Viral Identification

Question 1

Direct examination for detecting viruses (4)

Answer 1

1. Electron microscopy (incl immune electron microscopy)
2. Light microscopy
3. Antigen detection (immunofluorescece, ELISA)
4. PCR

Question 2

Types of immune electron microscopy (2)

Answer 2

1. Classical (IEM)
   - sample treated with anti-sera. Viral particles present will agglutinate
2. Solid phase (SPIEM)
   - grid coated with anti-sera. Virus particles present will be absorbed on grid by antibody

Question 3

What is sensitivity of electron microscope

Answer 3

2nm

Question 4

Advantages and disadvantages of antigen detection (immunoflourescence and ELISA)

Answer 4

Advantages:
- Quick results

Disadvantages:

• Reduced sensitivity
• Reduced specificity due to CROSS-REACTIVITY
• Tedious, time consuming

Question 5

Advantages of PCR

Answer 5

Extremely sensitive

Easy to set up

Fast results

Question 6

Disadvantages of PCR

Answer 6

Need specific primers

Contamination

Need to set up quantitative curve

Latent viruses can be difficult to interpret

Question 7

T/F - if the virus is RNA, you cant run a straight PCR

Answer 7

True - need to do RT-PCR

Question 8

Primers for PCR

Answer 8

1. Random
2. Oligo-DT (good for viruses with Poly-A tail)
3. Sequence specific virus

Question 9

Virus isolation can be done in which 3 types of cell culture? What are pros/cons of each

Answer 9

1. Primary cells
   - Pro: very similar to host, viruses like
   - Con: obtaining tissue from animal, cells dont last as long
2. Semi-continuous cells
   - Cells last longer than primary and appear similar to host
3. Continuous: (immortalized cells)
   - Pros: commercially available
   - Cons: dont resemble host as well, not all viruses grow on them

Question 10

Disadvantages of cell culture

Answer 10

1. Takes a long time
2. Sensitivity depends on condition of specimen
3. Susceptible to bacterial contamination
4. Susceptible to toxic substances that may be present on specimen
5. Many viruses wont grow on cell culture

Question 11

What type of eggs are used for viral culture

Answer 11

EMBRYONATED!

Question 12

Why are neonates used for viral cultures

Answer 12

Immune systems are weak

Question 13

How do you use IgG and IgM to differentiate primary and secondary infections

Answer 13

Primary:
IgG: 4 fold increased between acute and convalescent infection.

Presence of IgM

Secondary:
IgG: 1-fold increase
IgM: not present!

Question 14

What do western blots detect

Answer 14

Antigen/antibody

Question 15

What is hemagglutination inhibition

Answer 15

Virus specific antibodies interfering with the capacity of the virus to agglutinate RBCs

Question 16

Which viruses hemagglutinate RBCs?

Answer 16

Pox
Parvo
Toga
Flavi
Orthomyxo
Paramyxo
Corona
Bunya
Rhabdo
Reo
Rota

Question 17

Virus neutralization assay. Do the results mean you are protected?

Answer 17

Identifies and quantifies neutralizing antibodies. You arent necessarily protected - a virus can induce an immune response but that doesnt mean youre protected

Question 18

Pros/cons of serology

Answer 18

Pros:

• Onset of c/s occurs with development of antibodies
• Viruses that produce clnical disease a while after antibodies (retroviruses)

Cons:

• Long period of time required for dx between acute and convalescent sera
• Mild local infections may not produce a detectable immune response
• Cross reactivity (false positives) **
• Immunocompromised patients gie reduced/absent humoral response **
• Blood recipients: false pos due to antibody transfer **
• Cant be used in unculturable viruses

Question 19

What is TCID50

Answer 19

Tissue culture infective dose 50 - measures virulence of virus in-vitro (only within same group)