Lecture 3 - Virus Assays Flashcards
What do particulate virus assays do?
Calculate number of virus particles
What do infectious virus assays do?
Estimate amount of virus based on relative infectivity
What is the difference between quantitative assays and quantal assays?
Quantitative assays provide an actual number at a given dilution (# of plaques)
Quantal assays provide a yes or no response at a given dilution i.e. does the particular dilution of virus cause hemagluttination or not> (hemagluttination)
Why are virus assays performed?
Need to be able to titrate a sample and find out how much virus is in that sample
Name 3 assays based on the particulate nature of viruses and whether they are quantitative or quantal
Electron microscopy - quantitative assay
Hemagluttination - quantal assay
Protein or genome quantitation - quantitative assay
What are the 3 advantages and 2 disadvantages of particulate virus assays?
Advantages: -simple and inexpensive -rapid, high specificity and sensitivity -high-throughput Disadvantages: -no measure of infectivity or virus quality -can be imprecise
What is electron microscopy in the sense of a virus assay
Particulate quantitative virus assay based on the biophysical property that counts the number of virus particles present in a purified sample
Describe what type of assay hemagglutination is, the biological property it is based upon and how the titre is calculated
- Quantal assay (end-point dilution assay with yes or no response) based on the biological property (hemagglutination) of some virus particles
- depends on the ability of some viruses to bind to the surface glycoproteins on red blood cells with their envelope spike protein
- virus particles are multivalent and can bind multiple red blood cells, creating a lattice that prevents RBCs from settling to the bottom of a rounded well
- Titre = 1/highest dilution of virus that gives complete hemagglutination
What 2 factors do infective virus assays depend upon?
Ability of the virus to infect a sample and cause a measurable change
What are 3 measurable changes we can look for in infective assays?
Mortality, morbidity and CPE
Name 3 advantages and 2 disadvantages of infective virus assays
Advantages: -highly sensitive - does not require purified virus - provides additional info Disadvantages - doesn't work for all viruses - can be time consuming
What kind of assay is an endpoint dilution assay?
Quantal assays based on the ability of the virus to infect and cause identifiable changes in the host (cells or whole organisms)
How does an endpoint dilution 50% assay work?
Serial virus dilutions in replicates in multiwell plates scored as “infected” or “uninfected” to get TCID50 units (tissue-culture infective dose
Why does endpoint dilution assay look at 50% instead of 10% or 100%?
Because 50% is the area on the S-curve where the relationship between dose (viruses) and response (CPE, mortality etc.) is linear and 50% avoids the potential of ambiguity for taking measurements in the extremes
What is an advantage and disadvantage an endpoint dilution assay?
Advantage: alternative for viruses that don’t form plaques in tissue culture
Disadvantage: poor precision that depends on the dilution steps
What is a plaque assay?
Based on ability of virus to infect a cell monolayer and cause cytopathic effect (CPE) but can take days-weeks
How does a plaque assay work?
Serial dilutions of virus plated on susceptible cells and agar used to restrict spread of virus from infected cells
Titre calculation and units for plaque assays
Titre (pfu/ml) = plaques x 1/dilution x 1/volume
What does the linear does curve imply?
What is usually the case for this in most viruses?
What are 3 factors that may confound the linear dose curve?
- The linear dose curve implies that 1 virus can create 1 plaque.
- For most viruses, the particle:PFU ratio is >1
- Mutations (quasispecies), defective virions and host cell defenses can affect the particle:PFU ratio
What is a focus forming assay?
An assay that looks for microscopically visible infection foci
How does a focus forming assay work?
Cells are infected with virus that expresses beta-galactosidase, stained with X-gal and then virus infected cells are detected with virus-specific antibodies
