CMB1003/L17 Assay of Animal Viruses

Question 1

Give 2 cell based assays.

Answer 1

Plaque assay
Tissue culture infectious dose (TCID50) assay

Question 2

Give 2 protein based assays.

Answer 2

Haemagglutination assay
Electron microscopy
Immunofluorescence
Enzyme linked immunosorbent assay (ELISA)

Question 3

Give 2 uses of detection and quantification of viable virus particles in the lab.

Answer 3

Diagnosis
Prognosis

Question 4

Give a use of detection and quantification of viable virus particles in research.

Answer 4

Measure amount of virus in stock solution
Tells you effectiveness of a new drug

Question 5

How can viruses be grown in the laboratory? (5)

Answer 5

Grow cells in tissue culture
Infect cells with virus
Incubate infected cells
Observe for effects of viral infection
Harvest cells/medium and quantify virus yield

Question 6

Define multiplicity of infection (moi).

Answer 6

Number of infectious viral particles used to infect 1 cell

Question 7

Why is MOI important? (2)

Answer 7

May want all cells to be infected
Synchronise infections
Some viruses act differently
Testing viability of stocks
Studying cellular responses to infection

Question 8

How is a plaque assay conducted? (8)

Answer 8

Perform serial dilution
Add to cells in appropriate volume
Leave to absorb
Remove inoculum
Add fresh media containing agar
Overlay with medium to keep moist
Wait
Fix and stain

Question 9

What information is required to calculate the concentration of viable virus particles in an undiluted stock solution from PFUs? (3)

Answer 9

Dilution factor
Amount of diluted virus stock added to plate
Number of plaques produced

Question 10

Give 2 limitations of the plaque assay.

Answer 10

Virus must cause visible CPE
Time required can be significant
Sterility must be maintained

Question 11

How is a focus forming assay different from a plaque assay?

Answer 11

No agar overlay

Question 12

After 24 hours, what is performed to complete the focus forming assay? (3)

Answer 12

Fix and immunostain for virus with fluorescence antibody
Counterstain with DNA stain to count cells
Count cells and infected cells using fluorescent microscope

Question 13

Describe an end-point dilution assay.

Answer 13

Sequential dilution of virus stock in a micro-titre plate format

Question 14

What is TCID50?

Answer 14

Virus concentration that kills 50% of cells in a culture system

Question 15

Describe how to perform a TCID50. (4)

Answer 15

Grow cells in 96 well plate
Serial dilution across plate
Leave to infect and kill cells
Score wells as infected (+) or not (-)

Question 16

Give 2 disadvantages to TCID50.

Answer 16

Time consuming
Medium needs to be changed regularly
Prone to drying out
Sterility due to length of incubation

Question 17

What type of quantification does a haemagglutination assay give?

Answer 17

Relative quantification

Question 18

What needs to be conducted following a haemagglutination assay to get absolute qunatification?

Answer 18

Compare results to standard virus suspension

Question 19

What is immunofluorescence used for?

Answer 19

To stain virus antigens on the cell surface or in sections of virus-infected host cells

Question 20

Describe how to perform an immunofluorescence stain. (4)

Answer 20

Add virus-specific antibody Y
Wash cells to remove unabsorbed antibody
Add FITC (fluorescein isothiosyanate) conjugated anti-rabbit IgG antibody
Wash & examine using UV microscope

Question 21

What colour does OPD turn in ELISA?

Answer 21

Amber to detect HRP

Question 22

What colour does TMB turn in ELISA? (2)

Answer 22

Blue when detecting HRP
Yellow with addition of sulfuric or phosphoric acid

Question 23

What colour does ABTS turn in ELISA?

Answer 23

Green when detecting HRP