Biological interferon assay.

The biological assay for IFN production was performed as described previously (73). Briefly, “IFN-competent” HeLa cells were infected with different BUNV mutants (MOI = 3) and incubated at 37°C for 24 h. The supernatant was clarified by a brief centrifugation and inactivated by UV irradiation. The UV-inactivated supernatant was 2-fold serially diluted and used to treat “IFN-incompetent” A549-Npro cells (expressing the bovine viral diarrhea virus NPro protein). Cells were infected with encephalomyocarditis virus (EMCV) at an MOI of 0.01, fixed with 8% formaldehyde, and stained with a methyl violet staining solution (5% [wt/vol]).