Abstract
Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.
Highlights
To date, plaque assay continues to be considered the “gold standard” virological technique for quantifying viral titers of lytic virions [1]
The staining with Hoechst was much weaker in the internal region of the plaque indicating a loss of chromatin staining in the zone stained by SYTOX Green
These results suggest that the staining with DNA fluorescent dyes with differential membrane permeability enables the visualization of different stages of the Viruses 2021, 13, x FOR PEER REVIEcWytopathic effect at a single-cell level within individual viral plaques and indicates6thofa1t7DNA staining does not interfere with viral replication of both RNA and DNA animal viruses
Summary
Plaque assay continues to be considered the “gold standard” virological technique for quantifying viral titers of lytic virions [1]. The typical plaque assay relies on the use of solid or semisolid overlays (i.e., agarose or carboxymethyl cellulose, respectively) to restrict viral infection and spreading to the surrounding cells of the monolayer. This allows the formation of distinct foci of cell death that grow in time as the replication-lysis-infection cycle continues, arising as discrete and countable plaques that are visualized after fixation and counterstained with colorimetric dyes such as neutral red or crystal violet [5,6]. This classical approach takes several days until large enough plaques can be visualized and present wide variations in plaque recognition and counting among different analysts [1]
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