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

Plaque assays remain the gold standard readout of virus infectivity; however, plaque counting from plate images is labor-intensive and prone to inter-operator variability. We present an end-to-end, computer-aided workflow for cytopathic effect-based virus titration directly from laboratory plaque assay images. The proposed approach combines two models derived from the Segment Anything Model (SAM): a SAM2-based well-segmentation module that localizes assay wells across heterogeneous imaging conditions, and a SAM-based plaque-segmentation model that detects and enumerates plaques within each well. The method was evaluated on a mixed dataset comprising private plaque assay images of Mayaro virus and Coxsackievirus B3, together with public Vaccinia virus images from the VACVPlaque dataset. The pipeline outputs per-well plaque counts, automatically computes plaque-forming units per milliliter (PFU/mL), and is integrated into a web-based platform that allows users to review results and organize experiments. On held-out plates (17 from MAYV/CVB3 and 22 from VACV), the workflow generalized across two plate formats (6-well and 12-well) and showed strong agreement with manual annotations (Pearson correlation coefficients of 0.92 for MAYV/CVB3 and 0.88 for VACV). Automated plaque counts were further compared with annotations from four independent experts, demonstrating high concordance. The proposed system will be open sourced and publicly released upon acceptance of this manuscript to enable reproducible, scalable, and audit-ready plaque assay analysis while substantially reducing manual annotation effort.

High-fidelity molecular docking simulations can produce biologically relevant estimates of epitope-receptor binding affinity but are computationally expensive and therefore limit the number of candidates that can be screened for vaccine design. In this work, we evaluate machine learning (ML) approaches where variants of active learning are used to classify instances of high binding affinity between 9-mer epitopes and a well-conserved swine leukocyte antigen (SLA) receptor in the context of Porcine Reproductive and Respiratory Syndrome (PRRS). We use an internally generated dataset of 80 epitope-SLA docking affinities, each requiring more than 48 hours of high-performance computing (HPC). Multiple model families (linear, MLP, CNN, and a small transformer) are trained under strict low-data conditions within a pool-based active learning loop. In each case, optimal model configurations are identified by conducting large-scale hyperparameter optimization over the combined space of model architecture, training configuration, acquisition policy, and ensemble decision rules. To mitigate the effects of data subsample selection, each candidate configuration is evaluated by averaging performance over many randomized and balanced training and validation data subsets. Across experiments, transformer-based sequence models consistently emerged as the best-performing architecture, with active incremental learning yielding significant improvement over a baseline random sample acquisition strategy. Under moderate training data availability (N=30), the optimized ML-model configuration outperforms a standard baseline trained on twice the amount of data. Under higher training data availability (N=60), the same configuration achieves a peak accuracy of 86.8%, consistent with an upper bound of 85% classification accuracy based on two independent estimates of conformational noise.

Label-free virtual staining offers a compelling, non-destructive alternative to standard histopathology; however, its clinical adoption is hindered by the computational bottlenecks inherent to processing gigapixel Whole Slide Images (WSIs). Current deep learning approaches require patch-based inference to avoid memory constraints, which disrupts global tissue continuity and introduces tiling artifacts--displaying visible seams and color shifts. To address this, we introduce the Consistency Memory Bank (COMB), a novel label-free virtual staining framework that enforces spatial and channel consistency across tiles without memory bottlenecks. COMB decouples context storage from computation, utilizing a dynamic retrieval mechanism to fetch feature representations from adjacent tiles. This enables a retrieval-based context integration strategy that adopts local padding to resolve spatial discontinuities and neighbor-aware channel attention to stabilize statistical drift. Further optimized with a sliding window schedule to ensure minimal memory overhead, our method demonstrates superior performance over state-of-the-art baselines, achieving significant improvements in both perceptual fidelity and tiling consistency, while suggesting its downstream utility in tumor segmentation. Code is available at https://github.com/dou0000/COMB.