The cellular response to WNV infection is unaffected by TLR3 status.

<p>(A) RT-PCR analysis of TLR3 expression in HEK293-Null and HEK293-TLR3 cells. RT negative reactions were performed as a control. (B) Immunofluorescence images of cells either mock- or WNV-infected are shown. Cells were stained with WNV E antibody (red) and DAPI (blue) and merged, deconvoluted images are shown at 630× magnification. (C–F) Western blots of phosphorylated IRF3 and beta actin in HEK293-Null and HEK293-TLR3 cells at various time points following WNV infection or post-treatment with the TLR3 agonist poly I:C. Mock infected cells and cells treated with inactivated WNV are shown as controls. (G, H) xCelligence analysis of cell viability following infection with serial dilutions of WNV NY99 (MOI = 5 to 0.00005). WNV-induced cytopathogenicity curves are shown for HEK293-Null (G) and HEK293-TLR3 cells (H). Reads were taken every 30 minutes for a period of 100 hours. (I) WNV-induced CPE kinetics were obtained by plotting the time of peak cell index for each sample against the concentration of WNV. HEK293-Nulls are represented by the blue line while HEK293-TLR3s are denoted by the red line. This panel shows a linear regression across 5 log dilutions. Shown are the individual cell indices at each dilution, the calculated robust linear regression lines (R² = 0.968) and in the gray shading the standard error (SE) band across the entire dilution range. (J) Viral load was determined by QPCR analysis for WNV envelope protein using a set of known WNV envelope standards. Panel J shows WNV copy number as determined by real-time qPCR using two biological replicate experiments (1 and 2) conducted one week apart. There was no significant growth difference.