METHOD USED IN VIROLOGY.pptx.pdf_Adoximenes

Virology involves a variety of methods to study viruses. Some common methods include:

1. **Cell Culture**: Growing viruses in living cells to observe their effects and replication.
2. **Microscopy**: Using techniques like electron microscopy to visualize viruses.
3. **PCR (Polymerase Chain Reaction)**: Amplifying viral DNA or RNA to detect and quantify viruses.
4. **Serology**: Detecting antibodies or antigens in blood to determine if someone has been infected.
5. **Virus Isolation**: Cultivating viruses from clinical samples to study their properties.
6. **Genetic Sequencing**: Determining the genetic material of viruses to understand their structure and function.
7. **Neutralization Assays**: Testing how well antibodies can neutralize viral activity.

These methods help in diagnosing infections, researching viral mechanisms, and developing vaccines and treatments.

1. METHOD USED
IN VIROLOGY

2. VIRUS IDENTIFICATION
▪Cytopathic effects
▪microscopic or macroscopic degenerative changes or
abnormalities in host cells and tissues
▪Serological tests
▪Detect antibodies against viruses in a patient
▪Use antibodies to identify viruses in neutralization tests, viral
hemagglutination, and Western blot
▪Nucleic acids
▪RFLPs
▪PCR

3. CYTOPATHIC
EFFECT
▪Observe on a high power
microscopy
▪Changes in the cell
▪Used to monitor the progress of
infection
▪Other viruses do not have the
obvious cytopathic effect
▪Arenaviridae
▪Paramyxoviridae
▪Retroviridae

4. VIRUS ISOLATION
AND CULTURE

5. CELL CULTURE
▪Used are from continuous cell lines derived
from humans and other animal species.
▪Examples
▪HeLa Cells
▪Taken from Henrietta Lacks, a woman with
cervical carcinoma
▪Important note for the cell medium
▪Maintain osmotic pressure
▪pH
▪High Carbon dioxide (by a special
incubator)

6. KINDS OF CELL CULTURE
▪Cell Monolayer
▪The cells are then suspended in culture medium and placed in plastic flasks or covered
plates. Attached to the plastic to form a monolayer
▪Commonly used cell lines double in number in 24 to 48 h in such media
▪E.g Epithelial cell and Fibroblastic cells
▪Primary culture
▪They have a limited life span, usually no more than 5 to 20 cell divisions.
▪Examples are monkey kidneys, human embryonic amnion and kidneys, human
foreskins and respiratory epithelium, and chicken or mouse embryos.
▪Used for vaccination preparation eg Poliovirus in monkey kidney cells
▪Continuous Cell line
▪a single cell type that can be propagated indefinitely in culture.
▪E.g HeLa Cells

7. CELL CULTURE

8. EMBRYONATED
EGGS
▪At 5 to 14 days after fertilization, a
hole is drilled in the shell and
virus is injected into the site
appropriate for its replication.
▪Virus propagation for influenza
virus
▪For vaccine production

9. LABORATORY
ANIMALS
▪Use of monkeys in the study of
poliomyelitis
▪Development of hepatitis B virus
in chimpanzees

10. ASSAY OF VIRUSES

11. TWO MAIN TYPES OF ASSAY
FOR DETECTING VIRUSES
▪BIOLOGICAL
▪PLAQUE ASSAY
▪END POINT TITRATION
▪Do not measure non infectious particles
▪Viral Titer
▪the concentration of a virus in a sample.
▪PHYSICAL
▪ELECTRON MICROSCOPY
▪IMMUNOLOGICAL METHODS
▪Measures both infectious and non infectious particles

12. BIOLOGICAL

13. PLAQUE ASSAY
▪Modified by Renato Dulbecco (1952)
▪To determine the titer of bacteriophage stocks for use of animal virology
monolayers of
cultured cells
are incubated
with a
preparation of
virus to allow
adsorption to
cells.
After removal of
the inoculum, the
cells are
covered with
nutrient medium
containing a
supplement.
When the
original infected
cells release
new progeny
particles, the gel
restricts the
spread of viruses
to neighboring
uninfected cell.
each infectious
particle
produces a
circular zone of
infected cells, a
plaque

14. PLAQUE ASSAY

15. MODIFIED PLAQUE ASSAY
▪FLUORESCENT FOCUS ASSAY
▪a modification of the plaque Two hits assay, can be done more rapidly and is useful
in determining the titers of viruses that do not form plaques.
▪INFECTIOUS CENTER ASSAY
▪used to determine the fraction of cells in a culture that are infected with a virus.
▪TRANSFORMATION ASSAY
▪useful for determining the titers of some retroviruses that do not form plaques.

