Viruses require living host cells to replicate and are cultivated using various methods. They can be grown in laboratory animals, embryonated eggs, or tissue/cell cultures. Animal cultivation allows studying pathogenesis but is expensive and difficult. Eggs are inexpensive and facilitate growth of viruses like influenza in specific areas. Tissue cultures, especially primary cultures and diploid cell lines, support growth of many viruses and are widely used. Isolated viruses are identified using techniques observing cytopathic effects, hemagglutination, interference, or immunofluorescence. Cell cultures are now the primary method for isolating and identifying viruses.
2. • Cultivation of virus
Virus are considered as the primary infection causing agents in human
Viruses are obligatory intracellular parasite
Host for viruses may be animals ,plants, human, bacteria,fungus, protozoan and
algae.
Hence virologist use any one of the host system for viral culture
Better treatment of infections needs proper diagnosis .Isolation or cultivation of
virus is one of part of diagnosis
3. TECHNIQUES IN CULTIVATING AND
IDENTIFYING ANIMAL VIRUSES
Viruses require living cells as their “ medium”
In vivo-laboratory bred animal and embryonic bird tissue
In vitro-cell or tissue culture method
4. THE PRIMARY PURPOSE OF VIRUS
CULTIVATION
To isolate and identify virus in clinical samples
To do research on viral structure, replication, genetics and effects on host cell
To prepare viruses for vaccine production
5. Viruses are cultured by using three
methods
Inoculation of virus into animal
Embryonated Egg
Tissue culture
6. INOCULATION OF VIRUS INTO ANIMAL
In 1932 used mice for viral cultivation
Here after animals like rabbit , guinea pig ,rat , suckling mice, hamsters ,and
monkeys are used for viral cultivation
Inoculation site may vary depends up on the type of virus and target site of
infection
Eg ; intra cerebral inoculation is performed on mice to cultivate Arboviruses
After inoculation observe morphological and physiological changes of animals
7. ADVANTAGES
All types of animal viruses are cultivated.
Used to understand pathogenesis.
Used to understand the immune response of the animal.
Used to study the efficiency of the vaccines
Used to develop vaccines
Used to study interaction of drugs
8. DISADVANTAGES
Expensive
Difficult to handle
Show biological diversity
May leads to latent infections
Require efficient maintainence
Objection from blue cross members
9.
10. EMBRYONATED EGG
Woodruff and goodpasteur (1931)used fertilized chicken egg for viral cultivation this
is a simpler technique than animal inoculation ,are inexpensive and easily available .
Eggs usually not interfere with virus multiplication due to absence of immune
response.
Suitable cells for the growth of viruses are available in embryo and its membrane
,which may facilitate the growth of virus.
8-11day old chick embryo is used for cultivation.
Incubate for 2-9days after inoculation .
Duration of incubation is also depends on the type of virus and the route
Of inoculation .
11.
12. VIRUSES ARE INOCULATED VARIOUS
SITES OF EMBRYONATED EGG
To prepare the egg for viral cultivation
The cell surface is disinfected with iodine
Make hole with the help of drill
Inoculate viruses in any one of the method mentioned
After inoculation is sealed with gelatin and the egg is incubated
Viruses reproduce only certain parts of the egg
13. TABLE SHOWS THE SITE OF RESPECIFIC
VIRUS FOR CULTIVATION
SITE OF INOCULATION
Chorio allontoic membrane
inoculation (CAM)
Aminotic inoculation
Allontoic inoculation
Yolk sac inoculation
VIRUS
Herpes simplex virus pox, virusrous,
sarcoma
Influenza virusMumps virus
Influenza virusmumpus virus. New
castledisease virus
Herpes simplex virus
14. VIRUS ARE INOCULATED INTO VARIOUS
SITE OF EMBRYONATED EGG
Viral suspension or virus containing tissue is injected into the fluid of the egg .
Virus is injected into the proper location in the egg( virus are able to reproduce
only in certain parts of embryo
Death of the embryo or formation of typical pocks (pock assay)
Haemagglutination or lesion on the membranes of the egg that result from viral
growth
After growth viruses are identified with the help of serological techniques .
Eg ; pox virus produce pock or lesion . Influenza virus produce haemagglutination.
15.
16. TISSUE CULTURE
Cultivation of tissue or organ for the growth of viruses.
Three types of tissue culture
Organ culture
Explant culture
Cell culture
17. TYPES OF TISSUE CULTURE
ORGAN CULTURE
are mainly clone for highly specialized parasite of organ Eg;tracheal ring culture
is clone for isolation of corona virus
EXPLANT CULTURE
fragments of tissue can grown as explant in plasma clot
this method is rarely used cell cuture
CELL CUTURE
it is mostly used for cultivation of viruses.
18. CELL CULTURE
Now a days cell cultures are mainly employed in cultivation /isolation of virus
Growth of cells disassociated from the parent tissue by mechanical process is
called cell culture.
Cell cultures are sole system for virus isolation
Cell culture was first successfully under taken by ROSS WARRION in 1907
ROUX In 1885 for the first time maintained embryonic chick cells in a cell culture
21. PRIMARY CULTURE
Primary cultures are derived from the normal tissues of an animal.
Can be sub cultured only once or twice
These are widely used as the best culture system because they support the
widest range of virus
Eg ; monkey kidey cells
Once the primary is subcultured it becomes knows as cell line
A characterized cell line derived by selection or cloning are called cell strains
22. DIPLOID CELL LINE
After the first subculture the pimary subculture and becomes known as diploid cell
line
Derived from human fatal tissue and can be subcultured 20-50times .eg; human
embroyonic kidney
These diploid cells are the most employed host of choice for the the production of
human vaccine virus
23.
24. Continuous cells
Continuous cell lines are capable of an infinite number of doublings.
Such lines may arise with the mutation of a cell strain or more commonly from the
established cell cutures from malignant tissue .many viruses which are difficult or
impossible to grow have been cultured in continuous lines.
25.
26. CUTURED VIRUS ARE IDENTIFIED BY
Cytopathic effect
Haemadsorption
Interference
Immuno fluorescence test