Virus Titer Determination

The traditional measurement of viral titers often involve cytopathic effect (CPE) quantification by plaque-based assays or tissue culture infectious dose (TCID50) assays.   However, CPE evaluation based on visual interpretation of cell culture deterioration  is subject, and there is no universally accepted standard on how CPEs are scored.  Also, these labor-intensive endpoint assays can only provide a mere snapshot of the infection process and therefore the overall kinetics information is difficult or impossible to derive using limited number of data points.  The xCELLigence Real-Time Cell Analysis (RTCA) technology presents a unique, impedance-based live cell assay; allowing for label-free and real-time tracking of CPE progression in virus-infected cells.

 

Citing the fact that the traditional CPE/plaque assays used for quantifying viral titers are labor intensive and time consuming, Reisen and colleagues evaluated the efficacy of xCELLigence RTCA for determining the titers of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV)2.

  • 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 below).
  • 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 below).
  • 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 patient treatments.

 

Using xCELLigence RTCA to determine viral titer.  Upper panels: Real-time monitoring of (A) WNV- and (B) SLEV-induced cytopathic effect in Vero cells. The normalized Cell Index is shown for E-Plate wells that were inoculated with a negative control (Ctrl) or different numbers of plaque forming units (PFU) of WNV and SLEV. Each curve is an average of two independent replicate wells. The horizontal line denotes the point at which Cell Index has dropped to 50% of its initial value (i.e. before virus addition). The time required to reach this point is referred to as “CIT50”.  Lower panels: By plotting CIT50 as a function of viral titer, a standard curve was produced, which can be used for determining virus concentration in diverse types of samples.  Figure adapted from reference 1.

Vaccine handbook

Handbook: Explore Viral Cytopathic Effect Assays for Virology and Vaccine Research

  • Viral Titer Determination
  • Detection and Quantification of Neutralizing Antibodies
  • Studying Anti-viral Drugs
  • Testing Virucides
  • Oncolytic Viruses
  • Assessing virus quality/fitness

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Virus webinar

Webinar on Demand: A Groundbreaking Technology for Vaccine Development: New Techniques in Viral CPE Assessment using Real Time Cell Analysis

On Thursday, Nov 29, 2018 at 10am EST, Loic Benair (Sanofi Pasteur, France) and Brandon Lamarche (ACEA Biosciences) will describe new approach aimed at quantifying viral cytopathic effects during vaccine development.

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App note 18

Application Note 18: A New Way to Monitor Virus-Mediated Cytopathogenicity

A demonstration on the experimental workflow and the power of real-time impedance-based technology to evaluate viral cytopathic effects. 

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xCELLigence instruments that are compatible with virology/vaccine applications:

Dual PurposeSingle PlateMulti PlateHigh Throughput
RTCA DPRTCA SPRTCA MPRTCA HT
3×16 wells1×96 wells6×96 wellsUp to 4×384 wells
  1. Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology. J Virol Methods. 2011 May;173(2):251-8.
  2. Robust real-time cell analysis method for determining viral infectious titers during development of a viral vaccine production process.  J Virol Methods. 2017 Nov 14;252:57-64.
  3. Development of a Real-Time Cell Analysis (RTCA) Method as a Fast and Accurate Method for Detecting Infectious Particles of the Adapted Strain of Hepatitis A Virus. Front Cell Infect Microbiol. 2018; 8: 335.
  4. Real-time cell analysis – A new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity. J Virol Methods. 2013 Nov;193(2):364-70.
  5. 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.