Direct measurement of viral cytopathic effects (CPE) using bright field imaging

Cytopathic Effects (CPE) are indicated by the changes in host cell morphology which are caused by the target infecting virus [REF Medical Microbiology. 4th edition. Chapter 44 Effects on Cells].  The common visual observations of the host cells are swelling or shrinkage, rounding, lysis, plaques, clumping, syncytia, and inclusions.  In general, the viruses that have the ability to cause degeneration of the host cells are called cytopathogenic.  There are varying degrees of CPE caused by different viruses, where some can completely and rapidly destroy host cell monolayer while other viruses alter the morphology of the host cells [REF Erica Suchman, Carol Blair. 2007. Cytopathic effects of viruses protocols].

viral cytopathic effect CPE

BSC40 cells (left), moi <0.01 pfu/cell 48 hours after infection (center), moi=10 pfu/cell (right)

What makes a good CPE assay?

There are several critical factors that need to be considered in order to set up and perform a good CPE assay.  The list below shows the factors and reasons why they are important for CPE assay development.

  1. Host cell seeding density – It is critical to seed enough host cells to quickly grow and cover the surface of the well, which allows better visualization of the CPE when it occurs. If the confluence of the host cells is not near 100%, then there will be some uncertainty as to whether or not the virus has induced CPE.
  2. Virus concentration – It is important to perform a viral titer experiment prior to the CPE assay to determine the proper range of the virus concentrations in order to obtain the appropriate TCID50 results.
  3. Throughput – Utilizing the plate-based image cytometry method, the assay throughput can be significantly increased from the standard 6 – 24-well plates to 96 and 384-well plates.
  4. Imaging – Image cytometry can rapidly scan whole wells of the entire plate to digitally capture bright field and fluorescent images for analysis. Unlike the conventional microscopy method, which requires manual observation of the CPE, and lacks digital records.
  5. Analysis – Image analysis algorithms can automate the identification of CPE via the destruction of cell monolayer, reduction in host cell count, and morphological changes at an individual cell level. In contrast, traditional CPE assays require trained technicians to visually inspect and identify, which is tedious, time-consuming, and generate a high level of uncertainties.

Directly analyze CPE using the Celigo Image Cytometer

The Celigo Image Cytometer is a sophisticated plate imager that can rapidly image the entire microplate in bright field and fluorescence.  Utilizing the powerful built-in image analysis software, the Celigo can quickly generate counts of plaques, foci, and individual infected cells, morphological measurements, as well as fluorescent intensity.  It can analyze the CPE using different image analysis and counting methods that is fit-for-purpose tailoring to the virus and host cell pairing.  Scientists can perform high-throughput automated imaging and analysis of the CPE in plates using bright field imaging without any labeling.

  1. Speed: Less than 10 min/plate with our automated image acquisition and analysis tools
  2. Innovation: Count every infected cell and total cells in every well without trypsinization
  3. Faster Results: Earlier plaque detection reduces assay duration
  4. Single system: Automated bright field and fluorescence-based counting for focus formation assay
  5. Convenience: Gain time back with integrating a plate stacker with up to 50 plates analyzed per day

Celigo Image Cytometer uses proprietary image analysis algorithms that have been optimized for a unique Virology Application Suite in the software.  There are 3 image analysis methods for quantifying CPE, such as the destruction of cell monolayer, reduction in host cell count, and morphological changes at an individual cell level.

1. Measure the host cell monolayer using confluence application

The pseudo-green color shows where cells are present and that area is quantified and compared between different viral treatments.

confluence application

Healthy cells (left) and infected cells (right)

2. Count the total number of cells in each well

The green outlines identify the counted cells in the well. CPE will be indicated here by an overall reduction in the number of cells.

confluence application

Healthy cells (left) and infected cells (right)

3. Measure the morphological changes of the host cells

The aspect ratios are measured from the counted cells to quantify the morphological changes. The rounding up of the host cells can be an indication of cells dying and this difference in morphology from the healthy cells can be quantified.

morphological changes

Healthy cells (left) and infected cells (right)

Morphology analysis using Celigo Image Cytometer

Morphological quantification can be conducted using the gating function in the Celigo software to specifically identify cells of different sizes, smoothness, aspect ratio, mean and integrated intensities.

Morphology analysis using Celigo Image Cytometer

Example image analysis using the gating function in the Celigo software to identify rounded and elongated cells

SARS-COV-2 virus causing CPE on Vero cells

Below is an example of Vero cells that have been infected by SARS-COV-2 for 48 hours, which show a loss of monolayer as well as rounding and swelling.

CPE induced by SARS-CoV2

Bright field imaging of the CPE induced by SARS-CoV2 shown in a plate fixed 48h post-infection.  All 3 image analysis algorithms can be applied to the experiment. Shown left to right, Confluence Analysis: loss of monolayer area, Cell Count Analysis: loss of cells, High-contrast and Round Cell Count: infected cells showing CPE.

A general CPE assay Protocol for analyzing confluence percentages between CPE positive and negative samples

  1. Seed the host cells in microplates and allow to incubate and adhere overnight
  2. Host cells are inoculated with positive control (virus) and negative control (media)
  3. The microplates are scanned and analyzed multiple times using Celigo in a time-course manner
  4. The CPE are quantified using host cell confluence % measurement
  5. Directly report CPE results in the Celigo software

Example CPE measurement performed using the Celigo Image Cytometer: