- The cultivation of viruses from material taken from lesions is an important step in the diagnosis of many viral diseases.
- Studies of the basic biology and multiplication processes of human viruses also require that they are grown in the laboratory under experimental conditions.
- Human pathogenic viruses can be propagated in three types of cell systems. They are as follows:
1. Cell culture
- Cells from human or other primate sources are obtained from an intact tissue e.g. human embryo kidney or monkey kidney.
- Small bits of organs (organ culture) from man and animal are maintained in tissue culture growth medium.
- Organ cultures are performed mainly for highly specialized parasites of certain organs like tracheal ring culture from isolation of corona virus.
- Explant culture is rarely done nowadays.
- The cells are dispersed by digestion with trypsin.
- Then, the resulting suspension of single cells is generally allowed to settle in a vessel containing a nutrient medium.
- The nutrient medium for this culture is basically a balanced salt solution and contains 13 essential amino acids, glucose salts, buffering system, protein supplement (lact-albumin hydrolysate), calf serum (5%), antibiotics (penicillin, streptomycin) and phenol red (indicator).
- The cells will metabolize and grow and after few days of incubation at 370C will form a continuous film monolayer one cell thick.
- These cells are then capable of supporting viral replication.
- Cell cultures may be divided into three types according o their history.
a) Primary cell culture
- These are normal cells obtained from fresh organs of animal or human being and cultured.
- These cells undergo mitosis once they get attached to the vessel surface until a confluent monolayer of cells covers the surface.
- These cells are capable of only limited growth in culture and cannot be maintained in serial culture.
- For primary isolation of viruses and in preparation of vaccines, these cell cultures are commonly used.
- The examples are: monkey kidney cell, Human amnion cell culture, etc.
b) Diploid cell cultures (semi-continuous cell lines)
- These cells of single type, usually fibroblasts, contain the same number of chromosomes as the parent cell and are
- The diploid cell strains can be sub-cultured for limited number of strains.
- The growth rate is rapid and after about 50 serial subcultures they undergo “senescence” and the cell strain is lost.
- They are susceptible to a wide range of human viruses.
- They are also used for isolation of some fastidious viruses and production of viral vaccines.
- The fibroblasts are usually derived from embryo tissues (human embryo lung strains).
c) Heteroploid cultures (continuous tumour cell lines)
- These are cells of single type capable of infinite growth in vitro.
- They are derived from immortalized cell lines (cancer cells), often of epithelial origin.
- These cells grow faster and their chromosomes are haploid.
- They are termed continuous cell lines as they can be serially cultivated indefinitely.
- The standard continuous cell lines have been derived from human cancers, such as HeLa (derived from cervical cancer of a lady, Hela by name), Hep 2 and KB cells.
- Continuous cell lines are maintained either by serial subculture or by storing in deep freeze at-700C so that these can be used when necessary.
- These are not used for preparation of viral vaccines, as vaccines prepared in cancer cells are considered unsafe for human use.
- Primary cell cultures are generally best cell lines for virus isolation and rhesus monkey kidney cell cultures are widely used.
- They are sensitive to a wide range of viruses.
Isolation of viruses
- Inoculation of cell cultures with virus containing material produces characteristic changes in the cells.
- The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate.
- This effect is seen as the shrinkage or sometimes as ballooning of cells and the disruption of the monolayer by death and detachment of the cells.
- The replicating virus then can be identified by inoculating series of cell cultures with mixtures of the virus and different known viral antisera.
- If the virus is same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum.
- Thus, CPE will not be apparent in that tube.
- Alternatively viral antisera labeled with a fluorescent dye can be used to identify the virus in the cell culture.
2. The chick embryo
- Fertile chicken eggs, 10-12 days old, have been used as a convenient cell system.
- This is used to grow a number of human pathogenic viruses which is inoculated by one of the several routes.
- After inoculation of chick embryo, it is incubated and examined daily for virus growth.
- Influenza viruses, for example, can be grown in the cells of the membrane bounding the amniotic cavity.
- Similarly, small pox virus will grow in the chorio-allantoic membrane.
- The growth of small poxvirus in the embryo is recognized by the formation of characteristic pock marks on the membrane.
- Influenza virus replication is detected by exploiting the ability of these particles to cause erythrocytes to clump together.
- Fluid from the amniotic cavity of the infected embryo is titrated for its haem-agglutinating
- Yolk sac inoculation is done for cultivation of some viruses as well as for some bacteria (Chlamydia and Rickettsiae).
3. Laboratory animals
- White mice and chimpanzees were inoculated with specimen for viral cultivation in the past.
- This type of inoculation has been largely replaced by the use of cell cultures.
- Suckling mice (less than 48 hours old) are very susceptible to toga and coxsackie viruses, which are inoculated by intra-cerebral or intranasal route.
- Growth of the virus is indicated by signs of disease or death of the inoculated animal.
Cultivation of human viruses