Cytopathic Effect (CPE) and Intracellular Inclusions
One of the primary proofs of viral reproduction is microscope-visible changes in infected cells. The nature of the cytopathic effect is specific to different viral groups, allowing for their preliminary identification.
Key variants of CPE include:
- Destruction — the most pronounced damage. It includes cytoplasmic vacuolization, mitochondrial destruction, and cell rounding. Eventually, cells detach from the glass and die.
- Syncytium formation — fusion of adjacent cell membranes forming giant multinucleated structures.
- Proliferation — temporary overgrowth of the cell mass, which ultimately still ends in destruction.
A distinct manifestation of CPE is the formation of intracellular inclusions. These can be aggregates of viral particles (virions), their individual components (proteins or nucleic acids), or reactively altered cellular material ("cellular graveyards").
Inclusions range from 0.2 to 25 µm in size, can be round or irregular, single or multiple. They are detected using light microscopy (stained with aniline dyes) or fluorescence microscopy (stained with fluorochromes).
The localization of inclusions has diagnostic value:
- Cytoplasmic — characteristic of the smallpox virus (Guarnieri bodies) and the rabies virus (Negri bodies).
- Intranuclear — observed during infections with herpesviruses and adenoviruses.
Plaque Assay (Negative Colony Method)
The plaque assay is used not only for indication, but also for quantification and differentiation of viruses. Plaques are localized foci of destroyed cells in a monolayer. Visually, they appear as clear 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, limiting the free diffusion of the virus. As a result, the new generation of virions exclusively infects adjacent cells.
The method is based on the principle of cloning — each plaque is formed by the replication of a single parental virion. This allows to:
- Determine concentration: counting the number of negative colonies makes it possible to calculate the viral titer in the sample.
- 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 called hemagglutinins. They can interact with receptors on the membranes of human, avian, or mammalian erythrocytes. Two indication methods are based on this property:
- Hemadsorption assay. 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 this method is that it allows virus detection even before cell destruction (CPE) has 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 cross-link (agglutinate) added erythrocytes together.
"Color" Reaction (Metabolic Inhibition Test)
This method records viral reproduction without microscopy by monitoring changes in the pH of the nutrient medium. A special indicator is pre-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), causing the indicator color to change.
Positive result (virus is replicating): Viral infection suppresses normal cellular metabolism and leads to cell death. Because the cells are dead, acidic products are not released, and the pH remains at baseline. As a result, the nutrient medium retains the original color of the indicator, signaling successful viral indication.