Cultivation Of Viruses -Methods and Conditions

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PHARMACEUTICAL MICROBIOLOGY-
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Cultivation Of
Viruses
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What are Viruses?
 Viruses are non-cellular microscopic entities which
are only visible under high resolution electron
microscope. These are one of the smallest known
parasitic entitities only beside prions and viroids.
 They have genetic material either DNA or RNA
covered by a proteinous coat which is further
enclosed in an envelope (capsid).
 The nucleic acids are composed of building blocks
called nucleotides that are constituted of three parts
i.e: Phosphoric acid, Pentose sugar, and any organic
base containing nitrogen.
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 The viruses are obligate intracellular parasites that are only active
inside a host cell or tissue, and thus they can not be cultured outside
of a living host cell either in culture media or in agar dishes.
 They are asexual and partially non-living entities lacking organ
systems.So, they can not reproduce themselves even if provided
compatible conditions outside a host.
 Viruses have no cellular structure which makes them impossible to be
grown by cell-division.
 Viruses don’t reproduce, they “replicate” by making their identical
copies rapidly inside the host cells.
 There are three types of viruses: Bacteriophages, Animal viruses, and
Plant viruses.
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What is Cultivation?
 “Cultivation” is the process to grow viruses in a rich
quantity from few available viruses by rendering favourable
conditions and inoculating them into the host cells.
 In this process, the virus infects the host cell, undergoes replication ,
resulting in the formation of several copies of that particular virus in
a substantial number.
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Why do we cultivate viruses?
 There are several reasons for cultivating
viruses:
1. To isolate and identify the viruses and
their propagation.
2. To use them for vaccine development.
3. To evaluate their clinical, pathogenic,
genetical ,replication, and structural
properties.
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Cultivation Of
Bacteriophages
 “Bacteriophages” are the simplest viruses.
They infect the bacteria by bounding on specific
receptors in bacterial cell wall and injecting their DNA or
RNA into their cells, where it replicates and multiple
copies are released out after multiplication.
 They are cultivated because they are considered to
be an effective anti-bacterial aid.
 These are the easiest viruses to cultivate as the
growth of bacterial cell-culture is common nowadays.
 The conditions required for cultivation of
bacteriophages are:
1. Living bacterial cells must be available.
2. Bacterial culture in agar medium with optimal
conditions for virus,
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Conditions for Cultivation

Method Of Cultivation
Preparing Bacterial Culture
 Prior to cultivating bacteriophages, firstly we
have to make ready a culture of their hosts- the
bacterial cells.
 Initially, a culture of susceptible bacterial cells
e.g: Escherichia coli, is prepared in an agar
medium or broth.
 The bacterial cells are arranged in form of
colonies all over the nutrient media.
 In adherent cultures (agar) enzymatic and
mechanical dissociation will necessarily occur at
the end whereas in suspension cultures (fluid
culture) it is not.
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Phage Inoculation
 After preparing a suitable bacterial culture, a phage virus is
introduced onto one of the bacterial cells.
 The phage adsorbs on the surface of bacterial cell wall,
capturing appropriate receptors, and injects its genome into
the susceptible bacterium.
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Replication and Extrusion
 The viral genome infects the bacterial genome, it replicates to
form multiples and even hundreds of identical copies.
 These all viral genomes get together and leave the bacterial
cell either by forceful extrusion, by budding (virions collect at
a particular point to form a bud in bacterial cell wall that is
later separated, taking them out), or by bacterial cell lysis.
 New virions are formed and released in the culture to infect
adjacent bacteria and the host cell is dead.
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Formation Of Viral Plaques
 The released virions infect the other bacteria in the vicinity resulting in
production of more viruses and destruction of host bacteria.
 This infection and destruction continues until the phages have
destroyed all the bacteria in a localized area.
 When this is done, a “plaque” is formed in that particular area where
viruses have overwhelmed the bacteria.
 Finally, two prominent zones are distinguishable in the agar plate:
i. Viral Plaques, which are opaque clear, visible, spot-like zones.
ii. Bacterial Lawn, groups of adjoined live, uninfected bacterial
colonies
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Plaque Assay
 “Pfu” stands for plaque forming units which refer to the number of initial
phage particles introduced in the agar plate. 1 Pfu is equal to 1 phage
particle. As the number of plaques formed indicates the number of phage
forming units. This quantitative analysis is known as “plaque assay”.
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One-Step Growth Curve
 It is a simple graphical depiction of the viral replication during cultivation
method.
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Whereas in fluid cultures, the “turbidity” shows the regions where phages have
been produced by killing the bacterial cells.
After this analysis, the phages may be identified and then isolated for further
examinations and desired pharmaceutical or scientific procedures.

