MICR workshop virology Flashcards
Where are viral antigens detected?
In blood and other body fluids after onset of symptoms of viral illness.
In an acute viral infection what happens to conc and when is it’s peak.
increase in 3-4 days and will decline after the adaptive immune system is activated
In a chronic, persistent viral infection virus what happens to virus?
- not cleared
- remains detectable for life
What are obligate intracellular parasites that require living cells in order to replicate and produce infectious progeny?
Viruses
Process of virus isolation
- Biological samples from infected humans
- Sample placed with living cells in sterile container with medium
- Medium has growth factors and other molecules for growth and support
- Inoculated cells observed for CPE as evidence for replication
- The culture medium (supernatant) harvested when PE visible. Otherwise it will all die
Several general types of CPE,
total destruction,
subtotal destruction,
focal degeneration,
swelling and clumping,
foamy degeneration (vacuolisation),
cell fusion (syncytium), and
inclusion bodies.
What do plaque assays measure?
measure the amount of infectious virus, known as the viral titre, in a sample.
How to perform a plaque assay?
- Virus stock diluted 10-fold
- 0.1ml aliquots inoculated onto cell monolayer
- Incubation
- Virus will attach to cells then a semi-solid medium like agar overlayed onto monolayer
- Overlayer will prevent spread of new infections
- will restrict virus spread so it will only spread to neighbours
- circular zone formed -> plaque
- Virus replicated then matrix removed and cells stains with crystal violet or methylene blue
- plaque counted as plaque forming units
How is plaque used?
quantify the infectiousness of the virus inoculum– as any plaque can be assumed to have been produced by the virus inoculum added.
Do all viruses induce CPE
No
The presence of infectious virus can be identified by labelling the virus with a virus-specific____ which in turn has been labelled with an____ label, or a____ label.
antibody
enzymatic
fluorescent
Process of labeling virus with 2 antibodies
- Virus inoculum added to monolayer
- incubation to allow attachment and enter cells
- overlay with semi-solid medium
- spread limited to neighbours
- Days pass and the overlay removed
- Anti-viral antibodies added
- 2nd antibodies added with enzyme/fluoro label
- enzyme substrate is added
- infectious virus units identified by colour change
Viral particles can be detected using fluorescent markers, including (i) safm or (ii) hsfnasd
specific antibodies labelled with a fluorescent marker
or
highly sensitive fluorescent nucleic acid-specific dyes
highly sensitive fluorescent nucleic acid-specific dyes examples
SYBR Green I, DAPI, and YOPRO-1)
Dye techniques are used in combination with which 2 processes and have significantly improved the detection and quantification of viruses
epifluorescence microscopy or flow cytometry
