Cytopathic effect (CPE): how do viruses get away with murder
Viruses, by definition, are invisible to our eyes. They are nanostructures that can only become visible using electron microscopy, a technique that is time-consuming and not accessible for everyone. As a consequence, a virologist has to find alternatives to be able to work and study its object of interest. A practical way of “seeing” and indirectly measuring a viral infection is by looking at the damage a virus causes to a cell. This suffering or damage is known as cytophatic effect (CPE) and its measurement is widely used in virology labs all over the world.
CPE is a very basic approach to understand how a virus infects a cell, but that does not mean it is just used in basic scientific research. Measuring CPEs can also be a very useful readout for pharmaceutical companies and diagnostic laboratories.
What is virus-induced cytophatic effect?
Viruses are parasites that need a host cell to replicate. Once inside the cell, they hijack the cellular machinery to produce its own proteins, nucleic acid and membrane (when needed).
Since the virus is occupying cellular factors that are otherwise used by the cell, its replication can alter the host cell´s basic functions or even destroy it. The set of cell changes or alterations resulting from a viral infection are known as CPEs. These are usually negative changes that can cause structural, metabolic or functional modifications in the cell that is being infected. Over time, CPEs can give rise to the pathologic effects of the virus (the disease).
Classical examples of the cytopathic effect
A well-known virus-induced-CPE is cell death (check out a video of a dying cell in ). Many viruses kill cells either by lysis or by inducing apoptosis. For example, HIV is known for killing CD4+ T lymphocytes, which is the main reason why infected individuals become immunocompromised.
In the laboratory, an easy way of killing a mosquito cell line (like C6/36) or Vero cells is by infecting it with any famous arbovirus, like Chikungunya, Dengue or Zika. After a few days of infection the cells just fall apart right in front of your eyes (Fig. 1).
Figure 1. Vero cells were infected with Zika virus. After 72 h of infection, a clear CPE is evident by the presence of cell debri as a result of cell death.
Another CPE example is syncytia formation. Cells infected by enveloped viruses express viral proteins on their plasma membrane, which are used by viruses to mediate fusion with the host cell. When these viral proteins bind to receptors on the surface of neighboring cells, cell–cell fusion takes place, resulting in syncytia formation (multinucleated giant cell).
Respiratory syncytial virus (RSV) is the major cause of viral pneumonia in young children. Its name is derived from the fact that it can form syncytia in cell culture but, remarkably, also in the lungs of infected patients. 
CPE-related changes in cell morphology also include rounding, detaching and/or clumping of adherent cells. In this case, infected cells remain metabolically active to replicate the virus, but their cytoskeleton is altered. The morphological changes can be explained by a down-regulation of the expression of surface adhesion proteins as a result of infection. Ebola virus, for example, is known to cause dramatic morphological changes in adherent cells by decreasing the expression of integrin β1. 
Do you really need so much drama (death, gigantism, deformation) for it to be considered a CPE? No. Cells can just “behave” differently when infected, but look the same. More subtle but still clear CPE-type of changes can include alteration of growth rate/kinetics, changes in a given metabolic activity, or changes in cell function. Among the different viruses that can induce these types of CPEs stands Zika virus. In 2015, this mosquito-transmitted virus was all over the news because it causes microcephaly, a severe neurological damage characterized by a reduced size of the brain in fetuses. Later it was described that microcephaly is the result of several Zika-induced cytopathic effects on neural cells, including cell hypertrophy, growth restriction, cell-cycle dysregulation, and cell death. A colony formation assay was used, for example, to identify which of the different Zika proteins interfere with cell proliferation. 
Cytopathic effects are the result of virus-host interactions
It is important to mention that the CPE is not only defined by the virus, but also by the host cell. It is dependent on how permissive the cells are to infection. Being a permissive cell, one that can support the growth of a virus. In other words, not all viruses can infect all cell types. To keep it simple, we can say that:
- Not permissive cell – Virus cannot infect
- Permissive cell – Virus can replicate, but does not cause obvious CPE
- Highly permissive cell – Virus replicates and induces an obvious CPE
- Screening and evaluation of antiviral compounds
- Can a drug candidate prevent CPE formation?
