Fig 2. Replication of rGI and rGIII in IFN-α and β knockdown DEF.

(A) DEF were infected with SH7, rGI, SH15 and rGIII at 0.1 MOI and harvested at the indicated time points. The replication titers in the supernatants were titrated with TCID₅₀ assays in BHK cells and tested by Student’s t-test. The significant differences between rGI and rGIII at different time points are labeled (**, p < 0.01; *, p < 0.05). The significant difference between rGI and SH15 at different time points is marked (#, p < 0.05). The significant difference between rGIII and SH7 at different time points is indicated (&&, p<0.01; &, p < 0.05). (B and C) DEF were infected with rGI, UV-rGI, rGIII and UV-rGIII at 0.1, 1 and 5 MOI and harvested at 24 hpi for detection of NS5 levels by western blotting with anti-NS5 antibodies (B) and for measurement of IFN-α and β production at mRNA level by qRT-PCR (C). (D to F) DEF were treated with siRNA for silencing IFN-α or β expression (siRNA+), or with scrambled RNA control (siRNA-) and incubated for 12 h. The transfectants were subsequently infected with rGI, rGIII, rGI/V372A-H386Y and rGIII/A372V-Y386H at 0.1 MOI and harvested at 24 and 36 h for analysis of IFN-α and β induction at the mRNA levels by qRT-PCR (D) and protein levels by ELISA I as well as for measurement of replication titers by TCID₅₀ assays in BHK cells (F). All data are presented as mean ± SD from three independent experiments. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, no significant difference, by Student’s t-test.