animal viruses

1. Cultivation of Viruses
By
DR.S.Dhiva
Assistant Professor,
Department of Microbiology,
Sree Narayana College, Alathur, Palakkad, Kerala.678682

2. General Characteristics
• Viruses are too small to be seen with a light Microscope
• viruses are obligate intracellular parasites, require a living host cell
for its replication
• Viruses have few or no enzymes of their own for metabolism
• Viruses takes host metabolic machinery for its multiplication
• It Cannot be cultured out side the living host cell
• Viruses may infect Bacteria, plants and Animals

3. Cultivation of Viruses
• Unlike bacteria, Viruses cant be grown on an artificial nutrient
medium
• Viruses can be grown
• in vivo (within a whole living organism, plant, or animal) or
• in vitro (outside a living organism in cells in an artificial
environment, such as a test tube, cell culture flask, or agar
plate).

4. Bacteriophage
• viruses which infects bacteria are called Bacteriophages
• It can be grown on bacterial lawn (dense layer of bacteria)
• 0.7 % soft agar is used to grow Bacteriophages in a Petri dish .
• soft agar allows the bacteriophages to diffuse easily through the medium. So that lysed bacterial
cells can be observed as a clear zone which is called as plaque.
• Based on the lysis of bacteria in the lawn, many plaques are observed.
• Concentrations of viral suspensions is measured by the number of plagues are called as plaque-
forming units (pfu)
• E.coli, Cornyebacterium sp, etc., can be used as a host for Bacteriophage cultivation

5. Need of virus cultivation
• Virus cultivation is needed for various reasons such as
• 1. Diagnosis of Plant diseases
• 2. for the production of viral vectors
• 3. Diagnosis of viral infections in clinical specimens,
• 4. Vaccine production and
• 5. other research studies.

6. Methods of cultivation
• In the Laboratory, three methods are commonly used for
culturing animal viruses.
• These methods involve:
• Embryonated eggs
• Living Animals
• Cell cultures

8. • Goodpasture in 1931. first discovered that Embryonated chick eggs can be used
for the cultivation of animal viruses
• Inoculation at different sites of the embryonated egg was developed by Burnet
• Fertilized eggs of birds such as (e.g., chicken, turkey) incubated about 6-8 days
after laying is used for inoculation
• Before inoculation the shell surface is first disinfected with iodine and a hole is
drilled in the shell of the embryonated egg
• Through the hole Virus is injected into various target sites of the egg such
Chorioallantoic membrane, amniotic cavity, allantoic membrane and Yolk sac
Inoculation on Embryonated Egg

9. Pock formation
• After inoculation, the drilled hole is sealed with gelatin and the egg is
incubated
• Virus growth begins with the death of the embryo, by embryo cell damage
or by the formation of typical pocks or lesions on the egg membranes
• This method is still used to produce some vaccines
• Persons allergic to egg will not be injected with this vaccine because it may
carry egg proteins during vaccine production and leads to allergic reaction.

10. Animal Inoculation
• Mice, rabbits, hamsters, newborn or suckling rodents are also
used.
• Mouse is most frequently used for viral cultivation and
replication
• Animal inoculation is mainly used for diagnostic purposes.
• Some human viruses cannot be grown in animals or it can be
grown and do not cause disease

11. • After inoculation the animal is
observe for the development of signs
and symptoms of the disease, or it is
killed for the tissue examination for
the development of inclusion bodies
• But due to ethical issues
Experimental animals are rarely used
for cultivation of viruses
• Animals can be inoculated through
Intracerebral, subcutaneous,
intraperitoneal, or nasal instillation
routes

12. Cell culture
•Cell culture has replaced embryonated eggs for the cultivation and
assays of viruses.
•Steinhardt and colleagues in 1913 first used tissue culture for
diagnostic purpose
•Because these cultures are generally rather homogenous collections
of cells and can be propagated and handled much like bacterial
cultures
•They are more convenient to work with than whole animals or
embryonated eggs

13. Cell culture
• Cell culture can be divided into three types;
1. Fragment cultures,
2. Cell cultures and
3. Organ culture

14. Fragment culture/ explant culture
• Maitland in 1928 introduced cut tissues to the nutrient media for cultivation
of viruses
• It is a simplest form of cell culture
• It consists of fragments of tissue or minced tissues suspended in a fluid
medium. Eg. plasma clot cultures
• Here the cell remain viable for several days to allow the sufficient growth of
virus
• Draw back behind this culture is fragments does not multiply.

15. Cell line preparation
• Cell lines are prepared by treating a slice of animal tissues with a proteolytic enzyme
usually trypsin or with the chelating agent versene (EDTA, 171 ethylenediamine tetra-
acetic acid, sequenstrene) that separate the individual cells.
• These cells are suspended in a solution that provides the osmotic pressure, nutrients
and growth factors needed for the cells to grow.
• Normal cells tend to adhere to the glass or plastic container and reproduce to form a
monolayer.
• Viruses infecting such a monolayer cause the cells to deteriorate as they multiply
• This cell deterioration is called cytopathic effect (CPE), it can be detected and counted
as plaques in bacteriophage assay

16. Cell Culture
•Cell culture can be divided into three types:
•Primary cell lines
•Diploid cell lines
•Continuous cell lines
•Primary cell lines:
•Are derived from tissue slices, tend to die out after only a few
generations.

17. Diploid cell lines:
•They are developed from human embryos, can be maintained for about
100 generations and are widely used for culturing viruses that require a
human host.
•Cell lines developed from embryonic human cells are used to culture rabies
virus for a rabies vaccine production called human diploid culture vaccine
•Genetically, diploid cell lines differ from continuous cell lines and appears
as similar to normal cells.
•Rhinoviruses and the cytomegalovirus can be easily cultivated using
Diploid cell lines

19. Continous cell lines:
• It is used Routinely for the cultivation of viruses
• They are cancerous (transformed) cells that can be maintained through an indefinite
number of generations, and they are sometimes called immortal cell lines.
• After years of laboratory cultivation many such cell lines have lost almost all the
original characteristics of the cell such as contact inhibition, etc.,
• However these changes facilitate the viral propagation, so that enough number cell
lines can synthesized for viral cultivation
• Eg. HeLa cell line which was isolated from a human cervical caocinoma, and the
HEp-2 cell derived from a carcinoma of Larnyx

22. Organ Culture
• Small bits of the organs are used for
organ culture
• To preserve its original morphology
and function they are maintained in vitro
for few days.
•Nowadays, organ culture is not used.

23. References
• www.researchgate.com
• www1.mans.edu.eg
• www.biologyreader.com
• www.courses.lumenlearning.com
• www.virusabc.weebly.com
• www.slideshare.net
• Tortora, Funke, Case, Microbiology An introduction, 8th edition, pg 411-414
• Lansing M.Prescott, John P.Harley and Donald A. Klein, Microbiology 4th edition pg.. 337-338

24. Thank you