Large scale semi-automated quantification of viral spread reveals multi-phasic structure of plaque formation

Plaque assays are typically quantified at an endpoint and fail to capture the temporal dynamics of viral infection and spread. In this study we introduce a high-throughput method utilising live-cell imaging and a recombinant vaccinia virus (VACV) engineered to express two fluorescent transgenes linked to distinct replication phases. This novel approach allows for real-time tracking of VACV plaques from their inception at a single infected cell through to multicellular expansion, enabling detailed kinetic analysis of plaque development. Using this analysis pipeline we categorise VACV plaque formation into three distinct phases: Establishment, Expansion, and Exhaustion, and quantitatively describe their dynamics. Our findings reveal significant variability in the initiation time of plaque spread (from single-cell to multicellular stages) and the growth rate of plaques, both between individual plaques and across different cell lines. This variability underscores the influence of host cellular factors on the kinetics of viral replication. Additionally, we reveal that the viral replication cycle dramatically accelerates through the early phases of plaque formation, which we hypothesise is driven by the changing multiplicity of cellular infection at the plaque edge. Finally, we demonstrate that differences in plaque sizes between two cell lines (BS-C-1 and HaCaT) can be largely attributed to variations in viral replication rate. This research reinforces the value of live-cell fluorescence microscopy in elucidating the complex spatiotemporal dynamics of viral infections, and contributes to a deeper understanding of the mechanisms driving viral spread and replication kinetics.