simIFN-I can restore the antiviral activity of IFN-I in the presence of neutralizing anti-IFN-I autoAbs. (A) Replication kinetics of five GFP-expressing viruses in A549 cells that were pretreated for 16 h with the conditions indicated (simIFNα used with IFNα-neutralizing plasma A1). Cells were inoculated with an MOI of 0.1 PFU/cell for H5N1-GFP, 0.01 FFU/cell for PIV5-GFP, 0.1 FFU/cell for PIV2-GFP, 0.03 FFU/cell for MeV-GFP, and 0.1 TCID₅₀/cell for RSV-GFP. The GFP signal was monitored with the IncuCyte live-cell imaging system as a surrogate readout for viral replication. Data represent mean values from n = 3 replicates, and error bars indicate standard deviations. (B) AUC values of data shown in A. Data represent mean values from n = 3 independent experiments, and error bars indicate standard deviations. (C) AUC values of H5N1-GFP replication in A549 cells that were pretreated with similar conditions as described in A, using additional IFNα-neutralizing donor plasma samples as indicated. Data represent mean values from n = 3 independent experiments, and error bars indicate standard deviations. (D) Fluorescence images of A549 cells infected with H5N1-GFP from the experiments described in C. Images are from 12 h after inoculation, where virus replication had reached peak total integrated intensity values. The scale bar represents 200 μm. For all data panels, results are representative of n = 3 similar experiments. Statistical significance between groups was determined by unpaired t tests (B and C): ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. See also Fig. S5. AUC, area under the curve.