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Article

A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging

1
Centro de Investigación en Enfermedades Tropicales (CIET), Facultad de Microbiología, Universidad de Costa Rica, San José 11501-2060, Costa Rica
2
Dulbecco Lab Studio, Residencial Lisboa 2G, Alajuela 20102, Costa Rica
*
Author to whom correspondence should be addressed.
Academic Editor: Allan Brasier
Viruses 2021, 13(7), 1193; https://doi.org/10.3390/v13071193
Received: 31 May 2021 / Revised: 16 June 2021 / Accepted: 17 June 2021 / Published: 22 June 2021
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. View Full-Text
Keywords: vesicular stomatitis; herpes simplex; yellow fever; animal viruses; plaque assay; real-time; live-cell imaging; automated image analysis; DNA fluorescent dyes; antiviral screening vesicular stomatitis; herpes simplex; yellow fever; animal viruses; plaque assay; real-time; live-cell imaging; automated image analysis; DNA fluorescent dyes; antiviral screening
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MDPI and ACS Style

Arias-Arias, J.L.; Corrales-Aguilar, E.; Mora-Rodríguez, R.A. A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging. Viruses 2021, 13, 1193. https://doi.org/10.3390/v13071193

AMA Style

Arias-Arias JL, Corrales-Aguilar E, Mora-Rodríguez RA. A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging. Viruses. 2021; 13(7):1193. https://doi.org/10.3390/v13071193

Chicago/Turabian Style

Arias-Arias, Jorge L., Eugenia Corrales-Aguilar, and Rodrigo A. Mora-Rodríguez 2021. "A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging" Viruses 13, no. 7: 1193. https://doi.org/10.3390/v13071193

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