Stepwise description on how to cultivate viruses and a brief introduction to the ways of viral quantification.

1. UNIVERSITY OF NAIROBI
ISOLATION OF VIRUSES
Prepared by
Kennedy O. Sigodo MLS

2. CONTENTS
 Introduction
 Purpose of virus cultivation
 Virus cultivation systems
 Tissue culture
 Embryonated eggs
 Laboratory animals
 Viral quantification.

3. INTRODUCTION
 Viruses are obligate intracellular organisms
 Must be grown in living cells.
 They can't be grown in culture media or on agar plates alone, they
must have living cells to support their replication.
 The easiest viruses to grow are bacteriophages…..WHY?
 Animal viruses – difficult, due to the properties of the animal host
 Under natural conditions, many viruses are relatively host-specific.
Moreover, they may show a marked predilection for certain tissues of
the host such as nervous tissue, epithelial tissue etc.
 The majority can be adapted to foreign hosts by passage

4. VIRUS CULTIVATION
 Also known as viral propagation or growth.
 Necessary to supply the virus with appropriate cells in
which it can replicate.
 Phages are supplied with bacterial cultures.
 Plant viruses may be supplied with specially cultivated plants
or with cultures of protoplasts (plant cells from which
the cell wall has been removed),
 Animal viruses may be supplied with whole organisms, such as mice,
eggs containing chick embryos, insect larvae or animal cells.
Viruses can be isolated from different specimens.

5. SPECIMENS USED TO ISOLATE VIRUSES
-Blood
specimens
EDTA
Heparin
Serum
-Stool
-Throat swabs
-Naso-
-Stools, rectal swabs
-Urine
-Saliva
-Cerebro-spinal fluid
-Biopsy
Skin (filoviridae)
Organs (fixation
with formaldehyde
10%)

6. 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.

7. VIRUS CULTIVATION SYSTEMS
Tissue culture system
Embryonated eggs system
Whole animal systems
a) Natural host
b) Experimental animals
c) Transgenic animals

8. TISSUE CULTURE SYSTEM
HISTORY OF CELL CULTURE
Cultured cells could only survive for a few days.
 In 1951, cells taken from Henrietta Lacks (cervical cancer
patient)
 Cell line was found to be remarkably durable and prolific.
 George Gey was able to isolate one specific cell, multiply it,
and start a cell line.
 Named the sample HeLa. First human cells grown in the lab
that were immortal.
 The use of the antibiotics, chemically defined medium and
use of trypsin greatly enhanced the cell culture technique.

9. HeLa cells cont….
 1.polio vaccine
 2. Gardisil developed by studying HeLa
cells
 3.MMR vaccine.
 4. Understanding of TB, HIV, HPV-HeLa
cells have been used to understand how
these diseases impact cells
 5. Human chromosome number and
mitosis-scientists used HeLa cells to
determine the exact number of
chromosomes in

10. TISSUE CULTURE SYSTEM cont….
 Use isolated cell from animal that are cultured invitro.
 It is the preferred type of growth medium for viruses.
• Three discoveries greatly enhanced the usefulness of cell cultures for
virologists and scientists
1. The discovery and use of antibiotics made it possible to prevent bacterial and
fungal contamination
2.The discovery of proteolytic enzymes (e.g. trypsin) can free animal cells from
surrounding tissues without injuring freed cells
3.This technique has also become possible by the development of growth media
for animal cells.

11. STEPS IN TISSUE CULTURE TECHNIQUE
 Cultivating animal viruses using tissue culture technique
involves following three main steps:
1. Monolayer preparation
2. Clonal cell line preparation
3. Infection with virus
The first two steps are summarised with the notes on cell
culture in the next slides.

