Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 113

One-step growth analysis, while simple, remains a powerful tool for studying virus reproduction. When cells are infected at a high multiplicity of infection, sufficient viral nucleic acid or protein can be isolated to allow a study of their production during the infectious cycle. The ability to synchronize infection is the key to this approach. Many of the experimental results discussed in subsequent chapters of this book were obtained using such one-step growth analysis. The power of this approach is such that it reports on all stages of the reproduction cycle in a simple and quantitative fashion. With modest expenditure of time and reagents, virologists can deduce a great deal about viral translation, genome replication, and assembly. It has long been assumed from such one-step growth analyses that the same steps of the viral reproduction cycle occur at the same time in every infected cell. However, results from analyses of single infected cells demonstrate that the same steps can take place at vastly different times in individual cells in the population. We now understand that results from population-based studies of viral reproduction comprise an average of events occurring in individual cells. One-step growth analyses with single cells have the potential of unraveling the viral and cellular basis for such individual heterogeneity.

Figure 2.22 Single-cell virology. (A) A microfluidic device with 6,400 wells is fitted with four separate sample inlets (green) and pneumatic control lines (red) that permit each well to be sealed and isolated. A small part of the device is magnified at the top, showing an array of 24 wells, and four wells are further magnified to the left. (B) The device can be used to measure real-time fluorescence in cells infected with a virus encoding a fluorescent reporter. The production of fluorescence is shown in the graph and illustrated in the views of single cells in individual cells above. There is a lag in the detection of fluorescence in an infected cell (t_i), followed by virus reproduction (t_j) and a decline in fluorescence caused by cell lysis (t_k). Reprinted from Guo F et al. 2017. Cell Rep 21:1692-1704, with permission.

From the humble beginnings of the one-step growth curve, many new methods have propelled our understanding of viruses and infected cells to greater depths and at unprecedented speed. An astounding array of technologies, including high-throughput sequencing, proteomics, and single-cell approaches, have been developed. These methods have already led to significant discoveries about viral evolution, reproduction, and pathogenesis. We are truly in a remarkable era, when few experimental questions are beyond the reach of the techniques that are currently available.