Assay of Human Viruses Flashcards
What are cell based and protein based assays?
Cell based:
- plaque assay
- tissue culture infectious dose (TCID50) assay
Protein based:
- haemagglutination
- electron microscopy
- immunofluorescence
- enzyme linked immunosorbent assay (ELISA)
How do we grow viruses?
They obligate intracellular spaces. To grow them in the lab, we grow cells in a tissue culture, then infect the cells with the virions. They get incubated and observed for cytopathic effect (viral infection).
What is multiplicity of infection (moi)?
This is the number of infectious viral particles used to infect 1 cell. so, an moi of 10 means 10 infectious viral particles per cell. An moi of 0.1 means 1 infectious viral particle per 10 cells - only 10% cells will be initially infected.
However, infecting a cell culture with an moi of 1, doesn’t mean all cells are infected with a single infectious particle. This is due to Poisson distribution which is the chance change of being hit by 0 or more viral particles, therefore it assumes normal distribution.
Increasing the moi increases the LIKELIHOOD of cells being hit by, multiple, not neccessarily every cell being hit. There is always a small number of unaffected cells.
Why is MOI important?
We may want all cells in the culture to be infected. Some viruses act differently upon different moi’s, for example; HSV - high m.o.i causes a lytic infection, a low m.o.i causes latency.
What is a plaque assay?
This detects infectious viral particles. A serial dilution of the viral culture is performed, and cells are added at an appropriate volume. It is left to absorb, the cells are then washed (removing the inoculum), fresh media containing agar is added. A medium is overlayed to keep the cells moist, then cells are fixed and stained.
What is a focus forming assay?
It is a variation of plaque assay where there is no agar overlay. After a day, an immunostain is fixed for the virus with its fluorescent antibody.
It is a counter stain with a DNA stain to count the cells, and it counts cells vs infected cells with a fluorescent microscope. FFU - focus forming unit.
What is an end-point dilution assay?
It is a sequential dilution of a virus stock in a 96 well plate format. This allows multiple replicants of each dilution.
How is TCID50 used by end-point assay?
It represents the virus concentration that kills 50% of the cells in a culture system.
What is tissue culture infectious dose (TCID50)?
Essentially, each TCID50 unit can infect 1 out of 2 cells. So, for example, 100 TCID50 units will infect 50 samples of 100 samples.
What is electron microscopy?
It helps to identify individual viral particles (not a measure of infectivity). It can be compared to the PFU.
It helps get a measure of particle infectivity ratio, as for many animal viruses, the majority of viral particles are non-infective.
You can get transmission electron microscopy and scanning electron microscopy.
What is a haemagglutination assay?
This is the clumping together of RBCs when infectious virus is present. Giving a measurement of viable and non-viable viral particles.
It gives relative quantification - you can compare 2 viral suspensions. To get absolute quantification, results need to be compared to a standard virus suspension containing a number of known viral particles per ml.
They are viewed in wells, a small dot from overhead and side view means no viral particles. When the view from the side is a v shape and from the top looks ‘full’ or RBCs, virus is present.
What is an immunofluorescence assay?
Stains virus antigens on cell surface OR sections of virus-infected host cells.
Antibodies are added to infected cells as well as FITC (fluorescein isothiocyanate) which is a conjugated anti-rabbit IgG antibody.
Wash to remove unabsorbed antibody (leaving just antibodies that are bound to viral cells), and examine cells with a UV microscope.
What is an enzyme linked immunofluorescence assay (ELISA)?
An antigen (virus) is bound to an antibody with a reporter enzyme (e.g. HAP or AP) bound to it. We add in substrate, and if the antibody binds the antigen, a colour change will present.
An moi of 1.0, due to Poisson distribution means:
- 37% cells get one viral particle
- 37% cells get 0 viral particles
- 26% will get 1< particles
How is are the results of a plaque assay measured?
Each cleared area = 1 plaque forming unit (PFU) which is representative of one infectious virion.
By using the dilution factor, volume viral stock added to the plate, and number of plaques produced, you can calculate viable virus particles in the undiluted stock solution.
