CULTIVATION OF VIRUSES
Since viruses are obligate intracellular parasites, a living cell system is required to cultivate them. Three kinds of such systems have been developed.
• Laboratory animals
• Embryonated eggs
• Cell lines (Tissue cultures)
Laboratory Animals: The first method that was used to grow viruses was their inoculation into animals. This method has now gradually disappeared because of inherent disadvantages and availability of cell cultures which are simple to handle and much more versatile.
Embryonated Eggs: The chick embryo used are usually 8-11 days old. After inoculation these are incubated for 2-9 days, the duration depending upon the virus type and the route of inoculation. Viruses may kill the chick embryo or produce specific evidence of viral activity such as production of pocks on chorioallantoic membrane which helps not only in isolation and diagnosis of poxviruses but also in enumerating the same since one pock is the result of activity of one viable virus. Similarly, the hemagglutinating activity in amniotic or allantoic fluids may reveal the presence of influenza or related viruses. Despite many advantages with embryonated eggs, this method of cultivation does not permit a controlled study of virus-cell interaction and there are many viruses which fail to grow on primary inoculation into the eggs. However, the embryonated eggs remains one of the best host systems for influenza A virus for example.
Diagram: Viral Inoculation Sites into Chicken Egg
Cell Lines (Tissue Cultures): Various types of techniques and cultures (e.g. whole organ, explant cultures etc.) have been used in the past. Nowadays most of diagnostic and research work is carried out in cell cultures—usually in monolayers. Monolayers are produced from desired tissues by cutting them into small pieces and treating with a proteolytic enzyme, e.g. trypsin. The cells disperse into single cell suspension and these are settled onto the surface of glass or plastic container where nutrition to these is provided by a growth medium which contains all the essential nutrients and serum.
Types of Cell Lines
The cell lines have been classified according to the number of divisions which a cell line can undergo in vitro before dying out. At present three broad types of cell lines are in use:
Primary cell cultures. These cell cultures are capable of only 5-10 divisions at the most. Since these contain tissues taken directly from animals, a large variety of cells are present in it which makes this type of cell cultures very sensitive to the growth of numerous viruses. These cell cultures suffer from the drawbacks of having latent viruses present in the animal tissues; limited number of divisions, i.e. short life and high cost.
Diploid cell strains. These cells can undergo many more cell divisions than primary cell cultures before dying out. The number of divisions is roughly related to the life span of the species of animal from which it has been derived. The human diploid cells undergo around 50 divisions. These cells have been widely used for the diagnostic purposes as well as for the commercial production of some vaccines such as rabies vaccine.
Continuous cell lines. These cell lines also are known as immortal cell lines and these originate from malignant cells or by the spontaneous transformation of a diploid cell strain. These cell lines don’t match their cells of origin attributable to various mutations that these endure throughout their prolonged culture. Continuous cell lines are derived from monkeys (vero cell line), dogs, cattle, pig, cat, mouse, gnawer and rabbits. These carry the nice advantage of indefinite propagation by subculturing at regular intervals. At –196°C these stay viable for years. Once sizable amount of cells area unit is needed, the continual cells will be full-grown in suspension culture. Recently evolved helpful technique of growing cells on giant (an outsized/ an oversized) scale for vaccinum production is on plastic or Sephadex beads (microcarriers) maintained in suspension in large fermentation tanks
Uses of Cell Cultures
Cell cultures serve three important purposes:
1. Isolation of viruses from clinical samples for diagnosis
2. Production of vaccines and antigens
3. Biochemical studies of viral replication
Recognition of Viral Growth in Cells:
These can be detected by the following four techniques:
1. Cytopathic effect (CPE)
2. Haemagglutination and haemadsorption
3. Immunofluorescence
4. Interference
Cytopathic effect (CPE). Many viruses kill the cells in which they grow and some bring about detectable changes in the morphology of the host cells. All these effects are collectively called cytopathic effects. Some viruses, however, do not produce detectable CPEs. CPEs can be visualized under the microscope in both stained and unstained preparations.
Haemagglutination and haemadsorption: The property of adsorbing to erythrocytes is exhibited by Myxoviruses and Togaviruses and can be demonstrated after 24 hours of infecting the cell. Haemagglutination is a related phenomenon and virion as well as haemagglutinin are demonstrable in the supernatant fluid of an infected culture. Some viruses cause little or no CPE. However, during the process of infection the cell membranes become altered in such a way that erythrocytes of some animal species will firmly attach to them when added to the culture. This phenomenon is called as haemadsorption and is commonly seen with members of families Orthomyxoviridae and Paramyxoviridae.
Immunofluorescence. By this technique newly synthesized viral antigen can be detected by employing specific antiviral antibody coated with a fluorescent dye. Rabies antigen is quickly detected by this method in specimens of brain and spinal cord.
Interference. The phenomenon of interference is sometimes used for diagnosis of some viruses which do not multiply in the presence of another virus in the same host (interfering virus). However, now such cell lines are available which permit the growth of these viruses.