Fig. 2.

IFN-β pre-treatment resulted in decreased virus production. (a) DAOY and A549 cells were treated with human recombinant IFN-β at concentrations of 10, 100 and 1000 ng ml⁻¹, and after 12 h infected with TBEV (m.o.i. 5). MTT viability assays were performed at 5 d p.i., when CPE was observed in infected cells without IFN pre-treatment. The numbers represents the percentage of living cells normalized to the untreated control. The average with standard deviation from three independent experiments (in triplicate per experiment) is shown. *Represent a significant difference from the TBEV-infected control as calculated by Student’s t-test (*P<0.05; **P<0.01). (b) IFN-β- (10 ng ml⁻¹) or mock (no IFN-β)-treated DAOY cells were infected (m.o.i. 5) at 12 h post-treatment and viral titres (at 12, 24, 48 and 72 h p.i.) were determined by plaque assay. Time 0 stands for the initial infection input (6×10⁵ p.f.u.). The average with standard deviation from three independent experiments is shown. Significant difference from the control was calculated by Student’s t-test (****P<0.0001). (c) Cell lysates from (b) were used for the detection of viral NS3 levels by Western blot. GAPDH was used as a loading control. (d) DAOY and A549 cells were first co-transfected with p125Luc IFN-β promoter reporter expressing Firefly luciferase (500 ng well⁻¹) and pRL-CMV internal control expressing Renilla luciferase (2 ng well⁻¹). IFN-β promoter was stimulated following a secondary transfection of poly I:C (either 0, 1 or 10 µg well⁻¹) at 24 h post-first transfection. Relative luciferase activity was analysed at 24 h post-second transfection. The mean with standard error is shown for three independent experiments performed in triplicate. Data were normalized to cells co-transfected with p125Luc and pRL-CMV without poly I:C treatment. Significant differences from the control were calculated by Student’s t-test (**P<0.01).