16. END POINT DILUTION ASSAY
▪a measure of virus titer before the
development of the plaque assay.
▪the result of the assay can be expressed
in terms of 50% lethal dose (LD 50) per
milliliter or 50% paralytic dose (PD 50 )
per milliliter, end points of death and
paralysis,
▪End point is the dilution of virus that
affects 50% of the test units.

17. PHYSICAL

18. ELECTRON MICROSCOPY
▪Provide detailed views of the smallest bacteria, viruses, internal cellular
structures, and even molecules and large atoms.
▪Ultrastructure
▪Two types:
▪Transmission Electron Microscope
▪Scanning Electron Microscope

19. TRANSMISSION
ELECTRON
MICROSCOPE
▪Generates a beam of electrons that
ultimately produces an image on a
fluorescent screen.
▪Dense Area: Block electrons (Dark
Area)
▪Less Dense: Screen fluoresces more
brightly.
▪Uses Ultramicrotome (there is
sectioning)

20. HEMMAGGLUTINATI
ON
▪Members of the Adenoviridae ,
Orthomyxoviridae , and Paramyxoviridae
▪ Viruses bind on the rbc and can form lattice
formation
▪Example
▪Influenza virus
▪Due to glycoprotein called hemagglutinin

21. SEROLOGICAL
METHODS

22. VIRUS NEUTRALIZATION
▪ Virus is inoculated to animal and produce antibodies
▪Antibodies bind to virus particle and can cause neutralization
▪End point: The highest dilution antibody inhibits the development of cytopathic
effect in cells or virus.

23. HEMMAGGLUTINATION
INHIBITION
▪Antibodies against viral proteins with hemagglutination activity can block the
ability of virus to bind red blood cells
▪it is the method of choice for assaying antibodies to any virus that causes
hemagglutination.
▪Used in the Avian Influenza A (H7N9) in 2013

24. IMMUNOSTAINING
▪Antibodies can be used to visualize viral
proteins in infected cells or tissues.
▪Direct Immunostaining
▪an antibody that recognizes a viral protein is
coupled directly to an indicator such as a
fluorescent dye or an enzyme
▪Indirect Immunostaining
▪a second antibody is coupled to the
indicator
▪Sensitive as compare to Direct
Immunostaining

25. ENZYME IMMUNOASSAY
▪Detection of viral antigens or antiviral
antibodies can be accomplished by
solid-phase methods, in which
antiviral antibody or protein is
adsorbed to a plastic surface.

26. DETECTION OF
VIRAL NUCLEIC
ACID

27. POLYMERASE
CHAIN
REACTION
▪specific oligonucleotides
are used to amplify viral
DNA sequences from
infected cells or clinical
specimens.
▪Cycle
▪Annealing
▪Extension
▪Thermal denaturation

28. DNA MICROARRAYS
▪Th is approach provides a method for studying the gene expression profi le of a
cell in response to virus infection
▪in this method, millions of unique viral DNA sequences fixed to glass or silicon
wafers are incubated with complementary sequences amplified from clinical and
environmental samples.
▪Detected by using fluorescent molecules incorporated into amplified nucleic acids.

29. VIRUS PURIFICATION
•four commonly used methods
•differential centrifugation and density gradient
centrifugation
•precipitation of viruses
•denaturation of contaminants
•enzymatic digestion of cell constituents

30. DIFFERENTIAL
CENTRIFUGATION
• separates based
on size

31. DENSITY GRADIENT
CENTRIFUGATION
involves centrifuging particles (such as virions) or molecules (such
as nucleic acids) in a solution of increasing concentration, and
therefore density.

32. THANK YOU!!!