Lecture 2.

Cultivation, Purification and

enumeration of viruses
 Some important definitions
∙ A susceptible cell has receptor for a
given virus -­‐ the cell may or may not be
able to support viral replication
∙ A resistant cell has no receptor -­‐ it may
or may not be competent to support
viral replication
∙ A permissive cell has the capacity to
replicate virus -­‐ it may or may not be
susceptible
∙ A susceptible AND permissive cell is the
only cell that can take up a virus
particle and replicate it
 How can you grow and store viruses for experiment?

•Animal viruses could not be propagated in artificial

cells
•Most viruses were grown in laboratory animals
 Virus culture
A virus is a biological agent that reproduces inside the cells
of living hosts.
Viruses can't be grown in culture media or on agar plates
alone, they must have living cells to support their
replication. Because viruses are obligatory intracellular
parasites, they cannot reproduce outside a living cell.
An exception comes from the demonstration in 1991 that
infectious poliovirus could be produced in an extract of
human cells incubated with viral RNA.
Unlike most living things, viruses do not have cells that
divide; new viruses are assembled in the infected host cell.
When infected by a virus, a host cell is forced to produce
many thousands of identical copies of the original virus, at
an extraordinary rate.
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 Virus culture
Viruses, like bacteria, can be cultured in the laboratory.
Living host cells are required to grow viruses in the
laboratory.
A single type of cell line is not adequate, as specific
viruses need specific receptors on the cell surface to
which they attach to gain entry into the cell and to
initiate replication.

**The presence of specific cell receptors on the cell

surface determines which viruses will be able to infect
them, and this is called ‘viral cell tropism’.
Another problem in the laboratory is to maintain these
living cells in culture long enough to allow sufficient virus
growth.

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 Purpose of Virus Cultivation

The primary purposes of viral cultivation are:

1. To isolate and identify viruses in clinical specimens.
2. To prepare viruses for vaccines.
3. To do detailed research on viral structure,
multiplication cycles, genetics and effects on host cells.
 Virus Cultivation Systems

 Tissue culture system.

 Embryonated eggs system.
 Whole animal systems.
 Cell culture for virus growth
•Although human and other animal cells were first cultured
in the early 1900s, contamination with bacteria, mycoplasmas,
and fungi initially made routine work with such cultures
extremely difficult.
•For this reason, most viruses were grown in laboratory animals.
•In 1949, John Enders, Thomas Weller, and Frederick Robbins
made the discovery that poliovirus could multiply in cultured
cells.
•Nobel Prize was awarded in Physiology or Medicine in 1954, led
the way to the propagation of many other viruses in cells in
culture, the discovery of new viruses, and the development of
viral vaccines such as those against poliomyelitis, measles, and
rubella.
1954 Press Photo Dr Weller, Dr Robbins,
Dr Enders, Share Nobel Prize In Medicine
 Tissue culture system for Cultivation of viruses

•Animal and plant viruses are cultivated in cell cultures.

•A cell culture is prepared by encouraging cell growth outside
the animal or plant source.
•The cells are kept alive in a suspension of growth factors within
a Petridish.
•A thin layer of cells, or monolayer, is then inoculated with
viruses, and replication takes place. (Fertilized eggs and living
animals can also be used to cultivate viruses.)
•For research study, viruses can be cultivated in large volumes by
inoculations to tissue culture systems. After a time, the cells are
degenerated, and viruses are harvested.
 Virus culture – in plant cells
There are some methods of cultivation of plant viruses
such as plant tissue cultures, cultures of separated cells,
or cultures of protoplasts, etc. viruses can be grown in
whole plants.

Leaves are mechanically inoculated by rubbing with a

mixture of viruses and an abrasive. When the cell wall is
broken by the abrasive, the viruses directly contact the
plasma membrane and infect the exposed host cells.

A localized necrotic lesion often develops due to the

rapid death of cells in the infected area.

