The traditional measurement of viral titers often involves cytopathic effect (CPE) quantification by plaque-based assays or tissue culture infectious dose (TCID50) assays. In contrast to conventional endpoint assays, real-time cell analysis using the xCELLigence RTCA system offers a comprehensive and reliable evaluation of viral cytopathogenicity.
Typical methods vs xCELLigence RTCA
|Plaque Assay||Endpoint dilution assay (TCID50)||xCELLigence Viral CPE assay|
|Labor intensive counting of plaques formed by a virus at varying dilutions to obtain a low multiplicity of infection (MOI). Concentration of virus (titer) is calculated as plaque forming units (PFU) per unit of volume.||Indirect calculation to determine the point at which 50% of cells in a culture are infected. Wells are seeded with cells and infected at specific dilutions. Wells are visually scored for presence or absence of CPE.||Direct, real time, and quantitative kinetics for the entire virus life cycle. Cells are plated, infected with virus, and xCELLigence RTCA automatically monitors viral CPE in real time|
|Viral plaque formation can take days to weeks to be detectable.||Viral CPE observation can take days to weeks to be detectable.||Quantitative monitoring of both fast (hours) and slow (days to weeks) CPE.|
|Plaque assay provides no information about the onset of CPE or the kinetics of virus-mediated cytotoxicity.||Endpoint assay provides no information about the onset of CPE or the kinetics of virus-mediated cytotoxicity.||xCELLigence provides assessment and quantification of the full virus life cycle.|
|The manual counting of plaques by visual inspection can be highly subjective and inconsistent since plaque formation rates and sizes can vary dramatically.||Visual determination of CPE is highly variable and subjective, infected cells can be difficult to visualize due to poor Neutral Red contrast||Measurements are automatically recorded at a user defined frequency and are plotted by the xCELLigence software as Cell Index (CI). Accurate, precise, and highly reproducible method with less manual labor.|
Source: Smither SJ et al J Virol Methods. 2013 Nov;193(2):565-71.
The rapid, sensitive, and high throughput xCELLigence Real-Time Cell Analysis Assay is ideal for virus titer determination
Example: Determining West Nile Virus and St. Louis Encephalitis Virus titers
Figure reprinted from Journal of Virological Methods, volume 173(2), Fang, Y. et al., “Real-Time Monitoring of Flavivirus Induced Cytopathogenesis Using Cell Electric Impedance Technology,” pages 251–8. Copyright 2011, with permission from Elsevier.
Citing the fact that the traditional CPE/plaque assays used for quantifying viral titers are labor intensive and time consuming, Fang and colleagues evaluated the efficacy of xCELLigence RTCA for determining the titers of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV).
- Vero cells in suspension were incubated with serial dilutions of WNV or SLEV for 30 minutes, followed by addition of this cell/virus mixture to an E-Plate in which CPE development is monitored.
- Uninfected control cells grew to confluency and maintained a plateaued Cell Index.
- Virus-infected cells displayed a time- and dose-dependent decrease in Cell Index down to zero, indicating complete cell lysis (upper panels of Figures A and B above).
- Both viruses exhibited CPE kinetics that is extremely well correlated with the known titer of the virus. This is highlighted by plotting the CIT50 (time required for the Cell Index to decrease by 50%) as a function of virus titer (lower panels of Figures A and B above).
- WNV displays both an earlier onset of CPE and a more rapid rate of CPE progression than SLEV in the xCELLigence CPE assay, an observation consistent with the known cytolytic activities of WNV and SLEV,
Using this type of standard curve, it is easy to determine the viral titer in samples of unknown concentration. Beyond characterizing viral stocks being used for research or vaccine purposes, this approach can be applied in the clinic to quantify viral load before, during, and after treatments.
- Thieulent, Côme J. et al. Screening and Evaluation of Antiviral Compounds Against Equid Alpha-Herpesviruses Using an Impedance-Based Cellular Assay. Virology 2019 Jan, 526: 105–116.
- Gilchuk, P. et al. Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization. Immunity. 2020 Feb, 52(2): 388 – 403.
- 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]