 “Animal Viruses” are complex viruses that have the ability to
invade the eukaryotic animal cells , cause medical complications and
even death.
 These viruses are the most important to be cultivated in lab and
require sheer effort and focus for their growth.
 Primarily, they are cultivated for the development of vaccines and
studying their genomes, life-cycle and transmission.
 Theories suggest that they may be one of the prominent and lethal
weapons in any biological warfare in future.
 For millennias, these animal viruses have led to great pandemics and
have contributed to the extinction of species.7/3/2020
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Cultivation Of Animal Viruses

 The conditions required for cultivation of animal
viruses are:
1. Living animal cells must be available.
2. Appropriate enzymes to facilitate the life of cells.
3. Proper pH and medium to avoid any unwanted
cellular damage.
4. Animal serums
5. Sterilized equipments
6. Needed temperature for cell sustenance.
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 This is the most widely used method for cultivation of animal viruses. It is also
known as “cell culture”.
 In this method, firstly a tissue fragment or living cells are isolated from a live
organism with the aid of enzymes like trypsin or collagenase.
 A “cell suspension” is formulated which consists of a semi-confluent
monolayer of cells in liquid medium of basic nutrients and salts.
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Methods Of Cultivation
1) Tissue Culture
Then in an air-tight, sterile, flat-
bottomed glass apparatus, this cell
suspension is placed with a suitable
liquid media, usually animal serum
(usually Eagle’s serum or bovine
albumin).
The cells then attach at the surface,
grow , and divide in order to form a

Types Of Cell Culture
 There are three types of cell cultures on the basis of
chromosomal properties:
1. Primary Cell Lines: These are detached directly from a
specific animal organ which is considered extremely susceptible to
be a host for a particular virus. These are in form diploid cell lines
of intact living tissue and can not be retained as a culture for a
longer time. Though, these are helpful for the primary isolation of
viruses and assist in the preparation of vaccines.
e.g: Monkey’s kidney tissue culture, Human embryonic kidney cell
culture, etc.
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2. Secondary Cell Culture: These are derived from the
cultured primary cell lines. They are also diploid cell lines,
more homogenous but less susceptible than primary
cultures. Though, several subcultures can be made from
the, upto a maximum number of 50 until the cells
degenerate.
e.g: Cell culture isolated from monkey’s kidney primary cell lines,
human embryonic lung strain.
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3.Continuous Cell Culture: These are seperated from
carcinomic or sarcomic malignant tissues or simply from tumors.
As they are not normal cells, they can not be utilized for vaccine
development. They may be cultured countless times, therefore
they are known as “continuous cell lines”. They are stored by
deep freezing at below -70⁰C.
e.g: HeLa (Human carcinoma cervix cell lines), BHK-21 (Baby
Hamster Kidney cell line), etc.
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Cytopathic Effect (CPE) and Harvesting
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 The invasion of viruses upon cells cause visible morphological changes near the
site of infection in the monolayer tissue cells. This localized spoliation of these
cells is known as “cytopathic effect” (CPE).
 The cytopathic effect varies according to the cell type and viruses. General
cytopathic effects may be in the form of:
# CPE Type Observation Causative Virus
1 Total Cell
Destruction
Every cell leaves the surface and
destroys, confluent layer breaks
Enteroviruses.
2 Subtotal Cell
Destruction
Few cells leave the surface and are
destroyed, semi-confluent layer may
exist
Togoviruses.
3 Focal Degeneration Virus transfers from one cell to other
directly causing damage to particular
viral sites (Foci) in all cells
Poxviruses.
4 Foamy Degeneration Formation of numerous vacuoles
inside the cells
Retroviruses.
5 Syncytium Four or more cells fuse into each
other to form an enlarged cell
Herpesviruses.
6 Inflammation Infected cells are enlarged and may
form clumps
Flaviviruses
7 Inclusion bodies Formation of abnormal intracellular
structures that indicate the area for
Rabies Virus.

