Avian IRF1 and IRF7 Play Overlapping and Distinct Roles in Regulating IFN-Dependent and -Independent Antiviral Responses to Duck Tembusu Virus Infection

Figure 1. Transcriptomic analysis of differential gene expression in DEF and DF1 cells infected with DTMUV. The duck endoblastomal fibral cells (DEF) and the chicken stable cell line DF1 were infected with DTMUV at an MOI of 1 for 24 h. Total RNAs were extracted for transcriptomic analysis. The top induced genes in DTMUV-infected DEF are shown in (A) and DTMUV-infected DF1 cells are shown in (B).

Figure 2. Effects of IRF7-overexpression and -knockout on DTMUV replication and upregulation of IRF1, VIPERIN, IFN-β, IFIT5, and CMPK2. (A) Wild type (DF1) and IRF7-knockout (KO IRF7) cells were transfected with a Flag-tagged chicken IRF7 and infected with DTMUV at an MOI of approximately 2. Cells were harvested at 0, 8, 16, 24, 36, and 48 hpi, respectively, and total cell lysats were prepared in parallel. The chiIRF7 expression levels and the replication of DTMUV were assessed by Weston Blot with anti-Flag and anti-DTMUV antibodies. β-actin was detected for a loading control. (B) The genomic RNA levels of DTMUV in DF1 and KO IRF7 cells, as in (A), were determined by RT-qPCR and presented. (C) Viral titers were determined by TCID50 from the extracellular fluids of DF1 and KO IRF7 cells harvested, as in (A). Error bar represents the standard error of three replicate experiments. T tests were performed between the IRF7 overexpressed cells and the empty vector transfected cells in DF1 (blue and white) and KO IRF7 (red and black) cells. (D) The mRNA levels of IRF1, IFN-β, VIPERN, IFIT5, and CMPK2 from the cells harvested as in (A) were detected by RT-qPCR. T tests were performed between the…

Figure 3. Effects of IRF7-overexpression on the upregulation of IFN-β, IFIT5, VIPERIN, CMPK2, and IRF1 induced by DTMUV in IFNAR1-knockout DF1 cell (KO IFNAR1). DF1 and KO IFNAR1 cells were transfected with a Flag-tagged chicken IRF7 and infected with DTMUV at an MOI of approximately 2. Cells were harvested at 12, 24, 36, and 48 hpi, respectively. Total RNAs were extracted, and the induction levels of IFN-β, VIPERIN, IFIT5, CMPK2, and IRF1 were determined by RT-qPCR and presented. Error bar represents the standard error of three replicate experiments. T tests were performed between the samples with or without IRF7-overexpression in KO IFNAR1 (red and black) and DF1 cell (blue and slash). (ns, nonsignificant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 4. Inhibition of DTMUV replication and regulation of the expression of IFN-β, VIPERIN, IFIT5, and CMPK2 induced by overexpression of duck IRF1 in KO IRF7 and KO IFNAR1 cells. (A) DF1 and KO IRF7 cells were transfected with a Myc-tagged duIRF1, infected with DTMUV at an MIO of approximately 2, and harvested at 12, 24, 36, and 48 hpi, respectively. Total RNAs were extracted, and the copy numbers of the viral genomic RNA were determined by RT-qPCR and presented. (B) DF1 and KO IFNAR1 cells were transfected with a Myc-tagged duIRF1, infected with DTMUV at an MIO of approximately 2, and harvested at 12, 24, 36, and 48 hpi, respectively. Total RNAs were extracted, and the copy numbers of the viral genomic RNA were determined by RT-qPCR and presented. (C) DF1 and KO IRF7 cells were transfected with a Myc-tagged duIRF1, infected with DTMUV at an MIO of approximately 2, and harvested at 12, 24, 36, and 48 hpi, respectively. Total cell lysats were prepared, and the duIRF1 expression levels and the replication of DTMUV were assessed by Weston Blot with anti-Myc and anti-DTMUV antibodies. β-actin was detected for a loading control. The relative level of E protein was determined after…

Figure 5. Inhibition of poly(I:C)-induced IFN-β expression by DTMUV NS2B. DF1 cells were transfected with plasmids expressing Myc-tagged DTMUV NS1, NS2A, NS2B, NS3, NS4A, or NS4B, respectively, and treated with 1μg/mL poly(I:C). Cells were harvested at 24 h post-transfection, and the protein expression levels of NS1, NS2A, NS2B, NS3, NS4A, and NS4B in poly(I:C)-treated cells were determined by Western blot with anti-Myc serum. IFN-β induction at the mRNA level from above samples was determined by RT-qPCR and presented. Error bar represents the standard error of three replicate experiments. T tests were performed by comparing the vector-transfected sample (white) to samples transfected with each of the viral gene. (ns, nonsignificant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 6. The effects of DTMUV NS2B-overexpression on NDV replication and the induction of IFN-β, as well as IFN-reducible genes. (A) DF1 cells were transfected with plasmids expressing Myc-tagged DTMUV NS1, NS2A, NS2B, NS3, NS4A, or NS4B, respectively, and infected with NDV at an MOI of 1. Cells were harvested at 24 h post-infection of NDV, and the protein expression levels of NS1, NS2A, NS2B, NS3, NS4A, and NS4B in poly(I:C)-treated cells were determined by Western blot with anti-Myc serum. (B) IFN-β induction at the mRNA level from above samples was determined by RT-qPCR and presented. Error bar represents the standard error of three replicate experiments. T tests were performed by comparing the vector-transfected sample (white) to samples transfected with each of the viral gene. (C) NDV copy numbers in above samples were determined and presented. Error bar represents the standard error of three replicate experiments. T tests were performed by the vector transfected sample (white) to each of the viral gene transfected samples. DF1 cells were transfected with a plasmid expressing Flag-tagged DTMUV NS2B and infected with NDV at an MOI of 1. Cells were harvested at 12 and 24 hpi, and the…