High-throughput screening (HTS) is a powerful tool for identifying modulators of viral replication, but its success depends heavily on the robustness, scalability, and comparability of the underlying assay formats. In the context of vesicular stomatitis virus (VSV) production in HEK293F cells, we aimed to establish a reliable and scalable analytical platform to support compound screening for host-directed viral titer enhancement.

To this end, we developed a tiered analytical funnel comprising three complementary assays using a reporter VSV: a fluorescence-based readout measuring mean fluorescence intensity (MFI) in producer cells for primary screening, flow cytometry to quantify infected cells at single-cell resolution for secondary validation, and an already established method, kinetic infectious titer assay (KITA), to directly assess infectious virus output.

A key focus of this work was the systematic comparison of assay performance across 96- and 384-well formats. Miniaturization to the 384-well format enabled increased throughput and accelerated workflows, both critical for large-scale screening. Comparative analysis revealed consistent signal dynamics across formats, with the 384-well setup maintaining sufficient sensitivity to detect ≥5-fold changes in viral output. Importantly, strong correlations between MFI-based fluorescence readouts and infectious titers confirmed the reliability of the miniaturized assays.

This study highlights the importance of scale compatibility and analytical consistency in HTS assay development. Our optimized platform provides a scalable and validated framework for screening host-targeting compounds aimed at enhancing viral yields in biotechnological applications.