chapter 10

Question 1

Diagnosis of viral infections strategies

Answer 1

1. Observation of virus particles by electron microscopy
2. Culturing and propagating viruses
3. Immunoassays
4. Detection of viral nucleic acids

Question 2

1. Diagnostic Electron Microscopy pros

Answer 2

• No prior knowledge of virus necessary
• Identify general virus type
• Identify viruses that are not culturable
• Useful for identification of unknown viral agents in outbreaks
• Has resolution to discriminate between aggregated viral proteins and structured viral particles

Question 3

1. Diagnostic Electron Microscopy cons

Answer 3

• Expensive equipment, skilled scientists
• Need high virus concentrations
• Cannot identify virus specifically

Question 4

Immunoaggregation

Answer 4

• Used to increase the sensitivity of virus detection when low virus concentrations
• Viruses aggregated by binding to antibodies
• Able to identify viruses specifically and bind them to grids

Question 5

Immunolocalization

Answer 5

• Virus specific primary Ab and gold-labeled secondary antibody
• Ability to identify specific viruses/viral proteins within cells

Question 6

2. Culturing and Propagating Viruses

Answer 6

Patient sample applied to a cell culture known to support a specific virus replication

If cytopathic effect (CPE) is observed, then the culture is positive.
CPE = observed characteristic changes in cultured cells

Used to be the gold standard of diagnostic virology

Question 7

Virus propagation can be done in:

Answer 7

Cell cultures
Eg. Insect cells are used to produce flu vaccine for individuals with egg allergies (FluBlok).
Eg. Use of HeLa cell line (first human cell line developed) for Polio vaccines

Embryonated chicken eggs
Eg. Influenza virus

Question 8

2. Culturing and Propagating Viruses pros

Answer 8

Detects multiple viruses (depending on host cell used)
Able to study replication cycle, production quantities, CPE
Able to detect replicating viruses

Question 9

2. Culturing and Propagating Viruses cons

Answer 9

-Can be slow
-Expensive, skilled scientists
Virus needs to be kept infectious during transportation
–Only useful for culturable viruses
-some cultureable viruses require:
—-Specific cell type for infection
—Specific additions to the culture media

Question 10

Quantifying Virus Titers two methods

Answer 10

Plaque assays and Tissue Culture Infectious Dose 50 (TCID50) assays

Question 11

Plaque assays

Answer 11

Virus dilutions applied to cells
Can use a semi-thick gel overlay to localize virus infectious foci to restrict virus spread to neighbouring cells
Plaques (cell clearing) are quantified. Each plaque ~ 1 virus.
Can calculate plaque-forming units (PFUs)

Question 12

2. Tissue Culture Infectious Dose 50 (TCID50) assays

Answer 12

Similar serial dilution procedure
But scoring & calculations based on CPE and a math model (vs. counting plaques) Not as accurate as plaque assays, but useful for viruses that don’t form plaques

Question 13

ELISA

Answer 13

Enzyme-linked immunosorbant assay: common clinical practice
Detects viral antigens in patients or antibodies produced by patients against viral infections
Can be performed in virus infected tissues/cells, fluids, and patient serum

Question 14

ELISA pros

Answer 14

Quantifiable
Quick
Don’t have to culture virus
Sensitive

Question 15

ELISA cons

Answer 15

Need a monoclonal antibody against your antigen (for antigen detection protocols)
Need to have available viral antigen (recombinant or extracted from virus infected cells) for antibody detection protocols

Question 16

Nucleic Acid Detection pros

Answer 16

Specific for virus strain
Multiplex
Detect a number of different viruses at once
Very sensitive
Do not have to culture virus for detection

Question 17

Nucleic Acid Detection cons

Answer 17

Need genomic information (sequences)
Maybe too sensitive
Detect viruses not contributing to infection
No functional information