Cultivation of Viruses
Embryonated Egg
● Viruses do not fall in the category of
unicellular microorganism
● They are obligate intracellular parasites and
lack the machinery necessary for protein and
nucleic acid synthesis
● They depend on the host machinery for their
growth and survival
● Unlike other microorganism, complex
processes are involved in their multiplication
● Outside of the host cells, viruses are inactive,
Cultivation of Virus
● 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
Cultivation of Virus
● Viruses not only need living cells to grow in but
also they are specific about the type of cell they
infect and grow in
● There is no universal cell that will support all
viruses
● Viruses tend to be host specific; 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
Methods for Cultivation of Virus
● 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
Methods for Cultivation of Virus
● 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
Inoculation of Virus in Animals
● 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
Inoculation of Virus in Animals
● 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
Cell Cultures
● Prior to the advent of cell culture, animal
viruses could be propagated only on whole
animals or embryonated chicken eggs
● Cell cultures have replaced embryonated
eggs as the preferred type of growth medium
for many viruses
● Cell culture consists of cells grown in culture
media in the laboratory
● These cultures can be propagated and
handled like bacterial cultures; they are more
Inoculation of Virus in Embryonated
Eggs
● 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
Embryonated Egg
● Terms most often refer to eggs:
○ Embryonated: having an embryo
○ Unembryonated: not having an embryo
○ De-embryonated: having lost an embryo
● Embryonated egg, referring to an advanced stage of development and not
merely after fertilisation
Advantages
● An embryo is an early developmental stage of
animals marked by rapid differentiation of cells
● Birds undergo their embryonic period within the
closed protective case of an egg, which makes an
incubating bird egg a nearly perfect system for
viral propagation
● It is an intact and self-supporting unit, complete
with its own sterile environment and nourishment
● It furnishes several embryonic tissues that readily
support viral multiplication
● Defense mechanisms are not involved in
Inoculation of Virus
● 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
Inoculation of Virus
● The egg must be injected
through the shell, usually by
drilling a hole or making a small
window
● The viral suspension or
suspected virus- containing fluid
is injected into the fluid of the
egg
Detection of Viral Growth
● Viruses multiplying in
embryos may or may not
cause effects visible to the
naked eye
● The signs of viral growth
include:
○ Death of the embryo
○ Defects in embryonic
development
○ and localized areas of
damage in the membranes,
Detection of Viral Growth
● If a virus does not produce obvious changes in the developing embryonic
tissue, virologists have other methods of detection
● Embryonic fluids and tissues can be prepared for direct examination with an
electron microscope
● Certain viruses can also be detected by:
○ their ability to agglutinate red blood cells
○ or by their reaction with an antibody of known specificity
Parts of Embryonated Egg
● The air sac is important to the
developing embryo for
respiration and for pressure
adjustments
● The shell and shell membrane
function both as a barrier and
as an exchange system for
gases and liquid molecules
● The chorioallantoic sac and its
contents (allantoic fluid)
remove waste products
produced by the developing
embryo
Parts of Embryonated Egg
● The yolk sac is the source of nourishment for the developing Embryo
○ As the embryo develops, the yolk sac decreases in size until it is completely absorbed into
the digestive system of the mature embryo
● The amnion is a thin membrane that encloses the embryo and Protects it
from physical damage
○ It also serves as an exchange system and is best seen in the younger embryos
Routes of Viral Inoculation
● 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
Routes of Viral Inoculation
● The chosen route of inoculation and age of the embryo are determined by
the given virus selectivity for a certain membrane or developmental stage of
the embryo
○ For example Infectious bronchitis virus is propagated in the yolk sac of a 5-6 day old embryo
○ whereas Rous-sarcoma virus is inoculated on the chorioallantoic membrane of a 9-11 day
old embryo and will produce pocks 5-10 days post-infection
Candling of Egg
● Candling is the process of
holding a strong light above
or below the egg to observe
the embryo
● A candling lamp consists of
a strong electric bulb
covered by a plastic or
aluminum container that has
Candling of Egg
● Chick egg at day 6
● Chick embryo day 11
● Chick embryo day 16
Chorioallantoic Membrane (CAM)
● 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:
Amniotic Cavity Inoculation
● The virus is introduced directly into the
amniotic fluid that bathes the developing
embryo
● The volume of fluid in the infected
amniotic sac is small (1-2 ml)
○ The amniotic route is recommended for the
primary isolation of human viruses:
■ mumps virus
■ and influenza A, B and C viruses
○ has little application in veterinary virology
Allantoic Cavity Inoculation
● Many viruses such as Newcastle disease virus can grow readily
● Other viruses such as influenza, may require repeated amniotic passages
before becoming adapted to the egg and grown in the allantoic cavity
● Allantoic inoculation is a quick and easy method that yields large amounts
(8–15 ml) of virus-infected egg fluids
Yolk Sac Inoculation
● 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
