The plaque reduction assay (PRA) is the gold standard phenotypic method to determine virus susceptibilities to antiviral drugs. Cells are infected with a constant viral inoculum, the virus is then allowed to grow in the presence of serial drug dilutions for days before the cells are fixed and stained. The viral plaques are then visually counted. The drug concentration that reduces the cytopathic effects by 50% compared to negative (no drugs) controls is defined as the 50% effective concentration (EC50). However, this assay is laborious and prone to low reproducibility due to human subjectivity. The high reproducibility and exquisite sensitivity to virus-induced changes in host cell morphology and behavior makes the xCELLigence® technology very well suited for identifying and characterizing drugs that inhibit any facet of the virus life cycle.
Urs Greber and colleagues at the Institute of Molecular Life Sciences in Zurich aimed to identify a drug that could mitigate the effects of adenovirus in infected patients. Their screening assay involved growing HeLa cells to confluency and then infecting them with human adenovirus in the presence of different drug candidates. Most effective among these was flavopiridol, a semisynthetic flavonoid compound known to inhibit the cell cycle dependent kinase Cdk9. As seen in Figure A below, in the absence of the drug, adenovirus infection induces a robust CPE, with the impedance signal decreasing to zero (red curve). However, in a dose-dependent manner flavopiridol is able to significantly delay the onset of CPE (blue and orange curves). Importantly, these findings based on impedance were corroborated by microscopy analysis (Figure B).
Monitoring antiviral activity of flavopiridol. (A) HeLa cells were grown in an E-Plate to the point of confluence. Roughly 50 hours post seeding, cells were infected with human adenovirus strain C5 in the presence of different concentrations of flavopiridol. (B) Flavopiridol affords broad protection against adenovirus. Here WI38 lung fibroblasts were infected with human adenovirus strain D37 and four hours later flavopiridol either was or was not added. 48 hours post infection the drug is clearly seen to have prevented a cytopathic effect from occurring. Data adapted from reference 1.
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 (below 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 particular mutant strain of the virus having a mutation in its DNA polymerase, which is the target of acyclovir.
Comparing acyclovir sensitivity of WT and mutant HSV-1. Vero cells were grown to confluency in an E-Plate. 48 hours post seeding cells were infected with either WT or mutant HSV-1 for 90 minutes. Different concentrations of acyclovir were then added and impedance monitored every 30 minutes for an additional 100 hours. Note that much higher concentrations of the drug were required in order to prevent the cytopathic effect in the mutant. Data adapted from ref. 2.
xCELLigence instruments that are compatible with virology/vaccine applications:
|Dual Purpose||Single Plate||Multi Plate||High Throughput|
|3×16 wells||1×96 wells||6×96 wells||Up to 4×384 wells|
- Cell Cycle-Dependent Kinase Cdk9 Is a Postexposure Drug Target against Human Adenoviruses. ACS Infect Dis. 2017 Jun 9;3(6):398-405.
- 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.
- A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance. Sci Rep. 2016 Mar 15;6:22791.
- A Real-Time Cell Analyzing Assay for Identification of Novel Antiviral Compounds against Chikungunya Virus. Methods Mol Biol. 2016;1426:255-62.
- Antiviral effects of artesunate on polyomavirus BK replication in primary human kidney cell. Antimicrob Agents Chemother. 2014;58(1):279-89.
- Primary cultures of murine neurons for studying herpes simplex virus 1 infection and its inhibition by antivirals. Acta Virol. 2013;57(3):339-45.
- 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.