Theplaque reduction assay (PRA) is the gold standard phenotypic method to determine virus susceptibilities to antiviral drugs. Cells are infected with a constant viral inoculum and allowed to grow in the presence of serial drug dilutions for days. After this incubation period, the cells are fixed and stained, and the viral plaques are visually counted. The drug concentration that reduces the cytopathic effects by 50% compared to a negative control (no drug) is defined as the 50% effective concentration (EC50).
The plaque reduction assay is laborious and prone to low reproducibility due to human subjectivity. In contrast, real-time cell analysis using the xCELLigence RTCA system offers automatic and rapid results with high reproducibility for characterizing drugs that inhibit the virus life cycle.
Traditional (OR) Conventional methods vs xCELLigence RTCA
|Plaque Reduction Assay||xCELLigence RTCA Antiviral Assay|
|Considered the gold standard phenotypic method to determine virus susceptibilities to antiviral drugs. Cells are infected with the virus and treated with serially diluted anti-viral drugs. Plaques are manually counted to determine antiviral drug activity.||Direct, real-time kinetics to measure antiviral drug activity. Cells are grown to confluence and infected with the virus in the presence of different drug treatments. The xCELLigence RTCA automatically monitors antiviral drug activity in real-time.|
|Viral plaque formation can take days to weeks to be detectable.||Quantitative monitoring of both fast (hours) and slow (days to weeks) CPE.|
|PRA assay provides no information about the kinetics of the anti-viral drug activity.||xCELLigence provides quantitative kinetic data of the anti-viral drug effects on cells, captures the entire virus life cycle|
|Plaques are manually counted to determine the 50% effective concentration. The manual counting of plaques by visual inspection can be highly subjective and inconsistent since plaque formation rates and sizes can vary dramatically.||Accurate, precise, and highly reproducible method with less manual labor. Measurements are automatically recorded at a user defined frequency and are plotted by the xCELLigence software as Cell Index (CI). By plotting the Cell Index value at a given time point as a function of drug concentration, dose response curves are generated to calculate EC50|
The rapid, sensitive, and high throughput xCELLigence Real-Time Cell Analysis Assay is ideal for virus titer determination
Example Data: Using xCELLigence to evaluate drug sensitivity of wildtype and mutant HSV-1
Data adapted from the Journal of Clinical Microbiology, volume 54(8), Piret, J. et al. “Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus.” Copyright 2016, with permission from American Society for Microbiology.
Guy Boivin and coworkers at Laval University in Quebec analyzed the sensitivity of wild-type (WT) and mutant herpes simplex virus 1 (HSV-1) to the antiviral acyclovir. After being grown to confluency in E-Plates, Vero cells were infected with virus for 90 minutes before adding different concentrations of the drug. Although the CPE induced by both WT and mutant virus could be blocked by acyclovir, a much higher concentration of the drug was required for blocking the mutant strain than the WT strain (figure). By plotting the Cell Index value at a given time point as a function of drug concentration, dose response curves were generated (data not shown here), yielding EC50 values of 100 μM and 0.8 μM for the mutant and WT viruses, respectively. Importantly, these findings are consistent with this mutant strain of the virus having a mutation in its DNA polymerase, the target of acyclovir.
Antiviral Drug Studies Publications
- Caliaro, O. et al. Phenotypic testing of patient herpes simplex virus type 1 and 2 isolates for acyclovir resistance by a novel method based on real-time cell analysis. J Clin Virol. 2020 Feb, 125:104303. doi: 10.1016/j.jcv.2020.104303. [Epub ahead of print]
- Prasad, V. et al. Cell Cycle-Dependent Kinase Cdk9 Is a Postexposure Drug Target Against Human Adenoviruses. ACS Infect. Dis. 2017 Jun 9, 3(6), 398–405
- Watterson, D. et al. A Generic Screening Platform For Inhibitors of Virus Induced Cell Fusion Using Cellular Electrical Impedance. Sci. Rep. 2016 Mar 15, 6, 22791.
- Zandi, K. A Real-Time Cell Analyzing Assay for Identification of Novel Antiviral Compounds against Chikungunya Virus. Methods Mol. Biol. 2016, 1426, 255–62.
- Sharma, B. N. et al. Antiviral Effects of Artesunate on Polyomavirus BK Replication in Primary Human Kidney Cell. Antimicrob. Agents Chemother. 2014, 58(1), 279–89.
- Piret, J. et al. Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus. J. Clin. Microbiol. 2016 Aug, 54(8), 2120–7.
- Cymerys, J. et al. Primary Cultures of Murine Neurons for Studying Herpes Simplex Virus 1 Infection and Its Inhibition by Antivirals. Acta Virol. 2013, 57(3), 339–45.
- Witkowski, P. T. et al. Cellular Impedance Measurement as a New Tool for Poxvirus Titration, Antibody Neutralization Testing and Evaluation of Antiviral Substances. Biochem. Biophys. Res. Commun. 2010 Oct 8, 401(1), 37–41.
Featured xCELLigence RTCA Systems for Viral CPE Assays
|Dual Purpose||Single Plate||Multi Plate||High Throughput|
|3×16 wells||1×96 wells||6×96 wells||Up to 4×384 wells|