Cultivation of bacterial, plant and animal virus

1. Viral cultivation methods
Dr. Dhanya KC
Assistant Professor
Department of Microbiology
St. Mary’s College, Thrissur-680020
Kerala

2. Viruses - Obligate intracellular parasites
Cultivated within suitable hosts - a living cell
Virus cultivation is - to get sufficient amount of virus particles
• For studying virus - host interactions and diseases
• For studying the effectiveness of antiviral drugs
• For use as gene vectors in gene therapy
• For Viral pesticide production
• For vaccine production
Prescott−Harley−Klein: Microbiology, Fifth Edition

3. Viruses are host dependent
For cultivation
• Bacterial viruses - Bacterial culture
• Plant viruses – Plant tissue culture, whole plants
• Animal viruses - Embryonated eggs, tissue culture or
laboratory animals

4. Cultivation of Bacterial viruses
In agar culture -
Phage, bacteria, and nutrient media
mixed, plated and incubated
Bacteria multiply, replication of virus
lyses the bacteria
Form plaques, or clear zones
Each plaque develops from single
viral particle
Plaque assay for phage quantitation
Cultivated - Broth culture or Agar culture of Bacteria
In broth culture -
Phage added to broth culture of bacteria
Destruction of host cells due to viral multiplication
Turbid bacterial cultures clear rapidly
Prescott−Harley−Klein: Microbiology, Fifth Edition

5. Cultivation of Plant Viruses
For cultivation
Whole plants/ Plant parts
Plant tissue cultures - cells or protoplasts
Whole plants/ Plant parts
Inoculation - Leaves rubbed with virus + abrasive (Carborundum)
Cell wall breaks – virus reach plasma membrane and infect cell
Results in
Localized necrotic lesion due to the rapid death of cells
Infected plant show change in pigmentation or leaf shape
Some plant viruses transmitted only if a diseased part is grafted
onto a healthy plant
Plant tissue culture
Suspension culture or callous culture – inoculated with virus

6. Prescott−Harley−Klein: Microbiology, Fifth Edition
Growth of Tobacco Mosaic Virus on Nicotiana Plant leaves

7. Cultivation of animal viruses
For cultivation of animal viruses
i) Animals
ii) Embryonated Eggs
iii) Cell Culture
The system - free from bacteriological contamination
Achieved by
Specific Pathogen Free animals
By treatment with antibiotics e.g. Penicillin, streptomycin, etc
Passing through membrane filters (0.2 µm)
Viral replication destroys the infected living cells
Result in disease lesions or other abnormalities in the tissues

8. I) Inoculation into animals
Inoculation into human volunteers - Reed and his colleagues (1900)
- human volunteers - work on yellow fever
Monkeys - isolation of Poliovirus - Handsteiner and Popper in 1909
Currently Mice are most widely used
Infant mice susceptible to Coxsackie’s and arboviruses
Mice - inoculated through several routes
intracerebral, subcutaneous, intraperitonial, intranasal, etc.
Other animals – guinea, rabbits, ferrets, chicken
Should be germ-free - SPF (Specific Pathogen Free)

9. Experimentally inoculated/infected animals examined for
1. Clinical signs of disease, respiratory distress, CNS involvement,
and visible lesions on skin and membranes
2. Abnormal behavior of the animal
3. Blood samples for antibodies against viral antigen
4. Death of the animal
Biopsy material or tissue specimens should be examined for;
1. Microscopically for lesions (Cytopathic effects)
2. Histopathologically for pathological changes
3. Serologically for presence of specific viral antigens
4. By electron microscope, for identification of viral particles
Animal inoculation - disadvantage –
Immunity interfere with viral growth
Animals often harbor latent viruses
Difficult to handle and maintain

10. II) Embryonated eggs
First used by Good Pasteur and Burnet (1931)
Embryonated egg does not support all animal viruses but most of
the avian viruses grow
The eggs should be free from microbes
The major advantages are
1. Easily available, economical and convenient to handle
2. Relatively free from bacterial and many latent viral infections
3. Free from immune mechanisms

11. To inoculate the egg
Shell surface is disinfected and penetrated with a sterile drill.
After inoculation the drill hole is sealed with gelatin and the egg incubated
Developing chick embryo, 10 to 14 days after fertilization, provides differentiated
tissues, including
Amnion, Allantois, Chorion, Yolk sac, etc.
Serve as substrates for growth of a wide variety of viruses,
Orthomyxo viruses, Paramyxo viruses, Rhabdo viruses, Toga viruses, Herpes
viruses, Pox viruses, etc.

