Fig 1: Effects of NS5-V372A and NS5-H386Y variations on IFN-a and ß induction.(A) Schematic diagram of substitution mutant viruses used in this study. Viral proteins of GI and GIII strains are highlighted in blue and gray, respectively. (B) DEF were infected with the indicated substitution mutant viruses at 1 MOI and harvested at 24 hpi for measurement of IFN-a and ß production at the mRNA level by qRT-PCR. (C and D) DEF were transfected with the indicated plasmids and incubated for 24 h. The transfectants were mock-treated (-poly(I:C)) or treated with poly(I:C) (+poly(I:C)) for 12 h. The mRNA levels of IFN-a and ß in the cell pellets were determined by using qRT-PCR (C). The expression of Flag-NS5 and Flag-NS5 deletion mutants were analyzed with western blotting with anti-Flag antibodies (D). All data are presented as mean ± SD from three independent experiments. **, p < 0.01; *, p < 0.05; ns, no significant difference, by Student’s t-test.
Fig 2: Differences in IFN-a and ß induction and replication efficiency between the GI and GIII strains.ST, bEnd.3, and DEF cells were infected with GI (SH7 and SD12) and GIII (SH15 and SH19) strains at 0.1 MOI and harvested at 24, 36, and 48 hpi for measurement of IFN-a and ß production and viral replication. (A, C and E) The mRNA levels of IFN-a and ß in the cell pellets were examined by qRT-PCR. (B, D and F) The concentrations of IFN-a and ß proteins in the supernatants were determined by ELISA. (G, I and K) The replication titers of GI and GIII strains in the supernatants were titrated with TCID50 assays in BHK cells and the significant differences between the average titers of GI and GIII strains were tested at each time point. (H, J and L) The levels of viral NS5 were examined with western blotting with anti-NS5 antibodies. All data are presented as mean ± SD from three independent experiments. ***, p < 0.001; **, p < 0.01; ns, no significant difference, by Student’s t-test.
Fig 3: 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 4: 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 5: 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.
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