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Cultivation of Viruses
Viruses areobligate intracellularparasites; they cannot be grown on any inanimate culture medium. Three methods are employed for the cultivation of viruses – inoculation into animals, embryonated eggs and tissue culture or cell culture.
i. Animal Inoculation
The earliest method for the cultivation of viruses causing human diseases was inoculation into human volunteers. Monkeys were used for the isolation of the polio virus by Landsteiner and Popper (1909). The embryonated hen’s egg was first used for cultivation of viruses by Good pasture (1931). The embryonated egg offers several sites for the cultivation of viruses. Non human primates provide the only method for virus cultivation. Mice are most widely employed animals in Virology.
ii. Embryonated Eggs
a. Chorioallantonic Membrane (CAM)
Inoculation on the chorioallantonic membrane produces visible lesions (pocks). Different viruses have different pock morphology. Example: variola or vaccinia
b. Allantonic Cavity
Inoculation on the allantonic cavity provides a rich yield of influenza and some paramyxo viruses.
c. Amniotic Sac
Inoculation into the amniotic sac is for the primary isolation of the influenza virus.
d. Yolk Sac
Inoculation into the yolk sac is for the cultivation of some viruses like Chlamydiae and Rickettsiae. Allantonic inoculation is employed for growing influenza virus for vaccine production (Figure 10.3).
iii. Tissue Culture
First tissue culture in Virology was maintained by Steinhardt and colleagues (1913) for the vaccinia virus in fragments of rabbit cornea. Bacterial contamination was the major limitation. Different types of culture used are:
a. Organ culture
Small bits of organs can be maintained, used for the isolation of some viruses.
Example: Corona virus (respiratory pathogen) cultured on tracheal ring organ culture.
b. Explant culture
Fragments of minced tissue are grown as ‘explants’. This is also known as tissue culture.
Example: Adeno virus cultured on Adenoid tissue explants.
iv. Cell Culture
Tissues are dissociated into the component cells by the action of enzymes (trypsin) or by mechanical process and are suspended in a growth medium (amino acids, vitamins, salts, glucose) supplemented with fetal calf serum of antibiotics and indicator (Phenol red).
This media is dispensed in bottles, tubes or petridishes. The cells adhere to the glass surface and on incubation divides to form a confluent monolayer sheet of cells covering the surface within about a week. The cell culture is classified into three types.
a. Primary cell cultures
In this culture, normal cells are taken from the body and cultured. They are capable of only limited growth in culture. Example: Monkey kidney, Human embryonic kidney, Chick embryo cell culture.
b. Diploid cell strains
These are cells of a single type that retain the original diploid chromosome number and serotype during serial sub cultivation for limited number of times. Example: Human fibroblast.
c. Continuous cell lines
These are single type, derived from cancer cells that are capable of continuous serial cultivation.
Example: Cells derived from cancers, such as Hela, Hep-2 and KB cell lines.