells on a log₂ scale. Data represent the average of three independent samples and are expressed as the mean ± SEM. The dotted line indicates a 1.5-fold increase in expression. <b>C.</b> Venn diagram of the expression patterns of IFN-independent genes, based on microarray and qRT-PCR analyses. <b>D.</b> DKO mDC were treated with 25 µg/ml of an IFNAR-blocking antibody (MAR1-5A3) or an isotype control antibody (GIR-208) for one hour prior to infection with WNV-NY at an MOI of 0.1. Total RNA was isolated after 24 hours and relative gene expression was measured by qRT-PCR. Expression of the indicated target genes was normalized to <i>Gapdh</i> and is displayed as the fold increase compared to untreated cells on a log₂ scale. Data represent the average of four samples from two independent experiments and are expressed as the mean ± SEM. <b>E.</b> mDC from WT, <i>Irf5^−/−</i>, and DKO mice were infected with WNV-NY at an MOI of 0.1 and qRT-PCR was performed as in panel <b>D</b>. Data represent the average of 12 samples from four independent experiments, are displayed as the fold increase compared to untreated cells on a log₂ scale, and are expressed as the mean ± SEM. <b>F.</b> mDC from WT, <i>Irf5^−/−</i>, DKO, and TKO mice were infected with SeV at an MOI of 3 and qRT-PCR was performed as in panel <b>D</b>. Data represent the average of six samples from two independent experiments, are displayed as the fold increase compared to untreated cells on a log₂ scale, and are expressed as the mean ± SEM. <b>G.</b> mDC from WT and <i>Irf5^−/−</i> mice were infected at an MOI of 0.001 and viral replication was measured by focus-forming assay. Data represent the mean ± SEM of six independent experiments performed in triplicate. WT and <i>Irf5^−/−</i> titers were compared by two-way ANOVA and were not significantly different (<i>P</i>>0.05). The dotted line represents the limit of detection of the assay.