Cytopathogenic effect (CPE) and intracellular inclusions
One of the primary proofs of viral replication is changes in infected cells visible under a microscope. The nature of the cytopathogenic effect is specific to different groups of viruses, which allows for their tentative identification.
The following variants of CPE are distinguished:
- Destruction — the most pronounced damage. Includes cytoplasmic vacuolization, destruction of mitochondria, and cell rounding. Ultimately, they detach from the glass and die.
- Syncytium formation — fusion of the membranes of adjacent cells, forming giant multinucleated structures.
- Proliferation — transient proliferation of cellular mass, which nonetheless ultimately ends in destruction.
A distinct manifestation of CPE is the formation of intracellular inclusions. These can be accumulations of viral particles (virions) themselves, their individual components (proteins or nucleic acids), or reactively altered cellular material ("cellular graveyards").
Inclusions range in size from 0.2 to 25 µm, can be round or irregular in shape, single or multiple. They are detected using light microscopy (with aniline dyes) or fluorescence microscopy (with fluorochromes).
The localization of inclusions is of diagnostic significance:
- Cytoplasmic — characteristic of the smallpox virus (Guarnieri bodies) and rabies virus (Babes–Negri bodies).
- Intranuclear — observed during infection with herpesviruses and adenoviruses.
Plaque assay (negative colonies)
The plaque assay is used not only for indication, but also for quantification and differentiation of viruses. Plagues represent localized foci of destroyed cells in a monolayer. Visually, they appear as light spots against a solid background of stained living cells.
Technically, the method is implemented by adding agar to the nutrient medium over the cell monolayer. Agar acts as a physical barrier: it restricts the free diffusion of the virus. As a result, the new generation of virions infects exclusively neighboring cells.
The method is based on the principle of cloning — each plaque is formed as a result of the replication of a single parental virion. This allows to:
- Determine concentration: counting the number of negative colonies makes it possible to calculate the virus titer in the material.
- Differentiate pathogens: viruses form plaques that differ in appearance time, shape, and size.
- Perform selection: progeny can be isolated from a single plaque to obtain pure lines (strains) of viruses.
Interaction with erythrocytes
Many viruses possess surface proteins — hemagglutinins. They are capable of interacting with receptors on the membranes of human, avian, or mammalian erythrocytes. Two indication methods are based on this property:
- Hemadsorption reaction. Infected cells alter their surface properties and acquire the ability to bind (adsorb) erythrocytes directly onto their cell membrane. This is characteristic of influenza and parainfluenza viruses. The value of the method is that it allows the detection of the virus even when cell destruction (CPE) has not yet occurred.
- Hemagglutination assay (HA). A similar mechanism, but aimed at detecting viruses that have already been released from cells into the culture fluid. Viral particles agglutinate added erythrocytes together.
Color reaction (color test method)
This method allows recording the fact of viral replication without microscopy, by observing changes in the pH of the nutrient medium. A special indicator is preliminarily added to the medium.
Negative result (no virus): Healthy cells actively metabolize nutrients. During their life cycle, acidic metabolic products are released into the medium. This leads to acidification of the medium (decreased pH), resulting in a color change of the indicator.
Positive result (virus is replicating): Viral infection suppresses normal cellular metabolism and leads to cell death. Since the cells are dead, acidic products are not released, and the pH remains at the initial level. As a result, the nutrient medium retains the initial color of the indicator, which serves as a signal for successful virus indication.
Briefly: answers to topical questions
Which viruses cause syncytium formation in an infected cell culture?
Syncytium formation (giant multinucleated cells) in an infected culture is caused by paramyxoviruses: parainfluenza, mumps, measles, and RSV viruses. Syncytia are formed due to the activity of the F fusion protein. In measles, giant multinucleated cells may be represented by Warthin–Finkeldey cells.
Which specific stains are used to detect viral inclusions in light microscopy?
Aniline dyes and a number of special staining methods are used to detect viral inclusions in light microscopy.
- Romanowsky–Giemsa stain — used to detect Cowdry intranuclear inclusions (DNA viruses) and cytoplasmic Babes–Negri bodies (rabies virus).
- Hematoxylin and eosin stain — used to detect intranuclear viral inclusions in cytomegalic cells ("owl's eye" appearance).
- Mann, Turevich, and Muromtsev stains — specific methods for visualizing Babes–Negri bodies in smear-imprints or brain tissue sections.
To memorize the main indication methods, you can use the phrase: "Color Plaque Includes Hemo-CPE" (Color test, Plaques, Inclusions, Hemagglutination/Hemadsorption, Cytopathogenic effect).
Class and exam questions
What is the difference between hemagglutination and hemadsorption?
During hemadsorption, erythrocytes adhere to the surface of virus-infected monolayer cells. During hemagglutination, viral particles freely floating in the culture fluid agglutinate erythrocytes together.
Why does the preservation of color in the color test indicate the presence of a virus?
A replicating virus kills cells by halting their metabolism. Because of this, they stop releasing acidic metabolic products, the pH does not drop, and the indicator color remains unchanged.
Why is an agar coating used in the plaque assay?
Agar blocks the free spread of viral particles throughout the liquid medium. Due to this, the viral progeny can only infect strictly neighboring cells, forming a localized focus of destruction — a plaque.
What else to study on this topic
- Features of the application of fluorochromes in fluorescence microscopy of inclusions
- Mechanisms of cellular syncytium formation
- Specificity of hemagglutinins to erythrocytes of different animal species
- Technique for obtaining pure viral lines from plaques
- Differences between intranuclear inclusions of adenoviruses and herpesviruses
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