CULTIVATION OF VIRUS : Embryonated eggs

1. CULTIVATION OF
VIRUS : Embryonated
eggs

2. Introduction
▸ Viruses are obligate intracellular parasites so they depend on host for their
survival.
▸ They cannot be grown in non-living culture media or on agar plates alone, they
must require living cells to support their replication.
▸ In vivo-laboratory bred animal and embryonic bird tissue
▸ In vitro-cell or tissue culture method
▸ The primary purpose of virus cultivation is:
▸ To isolate and identify viruses in clinical samples.
▸ To do research on viral structure, replication, genetics and effects on host cell.
▸ To prepare viruses for vaccine production.
2

3. Cultivation of viruses
Inoculation into
embryonated egg
Woodruff and Goodpasture
(1931)used fertilized chicken egg for
viral cultivation. This is a simpler
technique than animal inoculation ,are
inexpensive and easily available .
Eggs usually not interfere with virus
multiplication due to absence of
immune response. Suitable cells for
the growth of viruses are available in
embryo and its membrane ,which may
facilitate the growth of virus.
Tissue Culture
Cultivation of tissue
or organ for the
growth of viruses.
Cell culture is most
widely used in
diagnostic virology for
cultivation and assays
of viruses.
3
Animal inoculation
Susceptible experimental animals
like Mice, Monkey, Rabbits,
Guinea Pigs etc. are used for the
cultivation of viruses. Virus sample
to be cultivated should be injected
into the experimental animal.
Intracerebral, subcutaneous,
intraperitoneal, or intranasal routes
are various routes of inoculation.
After inoculation, the animals are
observed for signs of disease or
death.

4. Embryonated eggs
▸ Woodruff and Goodpasture in 1931 first used the embryonated hen’s egg for the
cultivation of virus.
▸ The method further developed by Burnet was used for cultivation of viruses in
different sites of the egg.
▸ Many of these viruses cause well-defined and characteristic foci, providing a
method for identification, quantification, or assessing virus pathogenicity.
▸ The embryonated egg is also used for growing higher stocks of some viruses in
research laboratories and for vaccine production.
▸ Viruses can be cultivated in various parts of egg like Chorioallantoic membrane,
Allantoic cavity, Amniotic sac and Yolk sac.
4

5. Continue….
▸ Viruses are inoculated into chick embryo of 7-12 days old.
▸ For inoculation, eggs are first prepared for cultivation, the shell surface is first
disinfected with iodine, checked in ovoscope if it is alive and penetrated with a
small sterile drill.
▸ After inoculation, the opening is sealed with gelatin or paraffin and incubated at
36°c for 2-3 days.
▸ After incubation, the egg is broken and virus is isolated from tissue of 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
5

6. 6

7. Chorioallantoic Membrane (CAM)
▸ Inoculation is mainly for growing poxvirus.
▸ After incubation, visible lesions called pocks are observed, which is grey white area in
transparent CAM.
▸ Each infectious virus particle produces one pock.
▸ The morphology of the pocks may vary depending on the nature of the virus.
▸ The pox viruses, such as variola or vaccinia are identified by demonstration of typical pocks
on the CAM inoculated with the pox virus.
▸ Herpes simplex virus is also grown.
▸ This method is suitable for plaque studies.
▸ Nowadays, in a virology laboratory, chick embryo inoculation has been replaced by cell
cultures for routine isolation of viruses.
7

8. Allantoic cavity
▸ Is the most popular and simple method for viral inoculation.
▸ Inoculation in the allantoic cavity is used for serial passages and for obtaining large quantities of virus,
such as influenza virus, yellow fever, and rabies viruses for preparation of vaccines.
▸ For production of rabies virus, duck eggs were used due to their bigger size than that of hen’s egg. This
helped in production of large quantities of rabies virus, which are used for preparation of the inactivated
non-neural rabies vaccine. But they need a longer incubation period than embryonated hen’s egg.
▸ Most of avian viruses can be isolated using this method.
8
Amniotic sac
▸ Inoculation is mainly done for primary isolation of influenza virus and the mumps virus.
▸ Growth and replication of virus in egg embryo can be detected by haemagglutination assay.

9. Yolk sac inoculation:
▸ It is also a simplest method for growth and multiplication of virus.
▸ Mostly mammalian viruses are isolated using this method.
▸ Yolk sac inoculation is used for cultivation of Japanese encephalitis, Saint Louis encephalitis, and West
Nile virus.
▸ It is inoculated for cultivation of some viruses and some bacteria (Chlamydia, Rickettsiae)
▸ Immune interference mechanism can be detected in most of avian viruses.
9

10. Procedure :
▸ 1. Candling: For propagation of influenza virus,
pathogen-free eggs are used 11-12 days after
fertilization. The egg is placed in front of a light
source to locate a non-veined area of the
allantoic cavity just below the air sac. This is
marked with a pencil. After all the eggs have
been ‘candled’ in this way, a small nick is made
in the shell at this position using a jeweler’s
scribe.
10

11. Procedure :
▸ 2. Drill the hole: Next, a hole is drilled at the
top of the egg with a Dremel motorized tool.
If this is not done, when virus is injected, the
pressure in the air sac will simply force out
the inoculum.
11
Drilling pin- point hole.

12. Procedure :
3. Inject the specimen: After all the eggs
have been nicked and drilled, they are
inoculated with virus using a tuberculin
syringe. The needle passes through the hole
in the shell, through the chorioallantoic
membrane, and the virus is placed in the
allantoic cavity, which is filled with allantoic
fluid.
4. Hole sealed with paraffin wax: The two
holes in the shell are sealed with melted
paraffin,
5. Incubation: The eggs are placed at 37
degrees C for 48 hours. 12

13. Advantages
▸ 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
▸ They are free from contaminating bacteria and many
latent viruses.
▸ Defense factors are not involved in embryonated eggs.
▸ Widely used method to grow virus for some vaccine
production
▸ Chick embryos are packed in their shells and have
natural resistant against bacterial contamination.
▸ Chick embryo method is cheaper and easy to handle.
13
Disadvantages
▸ The site of inoculation for varies
with different virus. That is, each
virus have different sites for their
growth and replication.
▸ Some viruses do not show growth
on primary inoculation into the
eggs.
▸ Slight amount of bacterial
contamination in the inoculum may
kill the embryo.

14. THANK
YOU