Applied Veterinary Virology: The isolation and identification of viruses using embryonated
chicken eggs
Applied veterinary Virology:
The isolation and identification of viruses using
embryonated chicken eggs
Authors: Prof. Estelle H Venter
Licensed under a Creative Commons Attribution license.
INTRODUCTION
Viruses can only grow in living cells but they are particular about the type of cell they infect
and grow in – there is no universal cell that will support all viruses. Viruses tend to be hostspecific; therefore human viruses grow best in cells of human origin, bovine viruses in bovine
cells, canine viruses in canine cells, while some viruses will not grow in vitro at all. Therefore
in the laboratory the suspected virus must be grown in a culture method known to support its
growth.

animals are used for studying viruses which do not grow in cell cultures or eggs, and
for testing vaccines,

eggs support a fairly wide range of animal and human viruses – hence their importance
in the diagnostic service,

cell cultures; different types of cell lines will support different types of viruses.
The growth of viruses in eggs
Embryonated hen’s eggs will support the growth of some viruses. Not all viruses will grow in
the tissues of embryonated eggs initially but many can be adapted to growth in eggs without
much difficulty. Eggs provide a suitable means for the primary isolation and identification of
viruses, the maintenance of stock cultures and the production of vaccines.
The viruses grow in the cells of the embryo and membranes and can be detected in several
ways. These include mortality, deformity or haemorrhages in the embryo, lesions on the
membrane in the form of pocks, oedema of the developing membranes, inclusion bodies in
sections prepared from embryo tissues or the presence of viral antigens in the egg fluids.
An embryonated hen’s egg contains cells (the embryo and its membranes) that will support
the growth of some viruses. They grow either in the cells of the embryo or its membranes, or
in both and when these cells die they are liberated into the egg fluids. Collection of the virusinfected egg fluid is relatively simple, if somewhat messy.
1|Page
Applied Veterinary Virology: The isolation and identification of viruses using embryonated
chicken eggs
Eggs are inexpensive and easy to maintain. Eggs come in a usually sterile package
surrounded by a porous shell. As they should arrive clean in the laboratory, they should not
be washed or immersed in water as this may allow bacteria to enter the egg. Use a quickly
evaporating agent, such as alcohol, tincture of iodine or ether, to sterilize the eggshell at the
site re inoculation.
Eggs are freely available, especially hens’ eggs but ducks’ eggs have also been used. The
immune system of the embryo has not matured; therefore antibodies are not produced
against the inoculated virus. However, maternal antibodies are transferred from the hen to the
egg which implies that eggs should be obtained from non-vaccinated (especially against
Newcastle disease virus), mycoplasma-free flocks.
Advantages of eggs over animal host systems

Eggs are readily available, cheap and easy to maintain

Preliminary incubation of the eggs is carried out at 38 – 39°C and 60 – 70% humidity. The
eggs need to be turned at least twice a day or rolled continually in a specially designed
egg incubator

Once inoculated, the eggs are incubated at temperatures suitable for the growth of the
virus, but still maintaining a high degree of humidity

Eggs are easily manipulated under sterile conditions

Eggs come in a sterile package surrounded by a porous shell. They should arrive clean in
the laboratory. Do not wash the eggs or immerse them in water as this may allow bacteria
to enter the porous shell. To sterilize the site of inoculation, use a quickly evaporating
agent, such as alcohol, tincture of iodine or ether

Eggs are sheltered from the natural diseases often observed in laboratory animals, and
are relatively free from bacterial and many latent viral infections. However maternal
antibodies are transferred from the mother hen, therefore the eggs should be obtained
from non-vaccinated (especially Newcastle disease virus), mycoplasma-free flocks

Eggs are generally free from natural factors of defence, specific or non-specific, that
sometimes intervenes and prevents passage in adult animals. The immune system of the
embryo has not matured therefore antibodies are not made against the inoculated virus.
Also the embryo is sensitive of some viruses that are harmless to the adult bird

Eggs are easily identified and labelled with details of date, virus inoculated and
experimental procedure