Some plant viruses can be transmitted only if a diseased

part is grafted onto a healthy plant. 13
 Cell culture for animal viruses
⮚Cells in culture are still the most commonly used hosts for the
propagation of animal viruses.
⮚To prepare a cell culture, tissues are dissociated into a single-cell
suspension by mechanical disruption followed by treatment with
proteolytic enzymes.
⮚The cells are then suspended in culture medium and placed in
plastic flasks or covered plates.
⮚As the cells divide, they cover the plastic surface. Epithelial and
fibroblastic cells attach to the plastic and form a monolayer,
whereas blood cells such as lymphocytes settle, but do not
adhere.
⮚The cells are grown in a chemically defined and buffered
medium optimal for their growth.
⮚Commonly used cell lines double in number in 24 to 48 h in such
media. Most cells retain viability after being frozen at low
temperatures ( -70 to -196°C).
 Inoculation into embryonated egg
⮚Goodpasture in 1931 first used the embryonated hen’s egg for the cultivation of
virus.
⮚For inoculation, eggs are first prepared for cultivation, the shell surface is first
disinfected with iodine and penetrated with a small sterile drill.
⮚The process of cultivation of viruses in embryonated eggs depends on the type
of egg which is used.
⮚At 5 to 14 days after fertilization (chick embryo), a hole is drilled in the shell and
virus is injected into the site appropriate for its replication.
⮚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 pox or
lesions on the egg membranes
⮚Viruses can be cultivated in various parts of egg like chorioallantoic membrane,
allantoic cavity, amniotic sac and yolk sac.
⮚This method of virus propagation is now routine only for influenza virus.
⮚The robust yield of this virus from chicken eggs has led to their widespread use
in research laboratories and for vaccine production.
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A number of animal viruses have also been adapted to grow in
embryonated chicken eggs; reduced both the expense and the time
of virus assays.
 Animal Inoculation
⮚Viruses which are not cultivated in embryonated egg and tissue
culture are cultivated in laboratory animals (healthy and free from
any communicable diseases) such as mice, guinea pig, hamster,
rabbits and primates are used.
⮚Suckling mice(less than 48 hours old) are most commonly used.
⮚Suckling mice are susceptible to togavirus and coxsackie virues,
which are inoculated by intracerebral and intranasal route.
⮚Viruses can also be inoculated by intraperitoneal and
subcutaneous route.
⮚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.

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Growing Bacteriophages in the Laboratory
⮚Bacteriophages can be grown either in suspension of
bacteria in liquid media or in bacterial cultures on solid
media.
⮚Solid media makes possible the plaque method for detecting
and counting viruses.
⮚A sample of bacteriophage is mixed with host bacteria and
melted agar.
⮚The agar containing the bacteriophages and host bacteria is
then poured into a Petri plate containing a hardened layer of
agar growth medium.
⮚The viral particles are concentrated by precipitation methods
and purified by repeated centrifugations.
⮚Highly purified viruses can be obtained by crystallization and
concentration under established conditions.
 Types of cell culture
• There are three main kinds of cell cultures (Fig. 2.7), each with
advantages and disadvantages.
• Primary cell cultures are prepared from animal tissues. They
have a limited life span, usually no more than 5 to 20 cell
divisions.
• Commonly used primary cell cultures are derived from monkey
kidneys, human embryonic amnion and kidneys, human
foreskins and respiratory epithelium, and chicken or mouse
embryos.
• Such cells are used for experimental virology.
• They are also used in vaccine production: for example, live
attenuated poliovirus vaccine strains may be propagated in
primary monkey kidney cells.
• Primary cell cultures were mandated for the growth of viruses to
be used as human vaccines to avoid contamination of the
 Different types of cell culture used in Virus
cultivation.
Confluent cell monolayers

(A) Primary human B) mouse fibroblast (C) continuous line

foreskin cell line (3T3) of human epithelial
cells (HeLa)

