A) BALB/c mice were infected with 100 PFU ECTV WT in the footpad. At the indicated dpi, IFNs and Mx1 transcripts were determined by RT-qPCR in the D-LN. B) As in A, but the indicated transcripts were determined in the liver by RT-qPCR (left y axes) and virus titers were determined by plaque assay (open circles, right y axis, virus titers in upper and lower panels are identical data). C) BALB/c mice were infected with 100 PFU ECTV and at the indicated dpi T1-IFN in serum was determined using ISRE-Luc reporter cells. Mice infected intraperitoneally with LCMV were used as a positive control (2 dpi). D) BALB/c mice were infected with 100 PFU ECTV and at the indicated dpi T1-IFNbp in the sera was quantified using a sandwich ELISA and recombinant T1-IFNbp as standard. E) As in B, but SCID mice were infected with ECTV Δ166-GFP. All graphs show the mean ± SEM and are representative of two or three similar experiments with 3–5 mice/group in each experiment.

A) BALB/c mice were infected with WT ECTV. At 5 dpi livers were harvested and serial sections were stained with anti-T1-IFNbp or anti-EVM135 as indicated. Data are representative of three mice/group and of two independent experiments. The dot plot represents the size of all the foci found in five randomly selected microscopy fields as detected with the two antisera at 5 dpi. B) As in A, but showing serial sections at 7 dpi. Data are representative of three mice/group and of two independent experiments. C) As in A and B, but mice were infected with ECTV-Luc and livers sections stained with anti-Luc Ab to identify infected cells (green) and anti-T1-IFNbp (Red). Data are representative of 3 mice and two independent experiments. Original magnification was 200X.

A) ELISA plates were covered with recombinant T1-IFNbp and the presence of the protein was detected with the indicated mAbs or isotype control (IC). Data are representatives of 2 independent experiments with similar results. B) L929 cells (10⁵) were incubated with 10 ng recombinant T1-IFNbp at 37°C for 1 h, thoroughly washed, and the binding of mAb 10G7 (blue line) or 10F3 (green) to the cell surface was analyzed by flow cytometry. C) L929 cells were infected with ECTV-GFP (upper panels) or ECTV-Δ166-GFP (lower panels) at MOI of 0.05 for 16 h. GFP expression and the binding of IC, 10G7 or 10F3 mAbs were determined by flow cytometry as indicated. D) 10 ng of recombinant ECTV T1-IFNbp was incubated with 10 µl rabbit anti-IFNbp or 10 ng of indicated mAbs for 30 min at 37°C, incubated with 1U mIFN-α for another 30 min at 37°C. The cocktail was then added to L929 cells in 96-well plates. The cells were incubated with this cocktail for 24 h, infected with VSV-eGFP at MOI of 0.1 for 16 h and observed for GFP fluorescence under the microscope.

BALB/c mice were infected with 300 pfu of ECTV-Luc in the footpad, at 5 dpi, the mice were treated with indicated mAbs IP and imaged 2 dpt for light emission using a Carestream In vivo instrument for bioluminescence detection (left) The mouse on the top-left is an uninfected control. The bar graph (right) shows the mean ± SEM quantitative luminescence intensity of the two groups. The experiment is representative of two similar experiments.

L929 cells were left uninfected (top panels), infected with ECTV-GFP (middle panels) or ECTV Δ166-GFP (lower panels). 6 h after infection, the presence of T1-IFNbp at the cell surface was detected using anti-T1IFNbp (left panels) or naïve control sera (right panel). The data are representative of two similar experiments.

A) Mice were infected with 100 PFU ECTV-Luc. At 5 dpi they were treated with 200 µl (∼30 mg/ml protein) anti-T1-IFNbp or naïve sera. 16 h later the livers were harvested and stained with anti-rabbit IgG (red) and anti-luc (green). B) BALB/c mice were infected with 100 PFU of WT ECTV in the footpad. At 5 dpi, mice were treated i.p. with 200 µl rabbit antisera to the T1-IFNbp or, as a control, with naïve sera on the day of infection and monitored for survival. Data are representative of three similar experiments with groups of 10 mice. P values are vs. naïve sera. C) BALB/c mice were infected with WT ECTV. At 5 dpi they were treated with 200 µl anti-T1-IFNbp rabbit sera. Livers were harvested 2 dpt and virus loads determined by plaque assay. Graph shows the mean +/− SEM for 5 individual mice/group and two independent experiments. D) As in C but livers sections stained with H&E. Data are representative of five mice/group and two independent experiments.

A) BALB/c mice were infected with 100 PFU of ECTV in the footpad. At 5 dpi, mice were treated with the indicated mAbs i.p. One day later (a time when virus loads remain the same in all mice), the indicated transcripts in the livers were determined by RT-qPCR. Data are representative of two similar experiments with 3–5 mice/group. B) BALB/c mice were infected with ECTV-Luc in the footpad. At 5 and 6 dpi, mice were treated with the indicated mAbs. 2 dpt the livers were harvested and sections were stained with anti-Phospho-Stat1 (green), anti-Luciferase (red) and with DAPI to reveal nuclei. For the picture on the right (10G7 treated), the small rectangular area at the bottom is shown at higher magnification in the upper-left rectangular area. Data are representative of two similar experiments. C) As in A, but livers were harvested 2 dpt and viral load determined by plaque assay. Graph shows individual mice with the mean ± SEM and is representative of three similar experiments. D) As in A, but livers were harvested 2 or 3 dpt as indicated. Immunohistochemistry with anti-EVM135 sera (top and middle panels) and H&E staining (lower panels). E) Size of individual foci and the mean ± SEM 2 dpt in five randomly selected microscopy fields from D. F) BALB/c mice were infected with 100 PFU of WT ECTV in the footpad. At 5 dpi, mice were treated with indicated mAb i.p. and survival was monitored. Data are representative of 3 independent experiments each with 10 mice/group.

A) Hela cells were incubated with 10 ng recombinant VARV T1-IFNbp,UV treated supernatants from cells infected with VACV WR, or gamma-irradiated supernatants of cells infected with MPXV USA or MPXV Republic of Congo (RoC) strains as indicated. After 30 min the cells were thoroughly washed, incubated with mAb 10G7 (blue line) or IC (red line) for 1 h followed by FITC-anti-mouse IgG and flow cytometry analysis. All data are representative of 2 or 3 independent experiments with similar results. B) Tissue culture media (TCM, RPMI 10% fetal calf serum), 10 ng of recombinant VARV T1-IFNbp in TCM, 100 µl of supernatant of insect cells expressing recombinant VARV T1-IFNbp in TCM, or the indicated irradiated TCM supernatant from cells that had been infected with the indicated viruses were pre-incubated with the indicated amounts of hIFN-α for 1 h. The cocktails were then added to Hela cells in 24 well plates. Following 24 h incubation at 37°C, the cells were infected with VSV at a MOI of 0.1 for 24 h, fixed, and stained with crystal violet. Left and right panels correspond to the same plate but were separated to facilitate labeling of the figure. C) 10 ng of recombinant VARV T1-IFNbp or 100 µl of irradiated supernatant from cells that had been infected with MPXV USA were incubated with 10 ng mAb 10G7. After 30 minutes, 10 U/ml hIFN-α were added to the mixture and incubated for 1 h at 37°C. The cocktail was then added to Hela cells in 24 well plates. Following 24 h incubation at 37°C, the cells were infected with VSV at a MOI of 0.1 for 24 h, fixed, and stained with crystal violet. All data are representative of 2 or 3 independent experiments with similar results.