Viro-fluidics: Real-time analysis of virus production kinetics at the single-cell level.

Real-time visualization and quantification of viruses released by a cell are crucial to further decipher infection processes. Kinetics studies at the single-cell level will circumvent the limitations of bulk assays with asynchronous virus replication. We have implemented a "viro-fluidic" method, which combines microfluidics and virology at single-cell and single-virus resolutions. As an experimental model, we used standard cell lines producing fluorescent HIV-like particles (VLPs). First, to scale the strategy to the single-cell level, we validated a sensitive flow virometry system to detect VLPs in low concentration samples (≥10 VLPs/mL). Then, this system was coupled to a single-cell trapping device to monitor in real-time the VLPs released, one at a time, from single cells under cell culture conditions. Our results revealed an average production rate of 50 VLPs/h/cell similar to the rate estimated for the same cells grown in population. Thus, the virus-producing capacities of the trapped cells were preserved and its real-time monitoring was accurate. Moreover, single-cell analysis revealed a release of VLPs with stochastic bursts with typical time intervals of few minutes, revealing the existence of limiting step(s) in the virus biogenesis process. Our tools can be applied to other pathogens or to extracellular vesicles to elucidate the dissemination mechanisms of these biological nanoparticles.