Sendai virus

PBMCs were purified from citrated blood derived from three Babraham in-bred Large-white pigs by centrifugation over histopaque gradients. 3×10⁸ PBMCs were incubated at 4 °C for 15 min in an antibody mixture containing anti-CD3, anti-CD21 and anti-CD14 in 5% FCS/PBS. After washing twice in Ca/Mg-free PBS, anti-mouse IgG1 and anti-mouse IgG2 beads (MACS Miltenyl Biotec) were added and incubated and washed as before. The cells were passed through LS columns (MACS Miltenyi Biotec) and the flow-through was collected. After centrifugation, the cells were resuspended in DMEM+HEPES supplemented with 10% pig serum, 100 µg/ml streptomycin and 100 U/ml penicillin to a density of 5×10⁶ cells/well. Cells were infected with ASFV strains OUR T88/1 and OUR T88/3 at an MOI of 1 or mock infected. CpG ODN 2216 (InvivoGen) at a final concentration of 3.2 µg/ml or 2 hemagglutination units of Sendai virus (Charles River Laboratories International) were added as positive controls. Biologically active IFN was determined from cell supernatants 24 hours post infection using the MxCAT assay (Fray et al., 2001 (link)). The mock inoculum and the two ASFV strains were also tested for their ability to induce CAT expression in the MDBKt2 cells in the absence of the negatively-selected PBMCs.

Lipopolysaccharide (LPS), tumour necrosis factor-alpha (TNF-α) and interleukin-1 β (IL-1β) were obtained from Peprotech. Sendai virus was obtained from Charles River Laboratories, Wilmington, MA.

HEK293T and U87 cells were maintained in DMEM (Gibco) supplemented with 10% foetal bovine serum (FBS, LabTech) and 100 U/ml penicillin plus 100 μg/ml streptomycin (Pen/Strep; Gibco). THP‐1 cells were maintained in RPMI (Gibco) supplemented with 10% FBS and Pen/Strep. THP‐1‐IFIT‐1 cells that had been modified to express Gaussia luciferase under the control of the IFIT‐1 promoter were described previously (Mankan et al, 2014). THP‐1 Dual Control and cGAS^−/− cells were obtained from Invivogen. Lopinavir (LPV), darunavir (DRV), nevirapine (NVP) and raltegravir were obtained from AIDS reagents. STING inhibitor H151 was obtained from Invivogen. JAK inhibitor ruxolitinib was obtained from CELL guidance systems. PF‐74 was obtained from Sigma. Lipopolysaccharide, IFNβ and poly I:C were obtained from PeproTech. Sendai virus was obtained from Charles River Laboratories. Herring testis DNA was obtained from Sigma. cGAMP was obtained from Invivogen. Anti‐IFNα/β receptor and control IgG2A antibodies were obtained from PBL Interferon Source and R&D systems, respectively. For stimulation of cells by transfection, transfection mixes were prepared using lipofectamine 2000 according to the manufacturer's instructions (Invitrogen).

HEK293T, Vero, and mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Corning) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone), penicillin (100 U/ml), and streptomycin (100 μg/ml). Wild-type and Rig-i^−/− MEFs were prepared as described previously (42 (link)). Wild-type HSV-1 (KOS strain) and recombinant HSV-1 were generated and amplified in Vero cells as previously described (16 (link)), with viral titers ranging from 10⁷ to 10⁸ PFU/ml. Sendai virus was purchased from Charles River Laboratories.

HEK293T cells were maintained in DMEM (Gibco) supplemented with 10% fetal calf serum (FCS, Labtech) and 100 U/ml penicillin and 100 μg/ml streptomycin (Pen/Strep; Gibco). THP-1 cells were maintained in RPMI (Gibco) supplemented with 10% FCS and Pen/Strep. THP-1-IFIT-1 luciferase reporter cells express Gaussia luciferase under the control of the endogenous IFIT1 promoter have been described (Mankan et al., 2014 (link)). THP-1 CRISPR control, cGAS-/- and MAVS -/- knock out cells have been described (Mankan et al., 2014 (link)). Nup358 depleted HeLa cells have been described (Schaller et al., 2011 (link)). Lipopolysaccharide, poly I:C and TNFα were obtained from PeproTech. Sendai virus was obtained from Charles River Laboratories. Herring-testis DNA was obtained from Sigma. cGAMP was obtained from Invivogen. NF-ĸB Lucia THP-1 reporter cells were obtained from Invivogen. All cell lines were tested negative for mycoplasma.