Cytopathic Effects and Detection of Virus From Culture

Cytopathic effect

A cytopathic effect is the formation of the monolayer cells deterioration due to viral infection. This destruction of the tissue cells in monolayer allows for easy analysis of viral growth. When cells are in suspension the visualization is not possible. CPE is the visible result of viral infection. Cell death occurs by following ways:

• Multiplying viruses,
• Effect on permeability of membrane,
• Inhibition of DNA, RNA and protein synthesis.

CPE effect can be observed by focal degeneration, Syncytium, inclusion bodies, swelling, total destruction etc.

It is the structure changes in the host cell resulting from viral infection. CPE occurs when the infecting viruses cause lysis of the host cell or when the cell dies without lysis because of its inability to reproduce. If a viruses cause these morphological changes in the host cell, it is said to be cytopathogenic.

CPEs are an important aspect of viral infection in diagnosis. Many CPEs can be seen in unfixed, unstained cells under the low power of an optical microscope with the condenser down and the iris diaphragm partly close. However, with some CPEs, namely inclusion bodies, the cells must be fixed and stained and visualized under a light microscope. CPEs of some viruses are characteristic and therefore can be important characteristics that virologist may use to identify virus type. If CPE appears after 4-5 days in vitro then the viruses are considered slow whereas if the CPE appears after one to two days in vitro then the virus is thought to be rapid common example of CPEs include rounding of the infected cells, Fusion to adjacent cells to form syncytia and the appearance of the nuclear or cytoplasmic inclusions bodies.

Primary cell lines will divide and reproduce for a few generations and then will die then the viral culture will disappear along with these host cells at the time of death. This condition causes difficulty for the researchers. Continuous cell lines prefer cancer cells for culturing viruses. These cell lines can go through an infinite number of generations and are sometimes referred to as immortal. Thus continuous cell line is the method for maintaining a viral culture for purposes of research and study. A famous and first continuous cell line is called the HeLa cell line named after the patient Henrietta Lacks who provided these cancerous cells. She died in 1951.

Common types of CPEs

The cytopathic effect is due to viral infection. There are several types of cytopathic effect. They are as follows:

• Total destruction
• Sub- total destruction
• Focal degeneration
• Swelling
• Clumping
• Foamy degeneration
• Vacuolization
• Syncytium
• Inclusion bodies

This all cytopathic effect has its own different level of severity and effect to the tissue.

Total destruction

Total destruction of the host cell monolayer is the most severe type of CPE. To observe this process cells are seeded on a grass surface and a confluent monolayer of a host cell is formed. Then the viral infection is introduced. All cells in the monolayer shrink rapidly and become dense in a process known as pyknosis and detach from the glass surface within 3 days. This form of CPE is typically seen with enteroviruses.

Sub-total destruction

Sub-total destruction of the host cell monolayer is less severe than destruction. Similarly to total destruction, this CPE is an observed by seedling a confluent monolayer of host cell on a glass surfaces the introducing a viral infection. Sub- total destruction characteristically shows detachment of some but not all cells from the monolayer .It is commonly observed with some Tagovirus, Some picornavirus and some types of paramyxovirus.

Focal degeneration

Focal degeneration causes a localized attack of host cell monolayer. Although this type of CPE may eventually affect the entire tissue, the initial stage and spreading occur at localized viral centers known as foci. Focal degeneration is due to direct cell to cell transfer of virus rather than diffusion through the extracellular medium. This different mode of transfer differentiate it from total and subtotal destruction and causes the characteristic localized effects. Initially host cells become enlarged rounded and refractile. Eventually, the host cells detect from the surface the spreading of the virus occurs concentrically so that the cells surrounded by enlarging cells which are surrounded by healthy tissue.

This type of CPE is characteristic of Herpes viruses and poxviruses.

Swelling and clumping

It is a CPE where host cells swell significantly. Once enlarged, the cells clump together in a cluster. Eventually, the cell became so large that they detach. This type of CPE is characteristic of Adenoviruses.

Foamy degeneration (vacuolization)

It is also known as vacuolization. It is due to the formation of large and numerous cytoplasmic vacuoles. This type of CPE can be observed with fixation and staining of the host cells. Foamy degeneration is characteristics of certain retroviruses, paramyxoviruses, and flaviviruses.

Syncytium

It is also known as cell fusion and poly karyon formation. With this CPE, the plasma membrane of four or more host cells fuses and produce an enlarged cell with at least 4 nuclei. Although large cell fusions are sometimes visible without staining this types of CPE is typically detected after host cell fixation and staining. Herpes viruses characteristically produce cell fusion as well as other forms of CPE. Some paramyxoviruses may be identified through the formation of cell fusion as they exclusively produce this CPE.

Inclusion bodies

Inclusion bodies may be seen only with staining as they indicated areas of altered staining in the host cell. Typically, they indicate the areas of host cell where viral proteins or nucleic acid is being synthesized or where viron is being assembled. In some cases, inclusion bodies are present without an active virus. Inclusion bodies are present without an active virus. Inclusion bodies vary with the viral strain. They may be single or multiple small or large and round or irregular shaped. They may also be intranuclear or intracytoplasmic and eosinophilic or basophilic.

Example:

• Intracytoplasmic eosinophilic inclusion bodies

Negri bodies- in rabies virus

Guarnieri bodies- in vaccine, variola virus

Paschen bodies- variola

Bollinger- fowl pox virus

• Intranuclear acidophilic

Torroes- In yellow fever virus

Cowdry types B-In polio and adenovirus

• Both intranuclear and intracytoplasmic

Warthin- finkeldey- in measles

• Intranuclear basophilic

Owls eye appearance in cytomegalovirus (CMVs)

Detection of virus: The plaque assay

One of the most important procedures in virology is measuring the virus titer that is the concern of viruses in a sample. A widely used approach for determining the quantity of infectious virus is the plaque assay.

The plaque assay can be used to purify the clonal of virus or to determine viral titer as plaque forming unit per ml (PFU/ML) so that known amount of virus can be used to infect cells during subsequent work.

To perform a plaque assay cell monolayer is with a low ratio of viruses such that sporadic cells (infected). In the process 10 fold dilution of virus stock culture is prepared and 0.1ml of the sample volume is incubation period, to allow a virus to attack the cells, the monolayer is covered with a nutrient medium contained a substance usually agar that causes the formation of a gel. When the plates are incubated, the original infected cell release viral progeny, the spread of the new viruses is each infection particles produces a circular zone of infected cells called a plaque. Eventually, the plaque becomes large enough to be visible with naked eye. Dyes that stain living cell are often used to enhance the contrast between the living cells and the plaque.

The titer of a virus stock can be calculated in pfu/ml. when each infected cells produces virus and eventually lysis, only the immediately adjacent cells become infected. Each group of infected cells is referred to as a plaque. An uninfected cell surrounds the plaque. After several infection cycles, the infected cells in the center of plaque begin to lysis and the peripheral phenomenon causes the light passing through the infected cells to refract differently than the surrounding uninfected cells and the plaque can be visualized either by the naked eye or by light microscopy.

Each plaque represents a single virus. To minimize error, only plates containing a plaque between 10 -100 are counted. In the above example, there is 13 plaque on the plate made from 10^-5 dilution. Hence, the titer of the virus stock is 1.3*10⁷pfu/ml.

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries. Vol. Vol 2. ELBS London, 2007.

Tille, P. Diagnostic Microbiology. 13th. Elsevier, 2014.

D, Grenwood, Slack RCB and Peutherer J. Medical Microbiology. Dunclude Livingstone: ELBS, 2001.