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Finally, the viruses are harvested by taking out the sample from the
infected cell culture in bulk, and is then inoculated into test tubes.
These test tubes are incubated at 35-37⁰C for and then rotated in a
laboratory centrifuge to dissociate the viruses.
After centrifugation, assays are performed and the obtained viruses may be
used for vaccine production or other biotechnological and pharmaceutical
purposes.
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2) Embryonated Bird Eggs
 This is the second priority method for cultivation of
animal viruses.
 In this method, a fertilized bird egg , preferably of
chicken, duck or turkey , is used as a host for the
cultivation of viruses.
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It is a simple technique in
which virus is inoculated
inside the bird’s egg where
the virus infects the tissues
and replicate effectively.

 An embryo containing egg must be available.
 The egg must be acquired from a healthy organism.
 Needle-Syringes and Paraffin are needed.
 The environment and apparatus for performing this task must be
aseptic and free from pathogens.
 The egg must be free from specific micro-organisms that may
disrupt the replication of viruses.
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Preparing Egg for Inoculation
 Firstly, a fertilized egg is availed of a healthy bird.
 The egg is washed, disinfected with iodine and
sterilized to evade any pathogens.
 The egg is perforated by puncturing with the help
of a needle syringe at particular appropriate spots.
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Inoculation and Incubation
 The virus is injected into the embryo inside the egg at the
desired region.
 The hole is then sealed neatly with paraffin to avoid any
contamination of the internal contents.
 The egg is then incubated at 36⁰C for a period of 2-3 days.
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During this incubation period
and favourable environment,
the viruses are let to multiply.

 Specific embryonic regions are best suited for particular
viruses, The viruses can not replicate accurately in tissues
other than these specific tissues.
 We select the embryonic region to be inoculated that will be
productive for the virus
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The cultivation of viruses in an embryonated bird’s egg greatly
depends upon the embryonic region where the virus is ought
to be grown.

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Isolation and Assays
 The presence of viruses is identified either by the death of embryo,
by rupture in infected embryonic tissues, or by emergence of pocks
or lesions on membranes.
 After the incubation period, the egg is broken and the samples are
extracted from the tissues that were inoculated earlier.
 These samples are then isolated in a test tube and are then
observed to find any cytopathic effect caused by the viral growth and
are analyzed by performing plaque assays.
 There are certain new tests evolved with the development of
technology to observe the viral cultivation inside the egg such as
Egg candling, etc.
 At last the viruses are successfully collected and can be utilized for
pharmaceutical industry and research.7/3/2020
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3) Animal Inoculation
 This is the least commonly used technique for cultivation of
viruses.
 In this method, the virus is inoculated into an unvaccinated
healthy living organism. The virus itself finds the most
sustainable part of the body and there it infects the live
tissues.
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The host organism cops the viral
invasion with its immunity. If the
viral load is less than the virus
fails to replicate but if the viral
concentration is good enough to
defeat the host immunity, then
virus replicates and multiplies

Conditions For Cultivation
 The host organism must be approved for testing by the health
authorities.(usually rodents, mice, rabbits and non-human
primates)
 The animal must not have any pre-existing disease.
 Only those viruses should be cultivated via live animals
whose cultivation is impossible by the other two methods.
 The suspect animal must be isolated in lab for observation.
 All the ethical guidelines determined by the institution must be
obeyed.
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Inoculation
 The suspect host organism is taken and the virus is
inoculated through any of the four permitted administration
routes:
1. Intracerebral route
2. Intranasal route
3. Intraperitoneal route
4. Sub-cutaneous route
The selection of route varies with virus.
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Development Of Symptoms
 After inoculation, if the virus succeeds to infect the tissues,
then the host develops some , lesions, physiological
abnormalities, diseases or medical conditions that are
observable. These signs and symptoms help in detecting the
location and activity of virus inside the host. Sometimes the
host may die.
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Final Gain
 At the end, the viruses may be isolated by:
1. Picking out a whole infected tissue from the host, so it may be used
for cell culture.
2. Dissecting the dead host and preserving the obtained virus.
3. In case of immunity win over virus, the antibodies may be isolated for
vaccine preparation.
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 Advantages
1. It is a primary (first source)
isolation of viruses.
2. It helps in production of
anti-bodies.
3. It acts as a diagnostic tool
e.g: to distinguish a lethal
dose.
4. It provides for the systemic
study of oncogenesis,
epidemiology, immune
responses , and virology.
 Disadvantages
1. It is a labor-intensive
method. It needs skilled staff
for caretake.
2. It has a strict criteria for
selection and treatment of
hosts.
3. It may be contaminated by
host’s microbiome bacteria.
4. It requires a heavy financial
cost.
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 “Plant Viruses” are complex viruses that rot the living
plants, defect their growth and lead them to death.
 There are a number of plant diseases that are caused by
viruses such as tobacco mosaic disease, wound tumor
virus, potato yellow dwarf virus.
 Pharmacists also contribute to the drugs used for plants,
therefore we have to know about the cultivation of plant
viruses.
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Cultivation Of Plant Viruses

