FIG 2.

Knockdown of VDAC1 inhibits replication of IBDV. (A) Effects of VDAC1 RNAi on the expression of endogenous VDAC1. DF-1 cells were transfected with siRNA (RNAi#1, RNAi#2, or RNAi#3) or scrambled siRNA (Sc.). Cell lysates were harvested 48 h after the second transfection and examined by Western blotting with anti-VDAC1 antibody. Endogenous β-actin expression was used as an internal control. (B) Relative intensities of VDAC1 in knockdown cells. β-Actin was used as a protein loading control. Representative results are shown, with bars representing the intensities of VDAC1/actin normalized to the control condition. (C and D) Scrambled RNAi cells and VDAC1 RNAi cells were infected with IBDV at an MOI of 1. At different time points (12, 24, and 36 h) after IBDV infection, the infectious viral loads in the cell cultures (C) or supernatants (D) were determined by TCID₅₀ analysis using 96-well plates. (E) Scrambled RNAi cells and VDAC1 RNAi cells were infected as described for panels C and D. Cells were harvested 12 and 24 h after infection and examined by Western blotting with antibodies against VP1, pVP2, VP3, and VP5. Endogenous β-actin expression was used as an internal control. (F to I) Relative intensities of viral proteins VP5 (F), VP3 (G), pVP2 (H), and VP1 (I) in VDAC1 siRNA-treated cells. (J to L) Control RNAi cells and VDAC1 RNAi cells were infected as described for panels C and D. Cells were harvested 12 and 24 h after infection, and RNA levels of VP5 (J), pVP2 (K), and VP1 (L) were determined by qRT-PCR. Data are means and SD for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 versus control (two-way ANOVA).