Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 111

Much of virology research is carried out by using populations of cells in culture or in animals. However, as discovered by virologists in the 1950s, individual cells of the same type can behave very differently with respect to susceptibility and permissiveness to infection and the kinetics of virus production.

Figure 2.21 Interactions between human proteins and Nipah virus proteins. Network representation of interactions of Nipah virus and human proteins determined by affinity purification and mass spectrometry. Nipah virus proteins are shown in orange. Cellular proteins are shown in gray. Protein names (from UniProt) are shown. Adapted from Martinez-Gil L, Vera-Velasco NM, Mingarro I. 2017. J Virol 91:e01461-17, with permission.

As early efforts to study virus infections in single cells were hampered by technical difficulties, the field failed to progress. This situation has changed with the development of flow cytometry and microfluidics and the adaptation of highthroughput methods, such as genome sequencing and mass spectrometry, to single cells.

Initially, micropipettes were used to aspirate a single cell at a time from a population, using a microscope. This labor-intensive method was supplanted by fluorescence-activated cell sorting to allow isolation of up to millions of cells in a few hours, according to size, morphology, or synthesis of specific proteins. More recently, automated microfluidic devices have been developed to allow automated capture of single cells using integrated fluidic circuits. Infection, cell lysis, reverse transcription, and amplification are all performed in these systems before high-throughput sequencing.

The study of virus infections in single cells is expected to provide information that explains why some cells are not infected, why the kinetics of viral reproduction may be so different, and how genomes change in a single cell. An example is the study of poliovirus infection of single cells, using a microfluidics platform installed on a fluorescent microscope (Fig. 2.22). This approach revealed observations otherwise masked in population-based studies, including the unique and independent contribution of viral and cell parameters to reproduction kinetics, the wide variation in reproduction start times, and the finding that reproduction begins later and with greater speed in single cells than in populations. A study of influenza virus infection of single cells revealed a wide variation in the yield, from 1 to 970 PFU per cell. Furthermore, the amounts of viral RNAs within individual cells varied by three orders of magnitude.