CULTIVATION OF VIRUS

         As viruses are intracellular obligatory parasites, they always need living cells for their growth.

They cannot be grown on any artificial media.

There are three methods employed for the cultivation of animal viruses

                        1. Animal inoculation

                        2. Embryonated eggs or chick embryo method.

                        3. Tissue culture or cell culture.

1. Animal Inoculation –Susceptible experimental animals like Mice, Monkey, Rabbits, Guinea Pigs etc. are used for the cultivation of viruses.

Ø  Mice with less than 48 hours old are most commonly used.

Ø   or intranasal.

Ø  The growth of the virus in inoculated animals may be indicated by death, disease or visible lesion.

Ø  Antibodies against the specific viruses may also be identified in host.

Ø  Animal inoculation helps in study of primary virus isolation and used in study for immune responses, epidemiology and oncogenesis.

Ø  Disadvantages of animal inoculation are that immunity may interfere with viral growth.

Ø  The animal should be Specific Pathogen Free, because inter current infection can contaminate the propagated virus; animal must also have no prior imunity to the particular virus.

Ø  It is important to select specific host animal for particular viruses.

2. Embryonated Eggs or Chick embryo method

Good pasture and Burnet in 1931 first used the embryonated hen’s egg for the cultivation of virus (7-12 days).

            Embryonated egg provides several sites for the cultivation of viruses.

1.      Chorioallantoic membrane – poxvirus, Herpes simplex virus

2.     Allantoic cavity - influenza virus, yellow fever, rabies

3.     Amniotic sac – Influenza, mumps

4.     Yolk sac – chlamydia and rickttsiae

o   Different site is used for growth of different viruses.

o   Eg. Chorio-allantoic membrance is used for the cultivation of pox virus.

o   Allantoic cavity is employed for the Influenza virus.

o   Chick embryo method is cheaper and easy to handle.

Procedure:

o   At first, Disinfected with iodine

o   Then, Small sterile drill is made

o   After inoculation , the opening is sealed with gelatine or paraffin and incubated at 36oC for 2-3 days

o   After incubation, egg is broken and virus is isolated from tissue

o   Viral Growth and replication of virus indicated by

o   Embryos death

o   Embryo cell damage

o   Formation of typical pocks or lesions on membrane

o   This method is suitable for plague studies.

o   Growth and replication of virus in egg embryo can be detected by haemagglutination assay.

Advantages

            Widely used ideal substrate, cost effective and maintenance is much easier.

            Less labour is needed, free from contamination.

            Vaccine production.

3. Tissue Culture

Steinhardt and colleagues (1913), was the first who used bits of tissue or organ for the cultivation of viruses.

Cell culture is the process by which cells are grown under controlled conditions.

General method is as follows :-

Ø  Tissue like Monkey Kidney, Rabbit Kidney is taken and treated with proteolytic enzymes such as Trypsin and by mechanical shaking, tissues are dissociated into the component cells.

Ø  Trypsin, the proteolytic enzyme digest the binding material that binds the cells together in a tissue and results into free cells.

Ø  He La cells (i.e. human cells from cervical cancer region) are also commonly used cell system for the cultivation of viruses.

Ø  Cells are frown in vitro on glass or a treated plastic surface in a suitable growth medium.

Ø  At first growth medium, usually balanced salt solution containing 13 amino acids, sugars, proteins, salts, calf serum, buffer, antibiotics and phenol red are taken and the host tissue or cell is inoculated.

Ø  On incubation the cell divide and spread out on the glass surface to form a confluent monolayer.

Types of cell culture

3 types of cell culture

1.      Primary cell culture – normal cells freshly taken from animal of human body.

a.     Limited time cultivation and cannot be maintained in serial culture.

b.    Used for virus isolation and vaccine production.

c.     Ex-Monkey kidney, cell culture, human embryonic kidney, chick embryo cell culture.

2.     Diploid cell culture (semi continuous cell lines) – contains the same number of chromosomes as the parent cells.

a.     Sub-cultured up to 50 times by serial transfer.

b.    Used for isolation of fastidious viruses and production of viral vaccines.

c.     Ex- Human embryonic lung strain, Rhesus embryo cell strain.

3.     Continuous cell lines – derived from cancer cells.

a.     Serial cultured, maintained in deep freeze at – 70^oC.

b.    No vaccine production.

c.     Ex – HeLa cells, HEP-2 cells, BHK-21.

Detection of growth in cell culture

– cytopathic effects

– morphological changes in cells, observed microscopically.

-         Fluorescence antibody technique, Haemagglutination test, Nucleic acid amplification test, Polymerase chain reaction, Enzyme immunoassay.