Figure 2. DV1 replication and propagation in HUH-7 and shRIG-I cells.

(A) Flow cytometric analysis showed enhanced DV1 infection in shRIG-I cells as compared to HUH-7 cells. Cells were stained with anti-Dengue E or IBV S (an isotype control) antibodies and analyzed by flow cytometry. (B) Tissue Culture Infectious Dose 50 (TCID50) assay. Virus titers in 50% tissue culture infectious doses (TCID₅₀)/ml were determined according to Reed and Muench [45]. Symbols indicate significantly different from infected HUH-7 (**P<0.01). (C) Qualitative RT-PCR detection of IFN-related gene expression. Cells grown in 6-well plates were infected with DV1 for up to 72 h. Total RNA was extracted, used for cDNA synthesis and subjected to RT-PCR analysis. GAPDH was used as a control for equal RNA templates. (D) Real-time RT-PCR analysis of gene expression in mock and DV1-infected cells. Total RNA was isolated, used for cDNA preparation and subjected to real-time RT-PCR analysis. Bar histograms show the average difference in gene expression between mock and DV1-infected cells based on at least two independent experiments. IFN-β production was assayed using ELISA (lower panel). Symbols indicate significantly different from infected HUH-7 cells (**P<0.01). (E) Total cellular protein was extracted and analyzed by immunoblot. IRF3 dimerization was assessed by native PAGE with anti-IRF3 antibody as a probe. Tubulin protein level was assessed by SDS PAGE to ensure equal loading of cell lysate. Arrows indicate IRF3 dimer and monomer. Row below figure shows ratio of dimer: monomer analysed by densitometry.