HASHIM SIDDIQUE
70105507
PROPAGATION OF VIRUSES
Introduction:
• Viruses are important agents of many human
diseases, ranging from the trivial (e.g. common
colds) to the lethal (e. g . rabies)
• Viruses also play roles in the development of
several types of cancer.
• As well as causing individuals to suffer, virus
diseases can also affect the well-being of societies.
• Smallpox had a great impact in the past and AIDS
is having a great impact today.
Purpose of Growing Viruses in the
Laboratory :
• 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.
• Diagnosis of infection
• Research
Important Characteristics of Viruses
Related to Propagation:
• Viruses can not grow outside a living cell.
• The range of cell types in which they will replicate
is limited.
• A few viruses can not be grown at all (neither in
vivo nor in vitro)
Viruses can be propagated in:
• Animals
• Chick embryos
• Cell Culture
1. Propagation in Animals
• The first virus vaccines, smallpox
and rabies, were prepared in
animals.
• Smallpox on the skin of calves,
sheep or water-buffaloes.
• Rabies in the spinal cords of rabbits,
sheep or goats.
• Mice or other small animals are
sometimes used in research
laboratory.
• Viruses which are not cultivated in embryonated
egg and tissue culture are cultivated in laboratory
animals such as mice, guinea pig, hamster and
rabbits are used.
• The selected animals should be healthy and free
from any communicable diseases.
• After inoculation, virus multiply in host and
develops disease. The animals are observed for
symptoms of disease and death.
• After inoculation, virus multiply in host and
develops disease. The animals are observed for
symptoms of disease and death.
• Then the virus is isolated and purified from the
tissue of these animals.
• Live inoculation was first used on human
volunteers for the study of yellow fever virus.
Advantages of Animal Inoculation:
• Diagnosis, Pathogenesis and clinical
symptoms are determined.
• Production of antibodies can be identified.
• Primary isolation of certain viruses.
• Mice provide a reliable model for studying
viral replication.
• Used for the study of immune responses,
epidemiology and oncogenesis .
Disadvantages of Animal Inoculation:
• Expensive and difficulties in maintenance of
animals.
• Difficulty in choosing of animals for particular
virus.
• Some human viruses cannot be grown in animals ,
or can be grown but do not cause disease.
• Mice do not provide models for vaccine
development.
• Issues related to animal welfare systems.
2. Propagation in chick embryos:
• Good pasture 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
which is used.
• Viruses are inoculated into chick embryo of 712 days old.
• 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.
• Yolk sac inoculation: It is also a
simplest method for growth and
multiplication of 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.
• Chorioallantoic Membrane (CAM): Inoculation is
mainly for growing poxvirus.
• After incubation and , visible lesions called pocks are
observed, which is grey white area in transparent
CAM.
• Herpes simplex virus is also grown.
• Single virus gives single pocks
• This method is suitable for plaque studies.
3. Propagation in Cell and Tissue
Cultures:
• Cell culture is a collection of
dispersed living cells, either in
suspension or as continuous
(confluent) sheets adhering to
glass or plastic surfaces.
• Such sheets, one cell thick, are
called monolayers.It is the most
commonly used culture method.
• Culture mediaThese are nutrient rich media
and usually contain salts at physiological
concentrations, glucose, amino acids, essential
vitamins, and antibiotics.
• All ingredients are buffered at pH Fetal calf
serum (10-20%) is added to provide essential
growth factors.
• Cells in culture media are incubated at 37 C in
CO2 atmosphere.
Types of cell culture Based on the origin and the
chromosome property the tissue culture are
classified into 3 types.
1. Primary cell culture.
2. Diploid cell culture (Semi-continuous cell
lines)
3. Continuous cell lines
Virus Purification:
• After a virus has been propagated it is usually
necessary to remove host cells debris and other
contaminants before the virus particles can be:
• used for laboratory studies,
• for incorporation into a vaccine,
• or for some other purpose
Stabilization of Viruses:
By Salats:
• Many viruses can be stabilized by molar concentrations
of salts and cannot be inactivated even by heating at
50oC for 1 hour.
• The stability of viruses is important in the preparation
of vaccines.
• The nonstabilized polio vaccine must be stored at
freezing temperatures to preserve its potency.
• However, with the addition of molar MgCl2, the
potency can be maintained for weeks at room
temperature, even in the high temperatures of the
tropics
By pH:
• Viruses are usually stable between pH values
of 5.0 and 9.0.
• Because of the electrostatic forces in the
hemagglutination reactions, variations of few
tenths of a pH unit may be the deciding factor
in obtaining positive or negative results in this
test.
Inactivation of Viruses:
• By Radiation
 Ultraviolet, X-ray, and high energy particles inactivate
viruses.
• Vital Dyes
 Viruses are penetrable to a varying degree by vital dyes
such as toluidine blue, neutral red, and proflavine.
 These dyes unite with nucleic acid, and the virus then
becomes susceptible to inactivation by visible light.
 Photodynamic inactivation can be used for treating
herpetic lesions.
• Disinfectants
High concentrations of chlorine are required to
destroy viruses while formalin destroys
resistant viruses