12. 1) Chorioallantoic membrane (CAM):
For poxvirus and Herpes simplex virus
Visible lesions in transparent CAM - Pocks - from a single virus
Pock assay – for counting pock forming virus such as vaccinia
2) Allantoic cavity:
For influenza and some paramyxoviruses
3) Amniotic cavity:
For influenza virus and mumps virus
4) Yolk sac:
For Herpes Simplex virus – also for some bacteria like Chlamydiae
and Rickettsiae
https://www.researchgate.net/figure/Poc
k-lesions-on-CAM-inoculated-with-skin-
scab-suspension_fig2_274063624

13. The presence of viral growth in embryonated egg
1. Death of the embryo (Toga virus)
2. Deformities such as dwarf growth (Infectious bronchitis
virus)
3. Hemorrhages (New castle Disease virus)
4. Oedema and pock lesions on CAM (Cow pox, Herpes B-virus)
5. Intracytoplasmic inclusion bodies (Herpes virus)

14. III) Tissue culture:
Most widely used method for cultivation of viruses
• For primary isolation of viruses
• For infectivity assays
• For biochemical studies
• For production of viral vaccines
Advantages
1. Growth can be detected easily
2. Viruses can be grown in bulk
3. Cells can be stored for longer period of time
Disadvantages are
1. Requirement of good laboratory facility
2. More costly compared to the embryonated eggs
3. Chances for presence of latent viruses in the cultured cells

15. There are three types of tissue cultures
1) Organ culture
• Small bits of organs maintained in vitro
• Useful for the isolation of some viruses having organ specificity
• Tracheal ring organ culture – for corona virus, a respiratory pathogen
2) Explant culture
• Fragments of minced tissues embedded in plasma clots
• Adenoid tissue explant culture - for adenovirus
3) Cell culture
• Most routinely employed method
• For identification and cultivation of viruses

16. 3 Types of cell cultures
On the basis of
Origin of cells
Chromosomal characters of cells
Number of generations it can be maintained
1. Primary cell culture
2. Diploid cell culture
3. Continuous cell culture

17. Primary cell culture
• Normal cells from fresh organs of animals
• Attachment dependent, Contact inhibition
• Attach to vessel surface, mitosis, confluent monolayer of cells
• Capable of limited growth
Eg. Rhesus monkey kidney cell culture, Human amnion cell culture
Diploid cell culture
• Semi continuous cell lines - Derived from primary cell culture
• Possible up to 50 serial subcultures
• Then undergo senescence and the cell strain is lost
• Retain the original diploid chromosome number and karyotype
during serial sub cultivation
• Attachment dependent, Contact inhibition
Eg. Human embryonic lung strain (WI-38), Rhesus embryo cell strain
(HL-8)

18. 3) Continuous cell culture
• Permanent cell lines
• Derived from cancer cells
• Anchorage/attachment independent, no contact inhibition
• Grow as suspension culture
• Grow faster
• Chromosomes are haploid
• Continuous cell lines - maintained either
by serial subculture
by storing in deep freeze at -70°c.
Eg. HeLa cells derived from cervical cancer of a lady named
Henrietta Lacks, Vero (Vervet monkey kidney cell line), BHK (Baby
Hamster kidney cell line)

19. Procedure - primary cell culture
1. Tissue or organs are cut up in small fragments
2. Fragments mixed with Trypsin
Dissolve the connective tissue to separate cells (trypsinization)
3. Cells are cultivated in a suitable growth medium
(essential amino acids, vitamins, salts and glucose and a buffering
system and about 5% calf or fetal calf serum)
4. Antibiotics and phenol red are added
to prevent bacterial contaminants and as indicator
5. Incubation (24-48 hrs in a CO2 incubator at 37oC)
cells attach to the flask and divide to form a monolayer
6. Virus is inoculated and incubated at 37oC
https://biologyreader.com/animal-cell-culture.html

