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METHOD USED IN VIROLOGY.pptx.pdf_Adoximenes
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- 2. VIRUS IDENTIFICATION ▪Cytopathic effects ▪microscopicor 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 ona 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
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- 5. CELL CULTURE ▪ Usedare 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 CELLCULTURE ▪ 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
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- 8. EMBRYONATED EGGS ▪ At 5to 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 ofmonkeys in the study of poliomyelitis ▪ Development of hepatitis B virus in chimpanzees
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- 11. TWO MAIN TYPESOF 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
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- 13. PLAQUE ASSAY ▪ Modifiedby 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
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- 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 DILUTIONASSAY ▪ 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.
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- 18. ELECTRON MICROSCOPY ▪ Providedetailed 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 abeam 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 ofthe Adenoviridae , Orthomyxoviridae , and Paramyxoviridae ▪ Viruses bind on the rbc and can form lattice formation ▪ Example ▪ Influenza virus ▪ Due to glycoprotein called hemagglutinin
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- 22. VIRUS NEUTRALIZATION ▪ Virusis 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 againstviral 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 canbe 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 ▪ Detectionof viral antigens or antiviral antibodies can be accomplished by solid-phase methods, in which antiviral antibody or protein is adsorbed to a plastic surface.
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- 27. POLYMERASE CHAIN REACTION ▪ specific oligonucleotides areused to amplify viral DNA sequences from infected cells or clinical specimens. ▪ Cycle ▪ Annealing ▪ Extension ▪ Thermal denaturation
- 28. DNA MICROARRAYS ▪ This 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 • fourcommonly used methods • differential centrifugation and density gradient centrifugation • precipitation of viruses • denaturation of contaminants • enzymatic digestion of cell constituents
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- 31. DENSITY GRADIENT CENTRIFUGATION involves centrifugingparticles (such as virions) or molecules (such as nucleic acids) in a solution of increasing concentration, and therefore density.
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