12. 1.Monolayer and clonal cell line preparation
CELL CULTURE
 Cell culture refers to the removal of cells from an animal or plant
and their subsequent growth in a favourable artificial
environment.
 The cells may be removed from the tissue directly and
disaggregated by enzymatic or mechanical means before
cultivation, or they may be derived from a cell line or cell strain
that has already been already established.
 Can be classified under the following cell lines.
i) Primary culture
ii) Diploid cell lines
iii) Continuous cell lines

13. PRIMARY CULTURE
 Primary culture refers to the stage of the culture after the cells are
isolated from the tissue and proliferated under the appropriate
conditions until they occupy all of the available substrate (i.e., reach
confluence) (e.g. Primary monkey kidney, mice fibroblasts)
-Heterogeneous – many cell types
-Technical hassle
-5 to 20 cell divisions
-Normal chromosome number
-Contact inhibition
-need constant source
-Closest to animal

14. Making a primary cell line

15. DIPLOID CELL LINES
 After the first subculture, the primary culture becomes known
as a Diploid cell line or subclone. E.g(human fetal lung)
-Lines-up to 100 cell divisions
-Homogeneous population of a single
type.
-Typically derived from tumors.
Remain diploid
-Further from animal
-Technically less hassle

16. CONTINUOUS CELL LINES
 When a finite cell line undergoes transformation and acquires the ability to
divide indefinitely, it becomes a continuous cell line.
 Become immortal through a process called transformation.
 Can occur spontaneously or can be chemically or virally induced.

-Immortal
-Most homogeneous
-Genetically weird – furthest from animal
-Hassle free
-Suspension or monolayer
-Aneuploid- abnormal in chromosome
morphology and number, Grow rapidly.
(e.g.various types of cancer cells - HeLa
cells, Hep 2 cells, or human amnion cells,
continual monkey kidney cell line, dog kidney
cell line, etc.).

17. Summary.

18. 3.Infection with virus
 The clonal cell lines suspended in suitable media
are infected with any desired virus which
replicates inside the multiplying cells. If the virus
is virulent, they cause lysis of cells and virus
particles are released in the surrounding medium.
 These newly produced virus particles (virions)
infect the adjacent cells. As a result localized
areas of cellular destruction and lysis (called
plaques) often are formed

19. CULTURE CONDITIONS
 Culture conditions vary widely for each cell type.
 The artificial media invariably consist of a substrate or medium that
supplies the essential nutrients (amino acids, carbohydrates,
vitamins,minerals), growth factors, hormones, and gases (O2, CO2).
 It also regulates the physicochemicalenvironment (pH, osmotic
pressure, temperature).
 Most cells are anchoragedependent and must be cultured while
attached to a solid or semi-solid substrate(adherent or monolayer
culture),
 Others can be grown floating in the culture (suspension culture)

20. SUSPENSION ADHERENT

21. Differences between Adherent and
Suspension Cultures
ADHERENT CULTURES SUSPENSION CULTURES
Most cells can be cultured this way Cells which are adapted to suspension
cultures or non-adhesive cultures
Passaging required at certain intervals Passaging is much easier, can dilute
culture to stimulate growth
Allows easy visualisation of cells Harder to view cells
Cells dissociated enzymatically or
mechanically
Not require enzymatic or mechanical
dissociation
Surface area limits growth Cell concentration in medium limits growth
Used for cytology, harvesting products Used for bulk protein production, batch
continuously and also research harvesting and also research applications
applications

22. CELL INCUBATOR

23. Viral detection:
Detection of a growth of a virus is observed by the changes in the
cell culture monolayer - cytopathic effect (CPE).
 CPE are Changes of morphology of cells e.g:
 1. Lysis of the cells 2. Vacuolation,
 3. Formation of syncytia 4. Presence of inclusion bodies
Uninfected Cell Culture Infected Cell Culture with CPE

24. Timecourse of polio infection. Note how cells round up and die

26. CPE cont..

27. Formation of multinucleated cells
syncytium = CPE

28. Syncytium formation induced by Murine leukemia
virus

29. Cont…
 As some viruses do not cause CPE in cell lines,
they can be detected by other techniques.
 Hemadsorption of erythrocytes onto cells
infected with viruses which do not form CPE and
contain hemagglutinin can be used in myxovirus
and paramyxovirus detection.
 Influenza viruses can be released into the culture
medium and then detected by hemagglutination.

