Viruses are obligate intracellular parasites which means they can only grow or reproduce inside a host cell. The primary purpose of virus cultivation: To isolate and identify viruses in clinical samples. To do research on the viral structure, replication, genetics, and effects on the host cell. To prepare viruses for vaccine production. Isolation of the virus is always considered a gold standard for establishing the viral origin of the disease topics covered CULTIVATION OF VIRUSES Animal inoculation Embryonated eggs CAM Allantoic cavity Amniotic cavity Yolk sac Tissue culture Organ culture Explant culture Cell culture Primary cell culture diploid cell culture Continues cell lines

1. VIVA MARIYA K V
MSC.BIOTECHNOLOGY
ST.MARY’S COLLEGE THRISSUR

2. Viruses are obligate intracellular parasites which means they can only grow
or reproduce inside a host cell.
The primary purpose of virus cultivation:
 To isolate and identify viruses in clinical samples.
To do research on viral structure , replication , genetics and effects on host
cell.
To prepare viruses of vaccine production.
Isolation of virus is always considered as a gold standard for establishing
viral origin of disease

3. CULTIVATION OF VIRUSES
Animal inoculation
Embryonated eggs
 CAM
Allantoic cavity
 Aminotic cavity
 Yolk sac
Tissue culture
Organ culture
Explant culture
Cell culture
 Primary cell culture
 diploid cell culture
 Continues cell lines

4. Mainly 3 methods are employed
1. Animal inoculation
2. Embryonated eggs
3. Tissue culture
CULTIVATION OF VIRUS

5.  Earliest method of cultivation of viruses causing human viral disease
was inoculation into human volunteers.
• Mouse is most frequently used for isolation of viruses by animal
inoculation.
• After inoculation, the animals are observed for signs of disease or death.
• The infected animals are then sacrificed and infected tissues are
examined for the presence of viruses by various tests, and also for
inclusion bodies in infected tissues.
• Furthermore, infant (suckling) mice are used for isolation of coxsackie
virus and rabies virus.

6. Guinea pigs Rabbits
Ferrets

7. Inoculation routes in
mice
 Intranasal
 Intracerebral
 Subcutaneous
Inoculation routes in
mice

8.  Animal inoculation may be used as a
diagnostic procedure for identifying
and isolating a virus for a clinical
specimen.
 Mice provides a reliable model for
studying viral replication.
 Gives unique insight into viral
pathogenesis and host virus
replication.
 Expensive and difficulties in
maintenance.
 Mice don’t provide models for vaccine
production.
 It will lead to generation of escape
mutants.
 Issues related to animal welfare
systems.

9.  1931 Good Pasture first used the embryonated hen’s egg for cultivation of
virus.
 The egg used for the cultivation must be sterile and the shell should be intact
and healthy.
 A hole is drilled in the shell of the embryonated egg, and a viral suspension or
suspected virus- containing tissue is injected into the fluid of the egg.
 Viral growth and multiplication in the egg embryo is indicated by the death of
the embryo, by embryo cell damage, or by the formation of typical pocks or
lesions on the egg membranes.

10. Routes of Egg Inoculation

11. Chorioallantoic membrane
(CAM)
Allantoic Cavity Aminotic Cavity Yolk Sac
Virus growth and replication in
the CAM is indicated by visible
lesions (pocks); grey white area
in transparent CAM..
Allantoic cavity Is the
most popular and simple
method for viral
inoculation.
The amniotic sac is
employed inoculated for
primary isolation of
influenza a virus and the
mumps virus.
It is also a simplest
method for growth and
multiplication of virus.
Each pock is derived from a
single virion. The morphology of
the pocks may vary depending
on the nature of the virus.
Allantoic inoculation is
employed for the growth
and replication of the
influenza virus for
vaccine production.
Other allantoic vaccines
include Yellow fever and
rabies vaccines.
Growth and replication
of virus in egg embryo
can be detected by
haemagglutination
assay.
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
 Under optimal conditions,
each infectious virus particle
can form one pock. Hence
this method is suitable for
plaque studies.
Mostly mammalian
viruses are isolated
using this method.

12.  Widely used method for the isolation of
virus and growth.
 Ideal substrate for the viral growth
and replication.
 Isolation and cultivation of many
avian and few mammalian viruses.
 Cost effective and maintenance is
much easier.
 Less labor is needed.
 The embryonated eggs are readily
available.
 Sterile and wide range of tissues and
fluids.
 Widely used method to grow virus for
some vaccine production.
 The site of inoculation for varies with
different virus. That is, each virus
have different sites for their growth
and replication.

13. Organ culture Explant culture
Cell culture
•Cell culture is most widely used in diagnostic virology for cultivation and
assays of viruses.
•Different types of tissue cultures are used to grow viruses. Tissue culture can
be of three different types as follows:

14. • This was used earlier for the isolation of some viruses, which appear to show
affinity for certain tissue organs. For example, coronavirus, a respiratory
pathogen, was isolated in the tracheal ring organ culture.
• In this method, small bits of the organs are maintained in vitro for days and
weeks preserving their original morphology and function.
• Nowadays, organ culture is not used.

