Cultivation Of Virus
Viruses are obligate intracellular parasites. They depend totally on their host cells for their existence. Their total host dependence makes it extremely difficult to get good insight of them natural conditions, because the internal characteristics of the host cells are likely to interfere with the observations. Due to these reasons, it has been found desirable that viruses are cultivated or grown in the laboratory itself.
It has two advantages:
There are three methods employed for virus cultivation:
A) Inoculation into animals:
The earliest method for cultivation of viruses causing human diseases was inoculation into human volunteers. Reed and his colleagues (1900) used human volunteers for their work on yellow fever. Due to serious risk involved, human volunteers are involved only when no other method is available and the virus is relatively harmless.
Monkeys were used for the isolation of Poliovirus by Handsteiner and popper (1909). Due to their cost, and risk to handlers, they have limitations. The use of white mice by Theiler extended the scope of animal inoculation greatly. Mice are still most widely used animals in virology. Infant mice are very susceptible to Coxsackie’s and arboviruses. Mice can be inoculated through several routes i.e. intracerebral, subcutaneous, intraperiotonial, intranasal.
Other animals such as guinea rabbits and ferrets are also used. The growth of virus in inoculated animals is indicated by death, disease or visible lesions. Animal inoculation has a disadvantage that immunity may interfere with viral growth and that animals often harbor latent viruses.
B) Embryonated eggs:
The Embryonated hen’s egg was first used for cultivation of viruses by Good Pasteur and Burnet (1931). Cultivation of viruses in organized tissues like chick embryo necessitates a different type of approach. Theses are live embryos so very beautifully packed in their shells. For all practical purposes they all themselves behave as tissue cultures. The process of cultivation of viruses in embryonated eggs depends on the type of egg which is used. The egg used for cultivation must be sterile and the shell should be intact and healthy.
Process for cultivation of virus in embryonated egg:
In order to cultivate viruses in eggs, the procedure adopted should be very simple. The eggs are kept in incubator and embryos of 7-12 days old are used. The egg containing embryo usually has an air apace at the larger end. The position of this sac is first determined. The shell over the air sac is then cut off and removed. The membrane adjacent to the shell is then pierced. Usually the hypodermic syringe is used for piercing the shell. At this stage, the embryonic fluid may ooze out. The shell membrane is then removed. The rest of the embryo then gets exposed and ready for use. Virus suspension to be cultivated is taken in dropper and gently spread over the exposed embryo. After inoculation is thus completed, the open area of the shell is sealed eggs are incubated for one week as in hatching. The virus particles infect the membrane at random and create pock marked appearance against the transparent background. This indicate viral basis.
The embryonated offers several sites for the cultivation of viruses:
1) Chorioallantoic membrane (CAM):
CAM is inoculated mainly for growing poxvirus. Herpes simplex virus is also grown. Virus replication produces visible lesions, grey white area in transparent Cam. Each pock is derived from a single virion. Pocks produced by different virus have different morphology. Under optimal conditions, each infectious virus particle can form one pock. Pock counting, therefore can be used for the assay of pock forming virus such as vaccinia.
2) Allantoic cavity:
Inoculation into the allantoic cavity provides a rich yield of influenza and some paramyxoviruses. Allantoic inoculation is employed for growing the influenza virus for vaccine production. Other allantoic vaccines include Yellow fever (17D strain), and rabies vaccines. Duck eggs are bigger and have a longer incubation period then hen’s egg. They therefore provide a better yield of rabies virus and were used for the preparation of the inactivated non-neural rabies vaccines.
3) Amniotic cavity:
The amniotic sac is mainly inoculated for primary isolation of influenza a virus and the mumps virus.
4) Yolk sac:
It is inoculated for the cultivation of some viruses as well as for some bacteria like Chlamydiae and Rickettsiae.
C) Tissue culture:
There are three types of tissue cultures:
1) Organ culture:
Small bits of organs can be maintained in vitro for days and weeks, preserving their original architecture and function. Formalin is used for the preservation. Organ culture is useful for the isolation of some viruses which appear to be highly specialized parasites of certain organs.
Example: Tracheal ring organ culture is employed for the isolation of cornovirus, a respiratory pathogen.