30. DISADVANTAGES OF CELL CULTURES
 Long period (up to 4 weeks) required for result.
 Often very poor sensitivity, sensitivity depends on a large extent on
the condition of the specimen.
 Susceptible to bacterial contamination.
 Susceptible to toxic substances which may be present in the
specimen.
 Many viruses will not grow in cell culture e.g. Hepatitis B,
Diarrhoeal viruses, parvovirus, papillomavirus.

31. ADVANTAGES OF CELL CULTURES

32. EMBRYONATED EGGS
INTRODUCTION
 The Embryonated hen’s egg was first used for cultivation of viruses by
Good Pasteur and Burnet (1931).
 Cultivation of viruses in organized tissues like chick embryo necessitates
a different type of approach..
 For all practical purposes they all themselves behave as tissue cultures.
 The process of cultivation of viruses in embryonated eggs depend on the
type of egg which is used.
 The egg used for cultivation must be sterile and the shell should be
intact and healthy.

33. Cont…
 Use embryonated chicken, duck or turkey for
inoculation of viral suspension
 Are used especially for the influenza viruses
isolation.
 7 - 10 days old embryonated eggs are used.
 The egg must be cleaned, the shell
decontaminated with a disinfectant and checked
in ovoscope if it is alive
 Ovoscope is the equipment used for candling.

34. SITES SUITABLE FOR CULTIVATION OF VIRUSES

36. Cont..
TISSUES VIRUSES CULTIVATED
Chorioallantoic
membrane
Herpes,Small pox
virus,Rous Sarcoma virus
Amniotic cavity Mumps virus ,Influenza
Yolk sac Yellow fever,Rabies
Embryo Influenza ,Mumps

37. PROCEDURE
 1. CANDLING

38. 2.DRILL THE HOLE

39. 3.INJECT THE SUSPENSION WITH SYRINGE

40. 4.HOLE SEALED WITH PARAFFIN WAX

41. 5. EGGS INCUBATE AT 36C FOR 2-3 DAYS

42. HARVESTING OF EMBRYOS

43. DETECTION OF VIRAL GROWTH
 The signs of viral growth include:
i) Death of the embryo,
ii) Defects in embryonic development, and
iii) Localized areas of damage in the membranes, resulting in
discrete, opaque spots called pocks (a variant of pox).
iv)The embryonic fluid and tissue can be prepared for
examination with an electron microscope.
v) Some can also be detected by their ability to agglutinate
red blood cells or by their reaction with an antibody of
known specificity that will affix to its corresponding virus, if
it is present.

44. POCK LESIONS ON CAM

45. HARVESTED EMBRYOS

46. ADVANTAGES
 Isolation and cultivation of many avian and few mammalian viruses
 Ideal receptacle for virus to grow
 Sterile & wide range of tissues and fluids
 Cost- much less
 Maintenance-easier
 Less labour
 Readily available

47. DISADVANTAGES

48. WHOLE ANIMALS
- using live animal eg.mice, rats, rabbits, guinea pigs, hamster, chickens, and monkey.
- the animal is exposed to the virus by injection of a viral preparation or specimen into the brain, blood,
muscle, body cavity, skin, or footpads.
- use in example research to study the immune system’s response to viral infections.
- HIV: immunodeficient mice grafted to produce human T cells and human gamma globulin.
- Only system for studying pathogenesis & immune responses
- Used if it’s the only method through which the virus can be isolated.

49. ANIMAL MODEL
 usually a purpose-bred animal
 Mouse model
 Advantages
• in-breed strains reduce genetic variability
• genetics are well understood
• Introduce, mutate or inactivation specific genes thought to control the immune response.