Different routes of inoculation for different viruses are available i.e.
2|Page
Applied Veterinary Virology: The isolation and identification of viruses using embryonated
chicken eggs
o
the amniotic and / or allantoic cavities,
o
the yolk sac,
o
the chorio-allantoic membrane,
o
intravenous.
Viruses that can grow in embryonated chicken eggs
Embryonated chicken eggs are not routinely used for the isolation of viruses. Viruses of
veterinary importance which grow in eggs are tabulated under disease caused, age and route
of infection, incubation temperature and outcome of infection.
Table 1: Viruses of veterinary importance that can be grown in embryonated chicken eggs
ANIMAL
VIRUS GENUS
Rhabodovirus
DISEASE
Vesicular Stomatitis
ROUTE OF
INOCULATION
Inoculate onto allantoic
Sac
OUTCOME OF
INFECTION
Embryo death
8 days old embryo
3 – 4 days incubation
Capripoxvirus
Lumpy skin disease
Inoculate onto CAM
7 – 9 days incubation 33,5
– 35°C
Cattle
Orthopoxvirus
Cowpox
Inoculate onto CAM
Can be distinguished from
pseudocowpox (parapox)
which does not grow on
the chorio-allantoic
membrane.
Orthomyxovirus
Swine influenza
Swine
Amniotic/allantoic
inoculation into 9-10 day
old embryonating eggs
Pocks / lesions on
membrane
Staining of membrane
Pocks / lesions on
membrane
Haemagglutinates
chick RBC
Incubate 48 hrs at 33.5°C
Iridovirus
African swine fever
Yolk sac inoculation
Causes death in 6 – 7
days
Orbivirus
Bluetongue
Yolk sac / IV
Causes death of
embryo. Further
processed for
presence of virus
10 – 11 days old
embryonating eggs
Sheep
and
Incubate 3-5 days at
33.5°C
goats
Flavivirus
3|Page
Wesselsbron disease
Yolk sac
Applied Veterinary Virology: The isolation and identification of viruses using embryonated
chicken eggs
Orthomyxovirus
Equine influenza
Amniotic/Allantoic
inoculation into 10 day old
embryonating eggs
Haemagglutinates
chick and pig RBC.
Incubate 48 hrs at 33°C
Horses
Reoviridae Orbivirus
African horse sickness
Yolk sac /IV
10 – 12 days old
embryonating eggs
Causes death of
embryo.
Incubate at 33°C for 3 – 7
days
Bornavirus
Dogs
CAM
Paramyxovirus
Canine distemper
CAM – needs adaptation
Rhabdovirus:
Lyssavirus
Rabies
Difficult, but can be
adapted
Paramyxovirus
Newcastle disease
Amniotic/allantoic
inoculation into 9-10 day
old embryonating eggs
Incubate 2 – 5 days at
37°C
Herpesvirus
Infectious
laryngotracheitis
Produces lesions
Causes death of
embryo.
Haemagglutinates
chick RBC.
CAM–plaque formation
and death of embryo
Amniotic/allantoic
inoculation. Incubate 5-7
days
Coronavirus
Infectious bronchitis
Amniotic/allantoic
inoculation
Incubate 30 hrs at 37°C
Effects on embryo include
death, dwarfing, and
curling, plus uratic
deposits in the
mesonephrons.
Poultry
Coronavirus
Turkey enteritis
(Bluecomb disease)
Orthomyxovirus
Avian influenza, Myna,
turkey, chicken, duck,
gull.
Amniotic inoculation–
grows in embryo intestines
or yolk sac, 3 – 5 days
incubation.
Some strains require
10 days incubate for
maximum virus.
Further processed for
presence of virus
Amniotic/allantoic
inoculation
Incubate 2 days at 35°C
Death of embryo within 48
hours.
Adenovirus type 1
Chick embryo lethal
orphan (CELO)
4|Page
Avian adenovirus
infection
Allantoic inoculation
Incubate 3-4 days
May need up to five blind
passages
Causes death of
embryo with necrotic
foci in liver and urate
accumulations in
mesonephrons
Applied Veterinary Virology: The isolation and identification of viruses using embryonated
chicken eggs
Pox: Avipox
Fowlpox
CAM
Produces focal or
diffuse pocks
Enterovirus
Avian
encephalomyelitis
Yolk sac inoculation
Muscular dystrophy
and paralysis.
Incubate 9 days at 37°C
Signs of
encephalomyelitis
observed after
hatching.
Enterovirus
Duck viral hepatitis
Turkey hepatitis
Miscellaneous
Allantoic inoculation into 911day eggs
Reovirus
Infectious bursal
disease
CAM
Orthopox
Ectromelia (mousepox)
CAM
Leporipox
Myxomatosis (rabbits)
CAM
Leporipox
Shope fibroma
Causes death of the
embryo
No lesions
Table 2: The use of eggs to distinguish between clinically similar diseases
VIRUS AND DISEASE
Newcastle disease virus
(NDV),
EFFECT ON: CHORIOALLANTOIC
EFFECT ON: CHICKEN
EMBRYO
HAEMAGGLUTINATIO
N OF CHICK EMBRYO
FLUIDS
No pocks
Lethal within 48-72 hours
HA positive
Pocks
No effect
HA negative
No pocks
Dwarfing
HA negative
a paramyxovirus of chickens
Laryngotracheitis,
a herpesvirus of chickens
and pheasants
Infectious bronchitis,
a coronavirus of chickens
5|Page