Diploid cell strains (e.g. WI-38, human embryonic

lung)
The WI-38 cell line is the first human diploid cell line to be used in human vaccine preparation.
WI-38 cells are isolated from the lung tissue of a 3-month-old, female, embryo. The fetus came
from the elective abortion of a Swedish woman in 1962, and was used without her knowledge or
permission.
 Different types of cell culture used in Virus
cultivation.
• Some viral vaccines are now prepared in diploid cell strains, which
consist of a homogeneous population of a single type and can
divide up to 100 times before dying.
• The most widely used diploid cells are those established from
human embryos, such as the WI-38 strain derived from human
embryonic lung.
• Continuous cell lines consist of a single cell type that can be
propagated indefinitely in culture. These immortal lines are usually
derived from tumor tissue or by treating a primary cell culture or a
diploid strain with**ethylmethane
a mutagenic chemical or a tumor virus.
sulfonate and N-methyl-N-nitrosourea that induce point mutations in DNA
• Such cell lines are often abnormal in chromosome morphology
and number ( aneuploid ), and can be tumorigenic.
• Examples of commonly used continuous cell lines include those
derived from human carcinomas (e.g., HeLa [Henrietta Lacks] cells;
Box 2.3) and from mice (e.g., L and 3T3 cells).
Cell cultures are generally of 3 types:-
1. Primary culture – These are
prepared directly from animal or
human tissues and can be subcultured
only once or twice e.g. chicken embryo
fibroblast.
2. Diploid cell culture – They are
derived from neonatal tissues and can
be subcultured 5-10 times. e.g. human
diploid fibroblasts cells.
3. Continuous cells – They are derived
from tumor tissues and can be
subcultured more than 10 times. e.g.
Vero, Hep2, HeLa.
 Suspension culture
• In contrast to cells that grow in monolayers on plastic
dishes, others can be maintained in suspension cultures ,
in which a spinning magnet continuously stirs the cells.
• The advantage of suspension culture is that a large
number of cells can be grown in a relatively small volume.

- Application of suspension culture:

This culture method is well suited for applications that
require large quantities of virus particles, such as X-ray
crystallography or production of vectors.
 Evidence of Viral Growth in Cultured Cells
⮚Some viruses kill the cells in which they reproduce, and the
infected cells may eventually detach from the cell culture plate.
⮚As more cells are infected, the changes become visible and are
called cytopathic effects (Table 2.1).
⮚Many types of cytopathic effect can be seen with a simple light
or phase-contrast microscope at low power, without fixing or
staining the cells.
⮚These changes include the rounding up and detachment of
cells from the culture dish, cell lysis, swelling of nuclei, and
sometimes the formation of a group of fused cells called a
syncytium (Fig. 2.8).
⮚The time required for the development of cytopathology varies
greatly among animal viruses. For example, enteroviruses and
herpes simplex virus can cause CPE in 1 to 2 days while
cytomegalovirus, rubella virus, and some adenoviruses may not
produce such effects for several weeks.
 cytopathic effect (CPE)

Development of cytopathic effect. (A) Cell rounding and lysis during

poliovirus infection. (Upper left) Uninfected cells; (upper right) 5.5 h after
infection; (lower left) 8 h after infection; (lower right) 24 h after infection
Table 2.1 Some examples of cytopathic effects of viral infection of animal cells
Cytopathic effect(s) Virus(es)

Morphological alterations
Nuclear shrinking (pyknosis), proliferation Picornaviruses
of membrane
Proliferation of nuclear membrane Alphaviruses, herpesviruses
Vacuoles in cytoplasm Polyomaviruses,
Syncytium formation (cell fusion) papillomaviruses
Margination and breaking of chromosomes Paramyxoviruses,
Rounding up and detachment of cultured coronaviruses Herpesviruses
cells Herpesviruses, rhabdoviruses,
adenovimses, picornaviruses
Inclusion bodies Adenoviruse
Virions in nucleus s Rabies
virus
Virions in the cytoplasm (Negri bodies)
Poxviruses
"Factories" in the cytoplasm (Guarnieri
Arenaviruses
bodies) Clumps of ribosomes in virions
Herpesviruse
Clumps of chromatin in nucleus
s
cytopathic effect (CPE)
- Viral glycoproteins on the
surface of an infected cell bind
receptors on a neighboring cell,
causing fusion.

