Fig 1: FMDV 3Cpro C142T is deficient in the negative regulation of type-I IFN signaling. The PK15 cells or LFBK cells were transiently transfected with wild-type FMDV 3Cpro, 3Cpro C142T, or control plasmid, following VSV-GFP (1 MOI) infection. (A,E) GFP expression, (B,F) wild-type FMDV 3Cpro and 3Cpro C142T expression by qRT-PCR, (C,G) GFP absorbance and virus titer, (D,H) IL-6 and IFN-a secretion was measured at indicated time points. (I) PK15 cells were transiently transfected with wild-type FMDV 3Cpro, 3Cpro C142T, or control plasmid and infected with PR8-GFP (3 MOI). Immunoblotting was performed with cells harvested at 0, 6, 12, and 18 hpi to detect phosphorylated (p-) TBK1, TBK1, p-IRF3, IRF3, p-STAT1, STAT1, p-I?B-a, IkB-a, and ß-actin. (J) PK15 cells were transiently transfected with wild-type FMDV 3Cpro, 3Cpro C142T, or control plasmid and PR8-GFP (3 MOI) infection, following total RNA extraction and qRT-PCR for respective antiviral genes as mentioned. Data are representative of three independent experiments each with similar results. FMDV, foot-and-mouth disease virus; IFN, interferon; IL-6, interleukin 6; TBK1, TANK binding kinase 1; IRF3, interferon regulatory factor 3; STAT1, signal transducer and activator of transcription 1; I?B, inhibitor of nuclear factor-kappa B. All the values are expressed as mean ± SD of at least two biological replicates. Student’s t-test; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
Fig 2: Replication of rGI and rGIII in IFN-a and ß 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-a and ß production at mRNA level by qRT-PCR (C). (D to F) DEF were treated with siRNA for silencing IFN-a or ß 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-a and ß 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.
Fig 3: Effects of NS5-V372A and NS5-H386Y variations on IFN-a and ß production and viremia in ducklings.(A, B and C) Two-day-old SPF domestic ducklings (n = 10) were intramuscularly inoculated with the indicated substitution mutant viruses. Blood samples were collected daily through the jugular vein from 1 to 4 dpi for measurement of the levels of IFN-a (A) and IFN-ß (B) production and viremia (C). (D) Detection of viral loads in tissues. Two-day-old SPF domestic ducklings (n = 12) were intramuscularly inoculated with the indicated substitution mutant viruses. Three ducklings per group were euthanized daily from 1 to 4 dpi for collection of tissues (spleen, lung, kidney, liver, heart, and brain). Viral loads in each tissue were titrated with TCID50 assays on BHK cells. Data are shown as means ± SD. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, no significance, by Student’s t-test.
Fig 4: Identification of the viral determinant of different IFN-a and ß induction.(A) Schematic diagram of parental and chimeric recombinant viruses used in this study. Viral proteins of GI and GIII strains are highlighted in blue and gray, respectively. (B and D) DEF were infected with the indicated recombinant viruses at a MOI of 0.5, 1, and 5 and harvested at 24 hpi for measurement of IFN-a and ß production. (C and E) DEF were infected with the indicated recombinant viruses at 1 MOI and harvested at the indicated time points for measurement of IFN-a and ß production. The mRNA levels of IFN-a and ß in the cell pellets were examined by using qRT-PCR (B and C). The concentrations of IFN-a and ß proteins in the supernatants were determined with ELISA (D and E). 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.
Fig 5: Wild-type FMDV 3Cpro negatively regulates type-I IFN pathway. Wild-type FMDV 3Cpro stably expressing or control Raw264.7 cells were infected with VSV-GFP (1 MOI) at indicated time points and, (A) GFP expression, (B) FMDV 3Cpro expression, (C) GFP absorbance and virus titer, and (D) IL-6, IFN-ß, IFN-a, and TNF-a secretion was measured. The same cell line was treated with (E) poly (I:C) or (F) 5'PPP-dsRNA and measured their IL-6 and IFN-ß secretion. HEK293T cells were transiently transfected with control plasmid or wild-type FMDV 3Cpro plasmid, and VSV-GFP (0.5 MOI) were infected. At indicated time points (G) GFP expression, (H) FMDV 3Cpro expression, (I) GFP absorbance and virus titer, and (J) IL-6, IFN-ß secretion was measured at indicated time points. Data are representative of three independent experiments, each with similar results. FMDV, foot-and-mouth disease virus; IFN, interferon; IL-6, interleukin 6; TNF-a, tumor necrosis factor-alpha. All the values are expressed as mean ± SD of three biological replicates. Student’s t-test; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
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