16. • In this method, components of minced tissue are grown as explants embedded
in plasma clots.
• Earlier, adenoid tissue explant cultures were used for isolation of adenoviruses.
• This method is now seldom used in virology

17. • Cell culture is now routinely used for growing viruses.
• In this method, tissues are dissociated into component cells by treatment with proteolytic
enzymes (trypsin or collagenase) followed by mechanical shaking.
• The cells are then washed, counted, and suspended in a growth medium containing essential
amino acids and vitamins, salts, glucose, and a buffering system. This medium is supplemented
by up to 5% of fetal calf serum and antibiotics.
• The cell suspension is dispensed in glass or plastic bottles, tables, or Petri dishes.
• On incubation, the cells adhere to the glass surfaces and divide to form a confluent monolayer
sheet of cells covering the surface within a week.
• The cell culture may be incubated either as a stationery culture or as a roller drum culture. The
latter is useful for growth of some fastidious viruses due to better aeration by rolling of the
culture bottle in special roller drums.

18. Primary
culture
Diploid cell
structure
Continuous
cell lines
• The cell cultures are classified into three different types based on their origin,
chromosomal characters, and number of generations for which they can be
maintained

19. Primary cell culture:
•These are normal cells freshly taken from animal or human body.
•They are able to grow only for limited time and cannot be maintained in serial culture.
•They are used for the primary isolation of viruses and production of vaccine.
•Example : Monkey kidney cell culture, Human embryonic kidney and Chick embryo cell
culture.
Diploid cell strains:
• Also known as semi-continuous cell lines.
• They are diploid and contain the same number of chromosome as the parent cells.
• They can be sub-cultured up to 50 times by serial transfer following senescence and the
cell strain is lost.
• They are used for the isolation of some fastidious viruses and production of viral
vaccines.
• Example :Human embryonic lung strain, Rhesus embryo cell strain.

20. Continuous cell lines:
• They are derived from cancer cells .
• They can be seriously cultured so called as continues cell lines.
• They can be maintained either by serial subculture or by storing in deep freeze at -
70 degree Celsius.
• Due to derivation from cancer cell they are not useful for vaccine production.
• Example : HeLa-Human carcinoma of cervix cell
HEP2-Human epithelioma of larynx cell lines
BHK-21 –Baby hamster kidney cell lines.

21. Recognition of Viral Growth in Cell Culture
 The growth of viruses in cell culture can be monitored by a number of biochemical
procedures indicative of the intracellular increase in viral macromolecules and virions .
 In addition, there are simpler methods that are more commonly used for diagnostic work
Cytopathic Effects
Hemadsorption and Hemagglutination.
Immunofluorescence
Interference.

22. Cytopathic Effects:
 Many viruses can be detected and initially identified by observation of the
morphological changes in the cultured cells in which they replicate.
 The CPE produced by different types of viruses are characteristic and help in the initial
identification of virus isolates.
 Nuclear shrinking, vacuoles in the cytoplasm, syncytia formation, rounding up, and
detachment are the examples of alteration of morphology of the cells.
 Most CPEs can be demonstrated in unfixed and unstained monolayer of cells under low
power of microscope.
 For example, adenoviruses produce large granular changes resembling bunches of
grapes, SV-14 produces well-defined cytoplasmic vacuolation, measles virus produces
syncytium formation, herpes virus produces discrete focal degeneration, and
enteroviruses cause crenation of cells and degeneration of the entire cell sheet.

23. Hemadsorption and Hemagglutination
 Cultured cells infected with orthomyxoviruses, paramyxoviruses, or
togaviruses, all of which bud from cytoplasmic membranes, acquire
the ability to adsorb erythrocytes. This phenomenon, know as
hemadsorption, is due to the incorporation into the plasma
membrane of newly synthesized viral protein that binds red blood
cells .
 Hemadsorption can be used to demonstrate infection with non-
cytopathogenic as well as cytocidal viruses, and can be demonstrated
very early, e.g., after 24 hours, when only a small number of cells in
the culture are infected.
 Hemagglutination is a different, though related phenomenon, in
which erythrocytes are agglutinated by free virus .
 Virions or hemagglutinin may thus be demonstrated in the
supernatant fluid of an infected culture.

24. Immunofluorescence
Newly synthesized intracellular viral antigen can be detected by staining the fixed cell
monolayer with specific antiviral antibody which has been labeled with a fluorescent
dye, or with an enzyme such as peroxidase.
Interference
 The replication of one virus in a cell usually inhibits the replication of another virus .
 The viruses of rubella and of the common cold were first discovered by showing that
infected cell cultures, which showed no cytopathic effect, were nevertheless resistant to
challenge with an unrelated enterovirus.
 Cell lines have now become available in which these viruses produce cytopathic effects,
but interference is still used for the diagnosis of bovine virus diarrhea virus.