The field shows a mixture of individual

small cells and syncytia, indicated by the
arrow, which are large, multinucleate cells.

Formation of syncytia
 DETECTION AND TITRATION
OF VIRUSES
Most viruses were first detected and studied by
infection of intact organisms

-expensive and time-consuming work

- ethically unacceptable when applied to humans.

Experimental laboratory animals such as suckling mice, in

which many animal viruses are able to replicate, were
adopted for use because they are relatively easy and
inexpensive to raise.
How can you detect and quantify viruses

∙ Infectivity
∙ Physical: virus particles and their components

https://www.youtube.com/watch?v=er2dwOPwSRo
The plaque assay arose from work
with bacteriophages

1930s: used to study multiplication of

bacteriophages
 Virus culture – in bacterial cells
Bacterial cells can be grown in liquid (broth) culture, where
densities of 108 cells/ml are reached during the exponential
(i.e., most rapid) phase of growth.
Bacterial cells can also be grown on solid or semisolid
surfaces, allowing formation of colonies or clones where all
cells are the descendants of one single cell.

• Such plates are used extensively for plaque

assays of bacterial viruses.
• Viral plaque assays determine the number
of plaque forming units (pfu) in a virus
sample, which is one measure of virus
quantity.

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 Plaque assay

∙ 1930’s plaque assays were used for studying bacteriophages

∙ 1952, Renato Dulbecco developed the first quantitative assays for animal
viruses, setting up a plaque assay to measure Western equine
encephalomyelitis (WEE) and poliovirus titers.
∙ Dulbecco’s first paper on polyomaviruses described the ability of
polyomavirus to cause plaques in mouse embryonic kidney cells in
culture.
∙ It was published in 1959, and it provided the first way to measure
polyomavirus titers.
∙ Nobel Prize, 1975
Plaque assay
 Plaque assay
1. A virus suspension was subjected to 10-fold serial dilutions by adding 1 mL of the
original suspension to 9 mL of a dilution buffer.

2. After mixing, 1 mL of that dilution was added to 9 mL of fresh dilution buffer and
mixed; these steps were repeated a total of eight times.

3. Each successive tube contains a 10-fold dilution of the contents of the previous tube.
The eighth tube therefore is diluted by a factor of 108 compared with the original
virus suspension.

4. One-mL aliquots from this 108 -fold dilution were applied to four different Petri
dishes of susceptible cells and plaques (bottom) were allowed to develop.

5. The dish is covered with a semi-solid medium, such as agar or carboxymethyl

cellulose, to prevent the virus diffusion after lysis of infected cells.

6. When a plaque assay is used to measure the infectious titer of a virus suspension,
the results are usually expressed as plaque-forming units (PFU) per mL of
suspension.

7. To determine the titer, the number of plaques on a plate is multiplied by the factor
by which the original virus suspension was diluted before an aliquot was applied to
the plate.
Plaque assay
Endpoint dilution assay
Tissue Culture Infectious Dose 50 

10-2
10-3
10-4

TCID50
 Particle-to-PFU ratio
Number of virus particles in sample/number of
infectious particles
⮚A single particle can initiate infection.
⮚The ratio of physical virus particles to infectious
particles can be much greater than 1.
⮚Many viruses the ratio of physical particles to
infectious particles can be 10, 100, or even 1000!
 There are several possible reasons for the
low infectivity of virus preparations

•Damaged: Not all virus particles may be intact

• Mutation: Some virus particles may contain
defective genomes
• Empty capsids that contain no viral genome can be
made in large numbers
• Cells have antiviral defense mechanisms