The patient’s blood is seropositive for H3N2 and blocked IFN-α function in vivo. (A) We tested for HAI assay by treating patient (Pt) and healthy donor (HD) blood with receptor destruction enzyme (RDE) and diluting 1:10 before mixing with the H1N1, H3N2, and H5N1 IAVs at the indicated titers, together with 0.5% Turkey red blood cells. Blood from naive mice and mice immunized with H1N1 (A/Victoria/4897/2022) or H3N2 (A/Thailand/8/2022) was used as negative and positive controls. Experiment was done once. (B) A549 cells were incubated overnight with 50 pg/ml exogenous IFN-α2 with or without anti–IFN-α2 monoclonal antibody (mAb), patient blood (Pt), healthy donor blood (HD1 and HD2), or APS-1 patient blood (APS-1) at indicated dilution, and then infected with influenza A/California/04/2009 virus expressing NS1-mCherry (CalNSmCherry) at an MOI of 1. The percentage of the cells infected was determined 24 h after infection with a Celigo (Nexcelcom) imaging cytometer. The percentage of infection was normalized against cells infected without IFN-α2 treatment. The dotted line at 26.5% indicates the mean percentage of cells infected after treatment with IFN-α2 only. Experiments were done twice and paired t test was performed (P values: ****<0.0001; ***<0.001; **<0.01; *<0.05; ns > 0.05).