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

TL;DR

This paper presents a label-free, continuous imaging platform for viral plaque dynamics that accelerates antiviral testing and provides detailed time-resolved infection data, surpassing traditional methods.

Contribution

It introduces a novel, scalable, high-dimensional PRA platform combining ultra-large-area imaging and deep learning for rapid, dynamic antiviral susceptibility testing.

Findings

Matched ground truth with zero false positives

Accelerated readout by approximately 26 hours

Revealed distinct temporal effects of drug concentration on viral inhibition

Abstract

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.