 Disadvantages
• Sometimes not infected-therefore virus has to be adapted or use a closely related surrogate virus
• Does not always cause same disease state
• Mice are not humans

50. TRANSGENIC MOUSE MODEL

51. DETECTION OF VIRAL GROWTH
 The signs of viral growth include
 i)death of the animal
 Ii) defects in animal development.
 The infected animal tissue can be prepared for examination with an electron
microscope

52. VIRAL QUANTIFICATION
 Virus quantification involves counting the number of viruses in a specific
volume to determine the virus concentration.
 It is utilized in both (R&D) in commercial and academic laboratories as well as
production situations where the quantity of virus at various steps is an
important variable
 The methods used include but not limited to:
i) Hemagglutination assay
ii) Plaque assay
iii)TCID₅₀

53. HEMAGGLUTINATION ASSAY
 A direct method to titre virus.
 Based on the ability of some viruses to agglutinate RBCs
 Virus is tittered by making serial two fold dilutions of the virus and
determining the highest dilution of virus that causes agglutination of RBCs.

54. PLAQUE ASSAY
 When cells grow as monolayers, they can be used to
quantify the number of viruses using plaque assay.
- The virus is serially diluted in a liquid medium.
- For each dilution a set amount is added to separate
plate containing monolayer of tissue culture cells
and the viruses in that solution are allowed to
attach to the tissue culture cells.
- After attachment has been allowed to occur, a semi
solid medium is added to restrict the movement of
new viruses produced so that only adjacent cells
will be infected.

55. CONT…
Where virus has infected the tissue culture cells, the infected
cells will die causing the formation of a clear zone amongst
the otherwise intact monolayer of cells
This clear zone is called a plaque and it theoretically
represents an area where one virus has infected a single
tissue culture cell, has multiplied and been released, and has
gone on to infect adjacent cells.
The number of plaque forming units (pfu)/ml can be
calculated based on the dilution of the original viral solution.
The term pfu/ml is used rather than the number of
viruses/ml because it is possible that occasionally more than
one virus infects a single cell.
Often the cells or plaques are stained to help in visualization
of the plaques.

56. SERIAL DILUTION

57. …..

58. PLAQUE ASSAY RESULTS

59. CALCULATION OF PFU/Ml
 Plaques are
enumerated
 Plaque Counts are
averaged over wells
 The average is then
divided by the
dilution times the
volume
(43+40+38)/3
(10-4 x 0.1)
= 3,730,000 pfu/ml
43 4 1 0
40 3 0 0
38 6 2 0
Plaques formed per well

60. TCID₅₀
 TCID50 is the measure of infectious virus titer.
 This endpoint dilution assay quantifies the amount of virus required to kill
50% of infected hosts or to produce a cytopathic effect in 50% of inoculated
tissue culture cells
 This assay may be more common in clinical research applications where the
lethal dose of virus must be determined or if the virus does not form plaques.
 When used in the context of tissue culture, host cells are plated and serial
dilutions of the virus are added. After incubation, the percentage of cell
death (i.e. infected cells) is manually observed and recorded for each virus
dilution, and results are used to mathematically calculate a TCID50 result.

61. TCID₅₀ calculation
 The outcome of a TCID50 determination can be used
 to estimate a virus titre in pfu, or vice versa, using the formula

100
 75
50
1 TCID₅₀ = 0.7 pfu
101 102 103 104 105 106
TCID₅₀

62. Other methods for TCID50 calculation
 Two methods commonly used to calculate TCID50 are:
i) Spearman-Karber
ii) Reed-Muench method

63. REFERENCES
 www.invitrogen.com/cellculturebasics.
 http://web.lfhk.cuni.cz/klinmikrob/en/laboratory_diagnosis.htm
 http://www.microbelibrary.org/component/resource/laboratory-test/2875-cytopathic-effects-of-
viruses-protocols
 http://www.slideshare.net/kimareew/hela-cells
 http://en.wikipedia.org/wiki/Virus_quantification
 Flint, S.J.; Enquist, W.; Racaniello, V.R.; Skalka, A.M. (2009). "Virological Methods". Principles of
Virology
 John Carter; Venetia Saunders.(2007) Virology principles and applications

64. sigodo©