Cultivation of human viruses

July 27, 2018

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.