Cultivation of Viruses: Viruses are obligate intracellular parasites, which cannot be grown on inanimate culture media. Three methods are used for the cultivation of viruses.
- Laboratory animals
- Embryonated eggs
- Tissue culture
1. Laboratory animals
It is one of the oldest methods for the cultivation of viruses. Reed and colleagues (1900) used human volunteers for their pioneering work on yellow fever. Due to the serious risks involved, human volunteers are used only when no other method is available and viruses are harmless. Landsteiner and Popper (1909) used monkeys for the isolation of the poliovirus. However, due to their cost and risk to handlers, monkeys find less application in virology. The poliomyelitis virus after intraspinal or intracerebral inoculation in monkeys causes a typical paralytic disease. The use of white mice, pioneered by Theiler (1903) extended the scope of animal inoculation. Mice are still the most widely employed animals in virology. Infant (suckling) mice are very susceptible to coxsackie and arboviruses.
Other animals such as rabbits, guinea pigs, and ferrets are used in some situations. Smallpox virus may be inoculated in the scarified skin or cornea of rabbits.
The growth of the virus in inoculated animals may be indicated by death, disease, or visible lesions. Sometimes immunity in an experimental animal may interfere with the growth of viruses in the animal. Animal inoculation is also used for the study of pathogenesis, immune response, and epidemiology.
2. Embryonated eggs
The embryonated hen’s egg was first used for the cultivation of viruses by Goodpasture (1931) and the method was further developed by Burnet. The embryonated egg offers several sites for the cultivation of viruses (Fig.1).
Fertile chicken eggs incubated for 5 to 12 days can be inoculated through the shell aseptically. The opening may be sealed with paraffin wax and the egg incubated at 36 oC for the time required for the growth of the virus. The duration of incubation may depend on the type of virus and the route of inoculation. Viruses may kill the chick embryo or produce specific evidence of viral activity such as the production of pocks on the chorioallantoic membrane and haemagglutinating activity in the harvested amniotic and allantoic fluid. These effects help in the identification of the virus.
Inoculation into the allantoic cavity provides a rich yield of influenza and some paramyxoviruses. Inoculation into the amniotic sac is employed for the primary isolation of the influenza virus. Yolk sac inoculation is used for the cultivation of some viruses, chlamydiae, and rickettsiae. Yellow fever (17 D strain) and rabies (Flury strain) are other vaccines produced from chick embryos and vaccinia and herpes viruses from the chorioallantoic membrane. The advantages and disadvantages of this method are summarised as follows:
Advantages of embryonated eggs:
- The eggs are much simpler to handle than animals.
- Eggs are very economical and easily available.
- They are clean and bacteriologically sterile.
- They do not need feeding and caging.
- They do not have an immune mechanism like animals to counteract virus infection.
- Chick embryo offers several sites for the cultivation of viruses.
Disadvantages of embryonated eggs:
- Some viruses do not show growth on primary inoculation into the eggs.
- A slight amount of bacterial contamination in the inoculum may kill the embryo.
- Eggs may be contaminated with mycoplasma and latent fowl viruses which may interfere with the growth of other viruses.
3. Tissue culture
The first application of tissue culture in virology was by Steinhardt and colleagues (1913) who maintained the vaccinia virus in fragments of rabbit cornea. Maitland (1928) used chopped tissues in nutrient media for the cultivation of the vaccinia virus. The turning point which made tissue culture, the most important method for the cultivation of viruses was the demonstration by Enders, Weller, and Robbins (1949) for poliovirus. Since then tissue cultures of human or animal cells are frequently used for the cultivation of viruses. There are mainly three types of tissue cultures:
(i) Organ culture: Organ cultures are useful for the isolation of some viruses which appear to be highly specialized parasites of certain organs e.g. tracheal ring organ culture is employed for the isolation of coronavirus.
(ii) Explant culture: Minced tissue may be grown as an explant embedded in plasma clots. This is not useful in virology. In the past, adenoid tissue explant cultures were used for adenovirus.
(iii) Cell culture: This is a very popular and useful technique routinely used for the cultivation of viruses. Tissues are dissociated into the component cells by the action of proteolytic enzymes such as trypsin and mechanical shaking. The cells are washed, counted, and suspended in a growth medium and distributed in Petri plates, test tubes, or bottles. The cells adhere to the glass surface and grow out to form a monolayer sheet. Based on their origin and characteristics, cell cultures are classified into three types.
(a) Primary cell cultures: These are normal cells freshly taken from the body and cultured. They are capable of only limited growth in a culture and can not be maintained in serial culture. They are useful for the isolation and cultivation of viruses for vaccine production e.g. rhesus monkey kidney cell culture, human embryonic kidney cell culture, human amnion cell, chick embryo fibroblast culture, etc.
(b) Diploid cell strains: Diploid cell strains are derived from primary cell cultures established from a particular type of tissue, such as a lung or kidney, which is embryonic in origin. They are of a single type and can undergo 50 to 100 divisions before dying. They possess the normal diploid karyotype (set of chromosomes). They are useful for the isolation of fastidious pathogens and also for the production of viral vaccines e.g. human embryonic lung cell strain (WI – 38), and rhesus embryo cell strain (HL-8).
(c) Continuous cell lines: They are a single type of cells mainly derived from cancer cells. These also can be grown in successive generations by transferring them from one test tube to another test tube without a change in the character of cells. Standard cell lines derived from human cancers, such as HeLa, Hep-2 and KB cell lines have been used in different laboratories throughout the world. These cell lines may be maintained by serial subcultivation or stored in the cold (– 70 oC) e.g. Human carcinoma of nasopharynx line (KB), Human epithelioma of larynx cell line (Hep-2), Human carcinoma of cervix cell line (HeLa), Baby hamster kidney cell line (BAK 21), etc.
Virus growth in cell cultures can be detected by cytopathic effect, transformation, metabolic inhibition, interference, hemadsorption, and immuno-fluorescence.
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