1. METHODS IN VIROLOGY

2. Recap Good or bad, virus is important Neither living nor non-living
Basic virus structure
Virus morphology
Properties of naked and enveloped virus
Chemical composition of virus
Classification of virus

3. Growth & Quantification of Viruses
• Viruses need to be grown in their host cell
– animal, plant, bacteria …..
• Animal viruses – cell culture – cells from
animals grown artificially in the laboratory
– Often in monolayers on glass or plastic
overlaid with suitable liquid media for the cells
to grow
Complex medium – amino acids, vitamins, salts, pH buffers, glucose, antibiotics, pH indicator ..

4. Growing Viruses
Bacteriophages form plaques on a lawn of bacteria

5. Cultivation of Viruses
First started in early 1900’s
Successfully done in 1949 in case of poliovirus.
3 main methods:
Embryonated chicken eggs
Tissue Culture
Animal model

6. Virus Cultivation (Cont)
Embryonated Chicken Method
Most economical and convenient method
Fertile eggs of 5-14 days inoculated through shell aseptically.
Opening sealed with paraffin wax
Incubated at 36oC until the virus grow
Different types of viruses are grown in different tissues of chick embryo
Can be used for vaccine production

7. Embryonated Chicken Method

8. Virus Cultivation (Cont)
Tissue Culture Method
Most common method for propagation of virus
Convenient
Economical for maintenance
Observable cytopathic effects
Choice of cells for their susceptibility to particular viruses

9. Virus Cultivation (Cont)
Preparation of tissue culture
Dissociate tissue into single cell
Mechanical disruption
Proteolytic enzymes
Cell suspensions are placed into plastic flask or plates
Cells grow in monolayer in presence of chemically defined media, with vitamins, coenzymes, and amino acids.

10. Isolation of Cells .

11. Cell Culture
Some cells will grow indefinitely – permanent cell lines; others will remain alive only for a short period – primary cell lines.

12. Tissue Culture Method- 3 types
Primary culture-Derived from normal tissues of animal or human, have limited life span. E.g. primary human foreskin fibroblasts
Diploid cell strain- Establish from developing embryo, possess diploid karyotype, can divide up to 100 times. E.g. WI-38, human embryonic lung
Continuous cell line- Propagate for indefinite time, derived from tumor tissues or cells treating with tumor virus. E.g. HeLa

13. Cell Line
Primary human fibroblast
NIH3T3
HeLa

14. Cytopathic Effect

15. Purification of Virus Particles
Concentration of Virus Particles by:
-(NH4)2SO4
-PEG
-Ultracentrifugation
-Haemagglutination
Virus particles can be separated by:
-Differential centrifugation
-Column chromatography

16. Measurement of Infectious Virus
Done by determining the titer in a sample.
Plaque assay
Fluorescent Focus assay
Infection center assay
Transformation assay
End point dilution assay

17. Plaque Assay Discovered by Renato Dulbecco, 1952.
Cell monolayer is incubated by virus and covered with nutrient media and agar.
Each infectious virus produce a zone of infected cells, known as plaque.
Plaques can be distinguished from other cells as the cells in plaque are lysed.
No. of plaques in a given dilution is proportional to the no. of infectious particles.
Can be done only for cytopathic viruses

18. Plaque Assay

19. Plaque Assay (Cont.)
One -hit- kinetics: when one infectious particle is sufficient to initiate infection. And the curve is linear.
Two- hit- kinetics: two different types of virus particles must infect a single cell to ensure infection. The curve is parabolic.
One-hit-kinetics
Titer of virus, p.f.u./ml
Two-hit kinetics
Time in hr

20. Fluorescent Focus Assay
Useful for measurement of non-cytopathic virus.
An antibody to the virus is added
A fluorescin conjugated secondary Ab is added
A foci of infected cells fluoresce under UV microscope.
Each focus is initiated by a single virus particle.
Titer of virus can be expressed as FFU/ml.

21. Fluorescent Focus Assay

22. Infectious Centre Assay
To determine the fraction of cells in a culture infected with a virus.
Cells from the first monolayer separated before the progeny virus is produced.
Cells are then seeded on another (indicator) susceptible cell line.
No. of plaques on indicator cells measures the no. of cells infected in the original culture.
Can be used to measure the persistently infected cells.

23. Transformation Assay
Useful to measure the titer of oncogenic retroviruses.
Oncogenic virus transform the cells.
The cells lose contact inhibition and heap upon one another.
The transformed cells form distinguishable small piles or foci.
The count/infectivity is expressed as focus forming unit/ml.

24. End Point Dilution Assay
Very old method, used for non-cytopathic viruses to determine the virulence.
Serially diluted virus stock is inoculated in replicate test units.
No. of test unit become infected is determined for each dilution.
The end point of a dilution affection- 50% of the test unit is the ID50/ml.
Virulence or pathogenecity is expressed as LD50/ml or PD50/ml.

25. Measurement of Virus Particles & Their Components
Electron Microscopy
Hemagglutination
Measurements of viral enzymes
Serological methods
Neutralization
Heamagglutination inhibition
Complement fixation
Immunoblotting
EIA
NA detection

26. Particle-to-PFU ratio
Number of virus particles in sample/number of infectious particles
~1 for many bacteriophages
High for many animal viruses
A single particle can initiate infection
High particle-to­ pfu ratio, not all viruses are successful- Damaged particles, mutations, Complexity of infectious cycle

27. A Viral One-Step Growth Curve
Figure 13.10

28. One-Step Growth Curve
Synchronous infection- key to one-­step growth cycle
Multiplicity of infection (MOI)- Number of infectious particles added per cell- Not the number of infectious particles each cell receives
Infection depends on the random collision of virions and cells
The distribution of virus particles per cell- Poisson distribution

29. What’s the chemical formula for poliovirus???
Question?
What’s the chemical formula for poliovirus???