Conditions For Cultivation
 Only a clean laboratory setting, a
trained staff, necessary tools, and an
infected plant or a viral suspension is
required. A hypodermic needle may be
needed or any abrasive.
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Methods Of Cultivation:
 There are two general methods for the cultivation of plant
viruses:
1. Inoculation: In this method a viral suspension is injected into
a healthy plant with the help of a hypodermic needle.
2. Scratching: In this method, an already infected leaf, or a
homogenous part of plant is scratched along the healthy plant
aided by an abrasive e.g: carborandum
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Detection and Isolation
 The viral infection is detected by visible patches, spots, lesions,
abnormal growth, inflammed plant tissue, or rotting of a certain
infected part of a plant.
 When detected, the virus may be isolated by three methods:
1. Crystallization: The infected part of the plant is crushed to obtain
fluid juice, which is then purified by crystallization to separate the
virions. This is much common method.
2. Cell Culture: By culturing the wall-less plant cells called
“protoplasts” and implementing cell culture technique over them.
In some cases, culturing the monolayer susceptible cells of those
insects which carry the virus as a vector and feed on plant.
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Facts
 William C. Stanley got a Nobel Prize in 1946 for introducing the crystallization method for the
isolation of plant viruses.
 Animal Cell Culture was first used in 1907 by Ross Harrison.
 The first virus discovered was a plant virus “Tobacco Mosaic Virus” (TMV)
 Out of three cultivation methods for animal viruses, only tissue culture is “in vitro”(in
glass/visible) technique. The rest two are “in vivo”.
 Hepatitis B virus can only be cultivated by animal inoculation method.
 Coliphages are isolated by centrifuging and filtering the sewage waste or human stool in
laboratory.
 The animal inoculation method was first performed on human volunteers in 1900 by Walter
Reed.
 The embryonated egg method was introduced in 1931 by Ernest William Goodpasture.
 The cultivation of COVID-19 is widely undergoing by embryonated egg method, just like other
Flu viruses.
 It is possible to cultivate drastically lethal viruses in laboratory. It is a certain theory that they
may be used as “biological weapons”.
 Stefen Lanka, a Ph.D microbiologist and virologist says:
“Viruses do not exist. All the images you see of viruses are either graphics or CG”.
 Currently a US Senate Election candidate , Dr V.A Shiva Ayyudarai , who is the inventor of
email and has doctoral works as a biologist and technologist, stated three times in public that
:
“Viruses do not exist”.
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References
 Pelczar, M.J., Chan, E.C.S. and Krieg, N.R.
(1993). Microbiology : Concepts and Applications. New
York: Mcgraw-Hill.pp. 436-444.
 Denyer, S.P. and Hugo, W.B. (2011). Hugo and
Russell’s Pharmaceutical Microbiology. Chichester,
West Sussex, Uk ; Hoboken, Nj: Wiley-Blackwell. pp.
70-72.
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https://microbiologyinfo.com/techniques-of-virus-cultivation/
https://courses.lumenlearning.com/boundless-
microbiology/chapter/culturing-viruses/
https://www.slideshare.net/utmang/plant-viruses
https://www.slideshare.net/islamsarakbi/cultivation-of-viruses
https://www.slideshare.net/RaNaMB1/cultivation-of-viruses-
97803294
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2892770/
https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microb
iology_(Boundless)/9%3A_Viruses/9.4%3A_Culturing_Viruses/9.4C
%3A_Inoculation_of_Live_Animals
http://peripheralmind.blogspot.com/2020/03/viruses-do-not-exist-
virus-hoax-created.html
https://www.goodreads.com/quotes/tag/virus
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