virology

Question 1

Ways to visualize virus/bacteria/parasite

Answer 1

light/electron microscope

Question 2

ways to detect protein

Answer 2

ELISA, IFA, IHC

Question 3

Ways to detect DNA or RNA

Answer 3

PCR, NGS

Question 4

electron microscopy advantages/disadvantages

Answer 4

advantages:
* allows visualization of virus
* rapid
* detects viruses that cannot be isolated
* live virus not required

disadvantages:
* expensive
* need high numbers of virus
* specialized training

Question 5

virus isolation

Answer 5

grow virus in cell culture
Advantages
* Sensitive
* Allows for further characterization of the virus
* Allows for identification of unknown viruses

Disadvantages
* No longer widely available
* Slow, time consuming
* Requires specialized training
* Costly to perform

Question 6

antibody-antigen based tests

Answer 6

detect viral protein
ELISA
Immunofluorescence (IFA)
Immunohistochemistry (IHC)

Question 7

Enzyme-linked immunosorbent assay (ELISA)

Answer 7

Antigen based (“antigen capture”)
* Conjugated primary or secondary antibody
* Substrate gives a color reaction
* SNAP tests
* detects viral proteins

Antibody based ELISA
* Dilutions allow for detecting antibody titers
* A second antibody can detect IgM, IgA or IgG

Question 8

Immunofluorescent antibody tests (IFA)

Answer 8

• Cell culture or tissues
• Antibody has a fluorescent tag and is read with a fluorescence microscope
• Requires high viral titer

Question 9

Immunohistochemistry

Answer 9

Uses paraffin-embedded tissues mounted on glass slides
Sections are incubated with specific antibodies tagged with a substrate
Substrate gives a color reaction
Allows detection of the antigen in the lesion or in specific cells

Question 10

antibody based tests advantages/disadvantages

Answer 10

Advantages
* Rapid
* Sensitive
* Easy to use
* Can detect different serotypes

Disadvantages
* Not available for all organisms
* Can be difficult to interpret

Question 11

types of Genomic Detection (DNA or RNA)

Answer 11

PCR
In-situ hybridization
Next generation sequencing

Question 12

Real time PCR

Answer 12

more DNA= more fluorescence sooner (earlier cycle)
actively replicating= more viral DNA/RNA= detected at an earlier threshold cycle

Question 13

In-situ hybridization

Answer 13

Allows for visualization of the virus in the tissue or at the site of infection
Allows for determination of which virus is responsible for pathologic lesions

Question 14

Next Generation Sequencing

Answer 14

Whole genome sequencing
Detection of unknown viruses

Question 15

DNA-based tests advantages/disadvantages

Answer 15

Advantages
* Rapid
* Sensitive
* Specific
* Relatively inexpensive
* Potentially applicable to all viruses

Disadvantages
* Sensitive – detecting DNA does not necessarily indicate causation
* Risk of DNA contamination (PCR, NGS)
* Have to know what you’re looking for (PCR)

Question 16

serology

Answer 16

Measurement of antigen-antibody interactions
Looks for antibodies to determine exposure/disease/immunity
Many different tests available
* ELISA
* Immunodiffusion
* Virus neutralization

Question 17

antibody titers

Answer 17

Definition: The highest dilution (lowest concentration) of serum need to detectably interact with the antigen
* Gives you the relative concentration of antibody

Method: Create a series of standard dilutions of the original serum samples and test against the antigen of interest
* Not all tests produce titers

Question 18

Immunodiffusion

Answer 18

• Antigen is loaded at the center of the gel
• Test serum is loaded on the periphery
• A charge is added and antibodies move towards the antigen and precipitate creating a band

Question 19

Virus Neutralization

Answer 19

Using antibodies in serum to inhibit virus replication
The** titer is the inverse of the highest dilution needed to neutralize the virus**
Highly specific
Slow or expensive because it relies on inhibition of virus growth in cell culture