Viral Cultivation/Detection Flashcards
How are viruses cultivated in the laboratory?
Viruses need a host system and they can be grown in:
1) Animals
2) Tissue (cell) cultures (preferred method)
3) Embryonated Eggs
Why are animals used for viral research? What are the drawbacks?
1) Understanding pathogenesis (how it causes disease)
2) Understanding host immune response
3) Vaccine Development
4) Antiviral drug development Drawbacks:
* Ethical issues, genetics of different species, expensive
Who developed a new way to culture/study viruses in the 1940’s?
Enders, Wellers, Robbins developed cell (tissue) culture technique
Who are Dulbecco and Eagle?
Developed Dulbeco’s Modified Eagles Media (DMEM) used to culture animal cells
What were the two immediate effects of cell culture development?
1) Development of the polio vaccine
* the first vaccine produced in cell culture (monkey kidney cell lines)
2) Advent of molecular virology:
* the exploitation of cell culture for the cultivation and study of viruses
What key discoveries in molecular biology are the result of studying viruses?
1) Discovery of enhancers
2) Discovery of transcription factors
3) RNA editing processes
4) Cancer Biology
What are cell cultures used for and how do we establish them?
Cell cultures used to propagate viruses
- Different Cell types are used to grow different viruses
- Grown in nutrient rich media (salt, AA, glucose, serum)
- Antibiotics added to prevent bacterial growth
What are the 3 main types of cell culture?
1) Primary cell cultures
- prepared from animal tissues
- have a limited lifespan
2) Continuous cell lines
- single cell type can be propagated indefinitely
- commonly derived from tumor tissue
- HeLa cells from cervical tumor of H. Lacks
3) Suspension cell cultures
- do not need to attach to a substrate to divide
- large number of cells can grow in small volume
What methods do we use to recognize that a viral infection has occurred in cell culture?
- Cytopathic effects (CPE)
* How cells change after infection - Hemadsorption
* Adsorb (bind) erythrocytes
What are Cytopathic effects?
Cytopathic effects (CPEs) are how the infected cell changes after infection -can be observed with inverted light microscopes
1) Rounding/detachment from plastic flask
2) Syncytia/fusion (grouping)
3) Shrinkage
4) Inclusion bodies
5) Cell lysis/death
What are the EHV-1 Plaques we discussed in class?
Equine Herpes Virus-1 cultured cause plaques to form where EHV-1 destroyed the cells it infected Larger the plaque = the more virulent the strain of virus
What are quantitative assays and what are the types we can use?
Used to figure out the numer of infectious particles (virions) present in sample
Types:
- Plaque Assays
- Hemagglutination Assays
How is plaque assay accomplished and read?
Sample is diluted at different orders of magnitude and then cultured to see the numbers of plaques that arise
Plaque Forming Unit (PFU): a single virus particle that gives rise to a plaque
Do all viruses form plaques?
Not all viruses form plaques, but you can do a transformation/focus assay
- Cells form piles called foci
- can count the foci present to see the number of virions present
What are the general approaches for laboratory diagnosis of viral infections?
- Direct detection
- Virus isolation and growth in cell culture
- Serology
What can be used for Direct Detection of Virus?
- Immuno-electron microscopy
- Immunoflourescence
Usually not done in clinical lab
-too expensive
What are some other methods available for virus detection besides Direct Detection?
Nucleic Acid Methods (Rapid Identification)
- PCR (DNA)
- RT-PCR (RNA)
- RNA→DNA→PCR
Used to detect viruses that are not cultivatable
Can be used to manage patients w/ HIV
- Monitor TH count + Viral Load
What viral serology options are available to diagnose viral infections?
Viral Serology detects specific antibodies generated against virus
Different tests:
- Enzyme Linked Immunosorbent Assay (ELISA)
- Western Blot Assay
How does ELISA work?
- Coat the wells
- With antigen when testing antibodies in patient’s serum
- With Antibodies when testing viral antigen in patient’s serum
- Incubate and wash unbound Antibodies/Antigens
- Add Secondary Antibodies that contain enzyme indicator
- Wash and Add substrate
- If substrate binds secondary antibody then color change occurs and positive result
What are some positive controls that can be used when doing an ELISA test for HIV?
- Add known HIV antibody/antigen to test well(s) to ensure binding occurs
- Add known non-HIV antibody/antigen to test well(s) to ensure binding doesn’t occur
How does Western Blot Assay work?
- Separation Gel (SDS-PAGE) to separate Viral Proteins
- Big at the top and Small at the bottom
- Proteins transferred to Blot in Blotting tank
- Make a copy of gel proteins on membrane and immuno-stain the blot (add antibodies)
- Autoradiography and develop bands
- The dark bands show you which proteins present in the sample have antibodies present for them as well
