Fig. 3

WNV-NTs Titer and IFN-I-NTAbs neutralizing type I IFNs in WNV-infected individuals. To characterize the antibodies present in serum samples: (A) WNV-neutralizing antibodies (WNV-NTAbs) were detected in serum samples from WNND patients and asymptomatic donors using a microneutralization assay. WNV-NTAbs were identified in 73% of WNND samples (8/11) and in all donor samples, with median titers of 1:20 and 1:10, respectively. Titers below 1:10 were considered negative, while those equal to or above 1:10 were considered positive. (B) Autoantibodies against IFN-α2, IFN-β, and IFN-ω were detected using an ELISA assay. A reading above 0.5 OD (dotted line) was considered positive, based on reference values from healthy donor serum/plasma. Each sample was tested once. (C) and (D) A luciferase-based assay was used to identify autoantibodies capable of neutralizing type I IFNs. Plasma samples were tested for their ability to neutralize IFN-α2, IFN-β, and IFN-ω at different concentrations. HEK293T cells were transfected with luciferase plasmids and treated with type I IFNs. Neutralizing activity was defined as a relative luciferase activity (RLA) below 15% of the control value (dotted line). Each sample was tested once.