Continuous quantification of viral plaque dynamics using ultra-large-area label-free imaging enables rapid antiviral susceptibility testing

The plaque reduction assay (PRA) remains the gold standard for antiviral susceptibility testing, evaluating drug potency by measuring reductions in plaque-forming units (PFUs). However, the traditional PRA is time-consuming, labor-intensive, prone to manual counting errors, and offers limited scalability. Moreover, its reliance on destructive fixation and chemical staining reduces the assay to a static, endpoint observation, obscuring the dynamic, time-resolved kinetics of dose-dependent viral inhibition. Here, we introduce a label-free, time-resolved PRA platform that transforms the conventional assay into a continuous, high-dimensional measurement of viral infection dynamics. Our system integrates a compact lens-free imaging setup with a custom-designed ultra-large-area (100 cm^2) thin-film transistor (TFT) image sensor and deep learning-based algorithms to autonomously quantify PFU dynamics within an incubator. Validated using herpes simplex virus type-1 (HSV-1) treated with acyclovir, the platform matched chemically-stained ground truth measurements with zero false positives while accelerating readout by ~26 hours. Crucially, our system revealed that increasing drug concentrations induce temporally distinct delays and suppress new PFU formation, enabling conclusive drug efficacy evaluations within ~60 hours post-infection. This scalable, label-free framework redefines antiviral susceptibility testing as a rapid, time-resolved and information-rich measurement framework, providing a generalizable platform for virology research, high-throughput drug screening, and clinical diagnostics.