HSV-1 progeny production by individual human keratinocytes spans three orders of magnitude.

A fundamental property of viruses is their burst size-the amount of progeny produced during one round of infection. Herpes simplex virus 1 (HSV-1) is a widespread pathogen, causing significant morbidity in the human population worldwide. Despite decades of investigation into the biology of HSV-1, its burst size at the single-cell level has not been rigorously assessed. Here, we characterized HSV-1 progeny production by individual human keratinocytes, the physiological target of HSV-1 infection. Using a fluorescently-tagged virus, high-throughput single-cell sorting, and miniaturized plaque assay, we have quantified the progeny released from over a thousand individual cells throughout infection, at two multiplicities of infection. We found that individual keratinocytes harbor a very small number of infectious viruses inside them, which necessitated physical separation of cells to account for both secreted and cell-associated progeny. The total progeny produced by individual cells spans three orders of magnitude, and this variability is observed at both high and low multiplicities of infection, with a small sub-population of super-producer cells responsible for the majority of produced progeny by the entire population. We further found that super-production is not a genetic trait of the virus, as amplification of viruses produced by low and high producer cells results in similar burst sizes. Our findings both shed light on the fundamental process of progeny production during HSV-1 infection and provide a novel target for antiviral development, aimed at blocking super-producer cells.IMPORTANCEViruses are defined by their ability to take over host cells and turn them into factories that produce new progeny. Recent advances in Biology allowed us to study viral infections at the single-cell level, illuminating the vast heterogeneity presented by genetically identical cells during viral infection. Despite these advances, for many viruses, we still lack even basic characterization of progeny production at the single-cell level, due to the highly demanding technical challenges associated with measuring it. Here, we present the first quantification of progeny production by individual human keratinocytes infected by herpes simplex virus 1, revealing most individual cells produce a low level of new progeny, while a few rare cells produce significantly more.