2) Explant culture:
Example: Adenoid tissue explant culture was used for the isolation of adenovirus.
3) Cell culture:
The cell culture is the method routinely employed nowadays for identification and cultivation of viruses. Cells of various types of tissues of animals may be cultivated. But more commonly, fibroblast and muscle epithelial cells are used for the propagation of virus.
The tissue is first removed from the organism concerned. This tissue is then broken down into its constituent cells by utilizing suitable physical means. Homogenization in a homogenizer is common method utilized. The complete tissue is then converted into many small pieces. The tissue fragments are washed with salt solutions. Sterile physiological saline or other types of solution like saline or other types of solution like hank’s solution or eagle’s solution are used. The pieces are converted into their constituent cells by a process called dispersion of the cells from tissue. It is done by breaking down the proteinaceous cementing material (i.e. Haluronic acid), joining the cells with the help of proteolytic enzymes like trypsin and mechanical shaking. This step is called as trypsinization.
The washed tissue fragments are then placed in a flask with sterile trypsin solution at 4°c for about 18 hours. During this period, the tissue fragments area gradually dispersed into their cellular components. Presence of chemicals like EDTA helps in dispersion of cells. The cells are then centrifuged and resuspended in washing medium. It is done repeatedly. The washed suspended cells are then cultivated in a suitable growth medium. The essential constituents of growth medium are physiological amounts of essential amino acids, and vitamins, salts and glucose and a buffering system generally consisting of bicarbonate in equilibrium with atmosphere containing about 5% calf or fetal calf serum. Antibiotics are added to prevent bacterial contaminants and phenol red as indicator. Such media will allow most cell types to multiply with a division time of 24-48 hrs.
Cultivation is done after adjustment of the number of cells per unit volume. The required number of cells is suspended in the growth medium taken in a tube or flask. The entire culture is then incubated at 36°c for 72hrs. The cells in culture multiply and cover the bottom of the glass container with a thin but continuous layer, which is often one cell thick. Such cell layers are called as monolayer. This technique was improved by Dr. Dulbecco and his associates in the U.S.A. They found that the monolayer can be developed on agar containing necessary nutrients. Virus particles grown on such monolayer are extremely uniform in growth.
Sometimes the dispersed cells are not allowed to settle down at the bottom of the container. Rather they are kept floating by shaking the flasks continuously on the mechanical shaker. This type of culture is called as suspension culture. A vigorously growing monolayer or suspension culture is then inoculated with the types of viruses to which it is susceptible. The inoculation is done by mixing or spreading the viral suspension with the cultivated host cells. The virus particle infects the host cells in due course. They multiply in number within the host cell and eventually come out by destroying the host cell. They are thus liberated into the surrounding medium and infect the neighboring cells. The cell culture looks disintegrated. The initially formed virus particles soon lead to the production of many more viruses. These areas appear to be completely disintegrated and take shapes of white patches called as plaques.
Types of cell cultures:
On the basis of origin, chromosomal characters, and the number of generations through which they can be maintained, cell cultures are classified in three types.
1) Primary cell culture:
Examples: Rhesus monkey kidney cell culture, Human amnion cell culture.
2) Diploid cell culture:
It is also called as semi continuous cell lines. These are subsequent cultures derived from primary cell cultures. These are cells of single type that retrain the original diploid chromosome number and karyotype during serial sub cultivation for a limited period of time. There is rapid growth rate and after 50 serial subcultures, they undergo senescence and the cell strain is lost. The diploid cell strains are susceptible to a wide range of human viruses. They are also used for isolation of some fastidious viruses and production of virus vaccines,
Examples: Human embryonic lung strain (WI-38) and Rhesus embryo cell strain (HL-8)
3) Continuous cell culture:
These are cells of a single type, usually derived from the cancer cells that are capable of continuous serial cultivations indefinitely. These cells grow faster and their chromosomes are haploid. They are also called as permanent cell lines. Permanent cell lines derived from a single separated cell are called as clones. One common example of such clone is Hela strain derived from cervical cancer of lady Hela, by name. Continuous cell lines are maintained either by serial subculture or by storing in deep freeze at -70°c so that these can be used when necessary. Some cell lines are now permitted to be used for vaccine manufacture.
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