Ifn α

Human or mouse B cells were cultured in 96-well flat-bottom plates (Corning, Tewksbury, MA, USA) at 5 × 10⁵ cells per well in 200 μl RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Gibco) and antibiotics (penicillin 100 μg/ml, streptomycin 10 μg/ml; Invitrogen Life Sciences, Carlsbad, CA, USA) in a humidified atmosphere of 5% CO₂ at 37°C. For stimulation treatment, B cells were divided into four to six groups: Control (grown in normal media), IFN-α (1000 U/ml, eBiosciences, San Diego, CA, USA), R848 (1 μg/ml, Enzo Life Science, Farmingdale, NY, USA), affiniPure F(ab')2 Fragment Goat Anti-Mouse IgM (5 μg/ml, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) plus anti-CD40 (2 μg/ml, eBiosciences), two joint stimuli (anti-IgM/CD40+R848) and three joint stimuli (anti-IgM/CD40+R848+IFN-α). Each group of B cells was stimulated respectively on the first day, and the culture medium was changed every other day. Mouse B cells without treatment as the control group were collected immediately after isolation from the spleen, and used to compare the target gene or protein expression level. In addition, mouse B cells were exposed to the stimuli after pretreated with inhibitor zVAD-fmk (Merck, Billerica, MA, USA), Necrostatin-1 (Merck) or Catalase (Beyotime, Nantong, China) for 1 h.

The levels of secreted IL-2, IL-15, IFN-α, and IFN-β in serum or culture supernatants were detected by ELISA kits IL-2 (eBioscience, San Diego, CA, USA, sensitivity 2 pg/ml), IL-15 (eBioscience, sensitivity 20 pg/ml), IFN-α (Invitrogen, San Diego, CA, USA, sensitivity 3.2 pg/ml), and IFN-β (PBL assay science and assay range 50–4,000 pg/ml), according to the manufacturer's instruction.

To investigate the induction of mViperin by IFN-α, Marc-145 cells were seeded onto 12-well plates for 24 h and treated with IFN-α (Peprotech, Rocky Hill, USA) at various concentrations of 0, 300, 1000 and 3000 U/mL, respectively. Twenty-four hours later, the cells were challenged with PRRSV at 0.1 multiplicity of infection (MOI). After being incubated for 12, 24, 36 and 48 h, the cells were harvested, and both mViperin and PRRSV were detected by real-time PCR and western blotting [41 (link)].