 Since the viruses are obligate intracellular

parasites, they cannot be grown on any

inanimate culture medium
 Viruses can be cultivated within suitable
hosts, such as a living cell
 The primary purposes of viral cultivation are:
1. To isolate and identify viruses in clinical
specimens
2. To prepare viruses for vaccines
3. And to do detailed research on viral structure,
multiplication cycles, genetics, and effects on
host cells
 Generally three methods
are employed for the virus
cultivation
1. Inoculation of virus into
animals
2. Inoculation of virus into
embryonated eggs
3. Tissue culture
 The earliest method for the cultivation of
viruses causing human diseases was
inoculation into human volunteers.
 Reed and colleagues (1900) used human
volunteers for their pioneering work on
yellow fever.
 Due to serious risk involved, human
volunteers are used only when no other
method is available and when the virus is
relatively harmless.
 Landsteiner and Popper (1909) used
monkeys for the isolation of polio virus.
 However due to their cost and risk to
handlers monkeys find only limited
application in virology.
 Theiler (1903) use white mice and
extended the scope of animal inoculation
greatly.
 Mice are still the most widely employed
animals in virology.
 Laboratory animals play an
essential role in studies of viral
pathogenesis
 Live animals such as monkeys,
mice, rabbits, guinea pigs,
ferrets are widely used for
cultivating virus
 Mice are the most widely
employed animals in virology
 The different routes of
inoculation in mice are:
◦ intracerebral
◦ subcutaneous
◦ intraperitoneal
◦ or intranasal
 After the animal is inoculated
with the virus suspension, the
animal is:
◦ observed for signs of disease
◦ visible lesions
◦ or is killed so that infected tissues
can be examined for virus
 Cost
 Maintenance
 Interference of immune system
 Individual variations
 Difficulty in choosing of
animals for particular virus
 Goodpasture and Burnet in 1931
first used the embryonated hen’s
egg for the cultivation of virus
 The process of cultivation of
viruses in embryonated eggs
depends on the type of egg being
used
 Eggs provide a suitable means for:
◦ the primary isolation and identification
of viruses
◦ the maintenance of stock cultures
◦ and the production of vaccines
 Terms most often refer to eggs:
◦ Embryonated: having an embryo
◦ Un embryonated: not having an embryo
◦ De-embryonated: having lost an embryo
 Embryonated egg, referring to an advanced
stage of development and not merely after
fertilisation.
 Chicken, duck, and turkey eggs are the most
common choices for inoculation
 The egg used for cultivation must be sterile
and the shell should be intact and healthy
 Rigorous sterile techniques must be used to
prevent contamination by bacteria and fungi
from the air and the outer surface of the shell
 An embryonated egg
offers various sites for
the cultivation of viruses
 The different sites of
viral inoculation in
embryonated eggs are:
1. Chorioallantoic
membrane(CAM)
2. Amniotic Cavity
3. Allantoic Cavity
4. Yolk sac
 This method has been widely used in
veterinary virology
 Many viruses grow readily or can be adapted
to grow on the CAM
 Viruses produce visible foci or ‘pocks’,
inclusion bodies, oedema or other
abnormalities
 Each infectious virus particle forms one pock
 Viruses which can be grown include:
◦ Herpes viruses
◦ and poxviruses
  Primary isolation of
influenza and mumps
viruses
 Growth of virus detected by
haemagglutination
Influenza Virus

Hemorrhagic lesions in the proventriculus, seen at necropsy in fowl with Mumps Virus
Most popular
Most of avian viruses
High titered virus
Simple technique
 It is also a simplest method for growth and
multiplication of virus
 Mostly mammalian viruses are isolated using
this method
 Immune interference mechanism can be
detected in most of avian viruses
 This method is also used for the cultivation of
some bacteria like Chlamydiae and
Rickettsiae
 There are three types of tissue culture:
- Organ culture.
- Explant culture.
- Cell culture.
 Organ culture:
- Small bits of organs can be maintained in
vitro for days and weeks, preserving their
original architecture and function.
- Organs culture are useful for the isolation
of some viruses which appear to be highly
specialised parasite of certain organs.
- For example, the tracheal ring organ
culture is employed for the isolation of
coronavirus, a respiratory pathogen.
 Explant culture:
- Fragments of minced tissue can be grown
as ‘explants’ embedded in plasma clots.
- They may also be cultivated in suspension.
- This method is now seldom employed in
virology.
- Adenoid tissue explant culture were used
for the isolation of adenoviruses.
 Cell culture:
- This is the type of culture routinely
employed for growing viruses.
- Tissue are dissociated into the component cell by the
action of proteolytic enzyme.

- The cells are washed , counted and suspended in a

growth medium.

- Such media will enable most cell types to multiply with

a division time of 24-48 hrs.

- The cell suspension is dispensed in bottles, tubes or

petridishes
- The cell adhere to the glass surface and on
incubation, divide to form a confluent
monolayer sheet of cells covering the
surface within about a week.
Based on their origin,
chromosomal characters and the number of
generation through which they can be
maintained, cells cultures are classified into
three types.
- These are normal cells obtained from fresh
organs of animals or human being and
cultured.
- They are capable of only limited growth in
culture and cannot be maintained in serial
culture.
- e.g. monkey kidney cell culture.
- human embryonic kidney.
- chick embryo cell culture.
- They are commonly employed for primary
isolation of viruses and in preparation of
vaccine.
- These are cells of single type, contain the
same number of chromosome as the parent
cells and are diploid.
- The diploid cell strains can be subculture
for limited number of times.
- After about 50 serial passage they undergo
senescence.
- They are also employed for the production
of viral vaccine. Eg. human embryonic lung
cell strain WI-38
- These are cells of single type capable of infinite
growth in vitro.
- They are derived from cancer cells.
- They are termed continuous cell lines as they
can be serially cultivated indefinitely.
- The continuous cell line have been derived
from human cancer such as HeLa, Hep 2 and
KB cells(human carcinoma of nasophyranx)
- Continuous cell line are maintained either by
serial subculture or by storing in deep freeze at
-70 so that these can be used when necessary.