From spark to wildfire: illuminating single-cell origins of virus infection spread and diversification.

When a virus encounters a living cell it triggers infection, cell death, and the release of virus progeny. In lab cultures, such progeny spread to near and distant cells, creating a plaque, a visible record of virus growth and cell death. Plaque-based assays have long enabled amplification and quantification of virus particles, but the earliest infections of a plaque remain poorly characterized. Here, we used a recombinant rhinovirus expressing a fluorescent reporter protein to track the start and spread of infection across HeLa cell monolayers at single-cell resolution. We analyzed reporter expression profiles from eight plaques and over 150 cells, reviewing, testing and applying methods of time-series analysis, clustering, and dimensionality reduction. We identified primary infections based on early reporter expression in isolated cells and secondary infections among their neighbors. Primary infected cells exhibited tightly clustered gene expression profiles, while secondary infected cells showed broader heterogeneity in the timing, intensity, and duration of reporter expression, without correspondence to their associated primary cells. This heterogeneity likely reflects a combination of factors, including noisy gene expression, viral genetic changes, and variation in local cellular environments. The rapid diversification of gene expression highlights the power of single-cell imaging to shed light on infection spread.