Test 1 - Virus Quantitation (6)

Question 1

_____ counts the number of viruses in a specific volume to determine the virus concentration.

______ is the lowest concentration of virus that still infects cell. Also defined as the number of infectious units per mL of the sample.

Answer 1

Virus quantifaction

The Virus Titer

Question 2

What are the two types of viral quantification tests?

Answer 2

1. Biological: depends on a virus particle initiating a successful replication cycle. E.g. plaque assays, pock assays, various endpoint titration methods.
2. Physical: do not depend on any biological activity of the virus particle. e.g. Electron microscopic particle counts, hemagglutination, immunological assays (ELISA), quantitative PCR assays, flow cytometry.

Question 3

What are the ways of directly counting viral particles?

Answer 3

1. By TEM - not ideal (expensive, not good for geometrically unique viruses, high error)
2. Virus counter 2100: Each sample is stained with 2 fluorescent dyes, 1 for nucleic acids and the other for protein. They are analyzed as they go through a laser beam. Measures intact virions through co-localized protein + nucleic acid

Question 4

What are the ways to assess viruses based on antigen concentration?

Answer 4

1. Hemagglutination - viral attachment to RBCs put in serial dilutions. The inverse of the greatest dilution that completely agglutinates the RBCs is the “HA titer” of the virus suspension.
2. ELISA
3. High Performance Liquid Chromatography (HPLC): virus quantification through UV anaysis of fractions generated during HPLC.
4. Single Radial Immunodiffusion (SRID): radial diffusion of viral particles through agarose gel seeded with polyclonal antisera against a viral antigen.

Question 5

How can a virus be quantified based on gene expression?

Answer 5

Quantitative Polymerase Chain Reaction (qPCR)

Uses PCR to amplify viral DNA or RNA to produce high enough concentrations for detection and quantification by fluorescence. Fluorescent probes are used in a conventional PCR system.

Question 6

What are the different traditional/biological assays?

Answer 6

1. Monolayer Plaque Assay
2. Pock Assay
3. Transformation Assay
4. Quantal Assay

Question 7

Describe the principle behind a monolayer plaque assay.

What is a plaque?

Answer 7

Only viruses that cause visible damage of cultured cells can be quantitated using this assay. It is a functional measurement and has no relation to the actual number of viruses.

Plaque = a circular zone of necrotic cells surrouned by viable cells in a monolayer.

Unit = Plaque formning units/mL (PFU)

Question 8

Describe a pock assay.

What is a pock?

Answer 8

A pock assay involves CAM inoculation followed by pock lesion assessment.

pock = necrotic area on chorioallantoic membrane [CAM] of embryonated eggs.

Unit = pock forming units/ml

Question 9

Describe a transformation assay.

Answer 9

This is a quantitative determination of titers of oncogenic viruses. Oncogenic viruses transform cells in culture. Transformed cells lose contact inhibition and become heaped upon one another.

Unit: Focus-forming units/mL

Question 10

Describe a quantal assay. What are some of the measurements that can be determined?

Answer 10

Measures the presence or absence of infection. It is used for certain viruses that do not form plaques or for determining the virulence of a virus in animals or eggs.

Endpoint (virus dilution that affects 50% of the test subjects), Tissue culture infectivity dose₅₀, Lethal dose₅₀, Embryo Lethal Dose₅₀, Paralytic Dose₅₀

Question 11

Define TCID₅₀.

Answer 11

The tissue culture infectious dose which will infect 50% of the cell monolayers challenged with the defined inoculum.

Question 12

What is the multiplicity of infection (moi)?

Answer 12

The average number of virus particles infecting each cell.