- Screening for neutralizing antibodies
- Can a given antibody inhibit virus-induced CPE?
- Surveillance of antiviral drug resistance (phenotyping)
- Can a given virus isolate induce CPE in the presence of antiviral drugs?
- Characterization of virus tropism
- Which cell types are preferentially infected by a virus? Why? (Check a recent publication with the infamous SARS-CoV-2 in .)
- Diagnosis of viral diseases
- Is a virus actively infecting a person? Is the clinical specimen collected from that person inducing CPE? Let’s keep in mind that most diagnostic methods (ELISA, RT-qPCR) do not detect infectious viral particles.
So if you add virus to your cells and nothing happens, do not worry. You just need to find the right virus-cell combination.
Readouts – How to measure CPE?
Virus-induced cellular changes like cell death or an altered morphology are visible by light microscopy. You can just put your plate under a standard inverted microscope and see if your cells died or somehow changed. A positive aspect of this approach is that it is “easy” because it does not require any processing of the sample, which avoids fixation or staining artifacts.
On the downside, it is qualitative and not quantitative, and it heavily relies on the training of the observer. Nowadays, this approach can be automated by doing real-time monitoring of virus-induced cytopathogenesis. For example, virus alterations of cell spreading and proliferation can be assessed through a wound-healing assay, where reduced collective and single cell migration speed can be detected together with severe changes in cell morphology.  Another quantitative approach is the monitoring of cell proliferation by automated image analysis. With this technology it is possible to generate growth curves of infected vs. non-infected cell cultures to properly quantify the cytopathic effect that a virus infection can have.
If death cells are what make your day, an alternative is the famous Plaque assay. This classical virology technique is used to quantify infectious viral particles and it is based on the principle that viruses can induce cell lysis. After infecting a monolayer of highly permissive cells, the cells are counterstained with crystal violet (living cells are colored, death cells remain unstained), resulting in beautiful plaques (areas of death cells) that can be easily quantified with the naked eye.
But you do not have to see your cells to know they are death. Cell viability post-infection can be measured by quantifying intracellular ATP concentration with firefly luciferase or with the traditional MTT assay.
Is your virus inducing a CPE? Can you clearly see or measure it? Great! You can use this CPE as readout of a successful viral infection or replication. A given CPE can serve as an indicator to find out how to prevent an infection, to characterize host or pathogen factors required for an infection to take place, to identify virus tropism, among others. In practical terms, this means that it can be used as readout for:
Although there are several ways of measuring virus infection, checking for CPEs in permissive cells remains as a widely used alternative. For it to be really useful, you need to find the right combination: a virus, a permissive cell, and an accurate readout. Once you find this combination, the applications can be quite diverse and useful either in an academic setting, in industry, or in clinical diagnostics.
- Cytopathic Effect: Herpes simplex virus 1 infected cells. https://www.youtube.com/watch?v=lwRX53Y6pAg
- Doms RW. (2016) Chapter 3 - Basic Concepts: A Step-by-Step Guide to Viral Infection. Viral Pathogenesis (Third Edition) From Basics to Systems Biology. https://www.sciencedirect.com/science/article/pii/B9780128009642000033
- Francica JR, et al. (2009) Requirements for Cell Rounding and Surface Protein Down-Regulation by Ebola Virus Glycoprotein. Virology. 383(2): 237–247. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2654768/
- Li G, et al. (2016) Characterization of cytopathic factors through genome-wide analysis of the Zika viral proteins in fission yeast. PNAS. E376–E385. https://www.pnas.org/content/pnas/114/3/E376.full.pdf
- Kräter M, et al. (2018) Alterations in Cell Mechanics by Actin Cytoskeletal Changes Correlate with Strain-Specific Rubella Virus Phenotypes for Cell Migration and Induction of Apoptosis. Cells. 2018 Sep; 7(9): 136. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162683/
- Chu H, et al. (2020) Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study. The Lancet Microbe. Volume 1, Issue 1, Pages e14-e23.