Fig 8.

Knockdown of GILZ inhibits IBDV growth by type I interferon. (A and B) GILZ is required for IBDV-induced suppression of type I interferon expression in host cells after TNF-α treatment. HEK293T cells were treated with GILZ-RNAi or RNAi-control (Ctrl-RNAi) constructs or medium only as an untreated control and were mock infected or infected with IBDV at an MOI of 10. Twelve hours after IBDV infection, cells were treated with TNF-α at a final concentration of 20 ng/ml. Twelve hours after TNF-α treatment, mRNA expression of IFN-α and IFN-β were measured by qRT-PCR using specific primers. The expression levels of mRNA were calculated in relation to that of GAPDH. Results are representative of three independent experiments. Data are represented as means ± SD; n = 3. ***, P < 0.001. (C and D) Knockdown of GILZ inhibits IBDV growth. DF-1 (C) or HEK293T (D) cells were treated with GILZ-RNAi or control-RNAi constructs or medium only as controls and were infected with IBDV at an MOI of 10. At different time points (12, 24, 48, and 72 h) after IBDV infection, the viral titers in the cell cultures were determined as TCID₅₀ using 96-well plates. The significance of the differences between GILZ-RNAi and controls was determined by ANOVA (P < 0.01). (E) Type I interferon mediates the inhibitory effect of GILZ-RNAi on IBDV growth. Anti-human IFN-α1 (4 × 10⁴ neutralizing units/ml) and anti-human IFN-β (5 × 10⁴ neutralizing units/ml) were added to GILZ knockdown cells or RNAi controls 3 h ahead of IBDV infection. Forty-eight hours after IBDV infection, the culture samples were freeze-thawed three times and centrifuged at 2,000 × g for 10 min. The viral titers were titrated using TCID₅₀ in DF-1 cells. Results are representative of three independent experiments. Data are represented as means ± SD; n = 3. ***, P < 0.001.