Type I IFN Bioassay for WNV Infection

Bioactive type I IFN in culture supernatant was analyzed by a previously described method (41 (link)) that measured IFN pre-treated protection against encephalomyocarditis virus (EMCV) in a susceptible cell line (L929, ATCC). Briefly, culture supernatant collected from WT and Tlr8^−/− mice BMDCs that were infected in vitro with WNV for 24 hr (MOI = 5) were UV-inactivated (10 minutes at 120 mJ/s). The crude, UV inactivated supernatant was added to monolayers of L929 cells (cultured in DMEM supplemented with 10% FBS and 1% Pen/Strep) in 96-well flat bottom plates. Following incubation for 14 h at 37°C, medium was removed and cells were infected with EMCV (MOI = 10) for 7 hr. ECMV-mediated cell death was measured using a CellTiter 96 aqueous cell proliferation assay kit (Promega) and an ELx808 ultra microplate reader (BIO-TEK Instruments, Inc.). The percentage (%) of protected cells was calculated as described (41 (link)), according to the following formula: (optical density at 492 nm [OD₄₉₂] of supernatant-treated EMCV-infected cells / OD₄₉₂ of non-EMCV-infected cells × OD₄₉₂ of EMCV-infected cells) / (OD₄₉₂ of non-EMCV-infected cells) × 100%).