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

Viruses

Rodrigo A. Mora-Rodríguez¹  Eugenia Corrales-Aguilar¹  Jorge L. Arias-Arias¹ 

[1] Centro de Investigación en Enfermedades Tropicales (CIET), Facultad de Microbiología, Universidad de Costa Rica, San José 11501-2060, Costa Rica;

关键词: vesicular stomatitis;    herpes simplex;    yellow fever;    animal viruses;    plaque assay;    real-time;   

DOI  :  10.3390/v13071193

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown