Fig. 2. RNA derived from FMDV infection has an MDA5-dependent immunostimulatory activity.

(A and B) SK6 cells were infected with FMDV O1BFS at an MOI of 5 or mock-infected and total RNA was extracted at different times after infection. a RNA isolated from cells 5 h after infection or mock infection was transfected into fresh SK6 cells (10 pg RNA/cell). Transfected cells were lysed 6 h later and the fold induction of porcine IFN-β mRNA in cell lysates was determined by RT-qPCR normalized to GAPDH. Data shown are mean ± SD of triplicates with comparison made to cells transfected with RNA from mock-infected cells. b Total RNA extracted from infected or mock-infected SK6 cells (6 µg) was used to transfect SK6 cells (5 × 10⁵) which had been transfected 24 h before with plasmids encoding DDK-tagged -MDA5, -RIG-I or -EV (1 µg/10⁶ cells). Cells were lysed 2 h or 6 hpt and the fold induction of porcine IFN-β mRNA in cell lysates was determined by RT-qPCR as in A. Data are mean ± SD of triplicates with comparisons made to cells transfected with RNA from mock-infected cells unless specified otherwise. The indicated proteins were analyzed by immunoblot. An anti-DDK monoclonal antibody was used for detection of DDK-tagged MDA5 and RIG-I. c SK6 cells were transfected with DDK-MDA5 and 24 h later mock-transfected or transfected with equal amounts of RNA fractions from the pulldown assay shown in Fig. 1 (1.5 µl/0.5 × 10⁶ cells) and corresponding to RNA immunoprecipitated with MDA5, RIG-I or 2CARD proteins. Cells were lysed 2 h later and the fold induction of porcine IFN-β mRNA was determined by RT-qPCR normalized to GAPDH. Data shown are mean ± SD of triplicates with comparisons made to mock-transfected MDA5-expressing cells. Significant differences using the Student´s t test are indicated (*p < 0.05; **p < 0.01).