Cultivation of human viruses

  • 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.

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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).

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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.

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Cultivation of human viruses