Fig. 3: Validation of multiplex Orthoflavivirus infection assay for antiviral studies.

a Validation using sera containing virus-specific antibodies (Ab). V-NIR cells were infected with virus mixture R5 (Fig. 2), preincubated with serial dilutions of sera containing anti-DENV-2 Ab (circle), anti-JEV Ab (square), or anti-YFV Ab (triangle), respectively, as described in Methods section (SNT). Representative neutralizing curves plotted using nonlinear regression. The data presented means ± SD of n = 3 independent experiments. b Pie charts showing the percentages of DENV-2-, JEV- and YFV-infected cells versus the total infected cells at indicated dilutions. c Correlation analysis of single-virus SNT (Supplementary Fig. 4) and multiplex-virus SNT. The correlation between the single-virus assay and multiple-virus assay is indicated by a black solid line, with the 95% confidence intervals indicated by gray shaded areas within the dashed lines. Next to Pearson correlation (left panel), the Bland–Altman test (right panel) was used to estimate the degree of agreement between assays, comparing differences in infection rates obtained by single-virus assay (Supplementary Fig. 4) and multiplex-virus assay with their respective average values. Dashed lines represent bias and 95% confidence intervals (lower and upper limits of agreements). d Validation using known Orthoflavivirus inhibitors. V-NIR cells infected with virus mixture R5 were treated with a dilution series of IFN-α [a universal host-cell targeting (orthoflavi)virus inhibitor], NITD008³³, or JNJ-A07^6,7. Representative dose-response curves plotted using nonlinear regression. The data presented means ± SD of n = 3 independent experiments. e Pie charts showing the percentages of DENV-2-infected, JEV-infected, and YFV-infected cells versus the total infected cells at indicated concentration of inhibitors. f Correlation analysis of single-virus antiviral assay (Supplementary Fig. 4) and multiplex-virus antiviral assay. Pearson correlation analysis (left) and Bland–Altman test (right) were used to estimate the degree of agreement between assays as in panel (c). IU/mL International Units/mL, cpd compound, pie charts: − untreated virus control (VC), + representative medium dilution/medium concentration, +++ representative low dilution/high concentration. Source data are provided as a Source Data file.