Viruses can only multiply within living cells and must be grown using one of three cultivation methods: animal inoculation, embryonated egg inoculation, or tissue culture. Animal inoculation involves injecting animals like mice and observing for disease signs. Embryonated eggs are commonly used by inoculating virus into areas like the chorioallantoic membrane or allantoic cavity. Tissue culture uses organ fragments, explants, or cell lines derived from tissues to provide an environment for virus growth.

2. As viruses multiply only in living cells, they cannot be
grown on inanimate culture medium.
Three methods are employed for the cultivation of
viruses:
Animal inoculation
 Embryonated egg inoculation
Tissue culture

3.  Animal inoculation:
Animal inoculation is used for:
1. Primary isolation of certain viruses
2. To study pathogenesis of viral diseases
3. To study viral oncogenesis
Infant {suckling} mice are used in the isolation of
arboviruses and coxsackie viruses, many of which do not
grow in any other systems. Animals may be inoculated by
several routes.
They are : Intracerebral
Sub-cutaneous
Intraperitoneal {Intranasal}

4.  After inoculation, animals are observed for signs of
disease or death.
 Later on, they are sacrificed and tissues are tested
for the presence of virus.
 The viruses are identified by NEUTRALISATION
TEST using antiviral sera.
 In some viruses, inclusion bodies may be observed
in stained smear.
 Besides mice, other animals such as guinea pigs,
rabbits and ferrets are also used in some
situations.

5. Embryonated egg inoculation
Goodpasture {1931} first used embryonated hen’s
egg for cultivation of viruses.
Embryonated hen’s eggs [7 to 12 days old] are
inoculated by one of the several routes:
Chorioallantoic membrane { CAM }
Allantoic cavity
Amniotic sac
Yolk sac
After inoculation, eggs are incubated for 2-9 days.

6. Chorioallantoic Membrane { CAM }
CAM is inoculated mainly for growing pox viruses.
It produces visible lesions (pocks). Each pock is
derived from single virion.
Pock counting therefore indicates the number of
viruses present in the inoculum.
Pocks produced by different viruses have different
morphology.

7. Allantoic cavity
Allantoic inoculation is employed for growing
influenza virus for vaccine production.
Other chick embryo vaccines include yellow fever [17
D strain] and rabies [flury strain] vaccines.
Duck’s egg being bigger, provide a better yield of
rabies virus and were used for the preparation of the
inactivated non-neural rabies vaccine.

8. Amniotic sac
Inoculation into the amniotic sac is mainly used for
the primary isolation of influenza virus.
Yolk sac Inoculation
It is inoculated for the cultivation of some viruses and
certain bacteria (chlamydia and rickettsiae).

9.  Tissue culture
Three types of tissue cultures are available:
1. Organ culture
2. Explant culture
3. Cell culture

10. 1. Organ culture
 Small bits of organs are maintained in tissue culture
growth medium.
 Organ cultures are useful for the isolation of highly
specialized parasites of certain organs e.g., tracheal
ring culture for the isolation of coronavirus, a
respiratory pathogen.

11. 2. Explant culture
 Fragments of minced tissue can be grown as
‘explants’ which was originally known as tissue
culture.

12. 3. Cell culture
 This type of culture is routinely employed for
diagnostic virology.
 Cell cultures are classified into three different types
on the basis of their origin, chromosomal characters
and the number of generations through which they
can be maintained.
- Primary cell culture
- Diploid cell culture
- Continuous cell lines

13. - Primary cell cultures
 These are normal cells freshly taken from the organs
of animal or human being and cultured.
 They are capable of very limited growth in culture
perhaps 5-10 divisions at the most.
 They are commonly employed for the primary
isolation of the viruses and their cultivation for
vaccine production.
 Common examples include monkey kidney cells,
human amnion cell and chick embryo cell cultures.

14. - Diploid cell cultures
 These are cells of a single type that contain the same
number of chromosomes as the parent cells and are
diploid.
 They can be sub cultured for a limited number. After
about 50 serial subcultures they undergo ‘senescence’
and the cell strain is lost.
 Diploid cells developed from human fibroblasts are
susceptible to a number of human viruses.
 They are useful for the growth of some fastidious
pathogens. They are also employed for the production
of viral vaccines e.g., rabies vaccine.

15. - Continuous cell lines
 These are a cells of single type that are capable of
indefinite growth in vitro.
 They are usually derived from cancerous tissue.
 These cells grow faster and chromosomes are
haploid.
They can be serially cultivated indefinitely, therefore,
they are termed Continuous cell lines.
 HeLa, HEp-2 and KB cell lines have been used in virus
laboratories throughout world.
 Some cell lines are now being used for vaccine
manufacture, for example Vero cell line for rabies
vaccine.

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