Optimized methods for the detection of flavivirus infections in hyperendemic areas are still needed, especially for working with patient serum as a starting material. The focus-forming assay (FFA) reveals critical aspects of virus-host interactions, as it is a quantitative assay to determine viral loads. Automated image analysis provides evaluations of relative amounts of intracellular viral protein at the single-cell level. Here, we developed an optimized FFA for the detection of infectious Zika virus (ZIKV) and dengue virus (DENV) viral particles in cell cultures and clinical serum samples, respectively. Vero-76 cells were infected with DENV-2 (16681) or ZIKV (PRVA BC59). Using a panel of anti-DENV and anti-ZIKV NS1-specific monoclonal antibodies (mAbs), the primary mAbs, concentration, and the optimal time of infection were determined. To determine whether intracellular accumulation of NS1 improved the efficiency of the FFA, brefeldin A was added to the cultures. Focus formation was identified by conventional optical microscopy combined with CellProfiler™ automated image analysis software. The FFA was used with spike assays for ZIKV and clinical specimens from natural infection by DENV-1 and DENV-2. mAb 7744-644 for ZIKV and mAb 724-323 for DENV used at a concentration of 1 μg/ml and a time of 24 hours postinfection produced the best detection of foci when combining conventional counting and automated digital analysis. Brefeldin A did not improve the assessment of FFUs or their digitally assessed intensity at single-cell level. The FFA showed 95% ZIKV recovery and achieved the detection of circulating DENV-1 and DENV-2 in the plasma of acutely ill patients. The combination of the two techniques optimized the FFA, allowing the study of DENV and ZIKV in culture supernatants and clinical specimens from natural infection in hyperendemic areas.

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