20. Primary and secondary cell lines are anchorage dependent
Surfaces of glass, plastics, natural polymers such as collagen, or other
support materials
In lab scale liquid cultures for anchorage dependent cells in
https://www.thermofisher.com/order/
catalog/product/139446
https://www.fishersci.co.uk/shop/products/bottle-tube-roller-
variable-speed-5-80rpm-digital-display-fisherbrand/15376617
https://www.laboratorynetwork.com/doc/cell
-culture-devices-cellroll-and-cellspin-0001
https://www.thermofisher.com/in/en/ho
me/life-science/cell-culture/cell-culture-
plastics/cell-culture-flasks/t175-
flasks.html
• T- flasks
• Spinner bottles
• Roller bottles
• Trays
T- flasks
Spinner bottles
Roller bottles
Trays

21. Roller bottles
Bottles are rotated with the cells adhered
to its sides
Microcarriers of DEAE or dextran
Cells grow on the surface of the
microcarriers
Monolayers or as multilayers
Hollow fiber reactors
Cells immobilized on external surfaces of
hollow fibres
Nutrients pass through the tubes
Other immobilization based reactors
Tubular ceramic matrix reactors
Microencapsulation
Gel encapsulation
Large scale reactors for anchorage dependent cells
http://www.
cellon.lu/roll
ercell-
40.html
https://link.springer.com/referenceworkentry/10.1007%
2F978-3-662-44324-8_1191
https://chemomete
c.com/counting-
cells-microcarriers/
Roller bottles
Microcarriers
Hollow fiber reactors

22. Cancerous cells are anchorage independent
Continuous cell lines grow as suspension cultures
Stirred tank reactors
Bubble column reactors
Perfusion Bioreactors
https://www.bioprocessonline.com/doc/protoc
ol-scale-up-cho-cells-stirredttank-bioreactors-
0001
https://www.mdpi.com/2227-9717/3/2/384/htm

23. Detection of virus growth in cell cultures
1. Cytopathic effects / cytopathogenic effects (CPE)
2. Metabolic Inhibition
3. Hemadsorption
4. Interference
5. Transformation
6. Immunofluorescence

24. Detection of virus growth in cell cultures
1. Cytopathic effects / cytopathogenic effects (CPE)
Morphological degenerative changes in cultured cells, seen under
microscope - can be used to identify viral infection
Common examples are
• Rounding of the infected cell
• Fusion with adjacent cells to form a syncytia
• Appearance of nuclear or cytoplasmic inclusion bodies
https://www.virology.ws/wp-
content/uploads/2012/03/002_3310_17.pdf
Rounding of cell
Formation of syncytia

25. Intracytoplasmic inclusion bodies
Negri bodies - Rabies virus
Molluscum Bodies - Molluscum contagiosum virus
Guarnieri bodies - Vaccinia virus
Intranuclear inclusion bodies
Acidophilic inclusion bodies - Papova virus
Intranuclear basophilic bodies- Adeno virus
Cowdry type A bodies - Herpes simplex virus, measles virus

26. Detection of virus growth in cell cultures
2. Metabolic Inhibition
No acid production in presence of virus
3. Hemadsorption
Influenza & Parainfluenza viruses, by adding guinea pig
erythrocytes to the culture
4. Interference
Growth of a non cytopathogenic virus can be tested by
inoculating a known cytopathogenic virus
Growth of first virus will inhibit the infection by second
5. Transformation
Oncogenic viruses induce transformation & loss of contact
inhibition - microtumors
6. Immunofluorescence
Test for viral Antigen in cells from viral infected cultures

27. References
• Prescott−Harley−Klein: Microbiology, Fifth Edition, © The
McGraw−Hill Companies, 2002
• http://www.brainkart.com/article/Viral-Pathogenesis-at-the-
Cellular-Level_18363/
• https://microbiologyinfo.com/techniques-of-virus-
cultivation/