Viral Assays and methods of Identification

Question 1

What are 8 ways to diagnose viral diseases?

Answer 1

Clinical signs +

1. virus detection
2. Isolation (isolation + immunological detection)
3. Quantitation (plaque assay, TCID50)
4. PCR
5. Hemagglutination (or HAI)
6. ELISA
7. Immunological detection (IH or IF)
8. Electron microscopy

Question 2

Name 3 types of viral titration

Answer 2

1. Plaque assay
2. Pock assay
3. Transformation assay

Question 3

What is a viral plaque? How will it appear?

Answer 3

Each infectious particle gives rise to a localized focus of infected cells
Appear as clear areas – counted with unaided eyes w/o staining

Question 4

How is the titer for a plaque assay preparation calculated?

Answer 4

From # of plaques produced + dilution of the sample

Question 5

What is ID50 and LD50?

Answer 5

ID50 – the infectious dose that will infect 50% of the population exposed
LD50 – the dose that will kill 50% of the population exposed. Only applies to those viruses that can kill their host

Question 6

What is TCID50?

Answer 6

The dose on culture that will infect 50% of culture wells

Used for viruses we can’t see plaques for

Question 7

What are the steps involved in viral isolation and identification?

Answer 7

1. Clinical History
2. Light Microscopic Examination
3. Detection of viral antigens
4. Detection of viral nucleic acid
5. Viral isolation
6. Other immunological assays for viral identification
7. Dx of viral diseases
8. Limitations of serology

Question 8

What type of microscopic examinations can be performed to isolate/identify virus?

Answer 8

1. Films and smears: inclusion bodies
2. Biopsy or autopsy material for histo examination
3. Electron microscopy
   
   • Direct
   • Immunoelectron microscopy
   • Immunogold microscopy

Question 9

6 ways we can detect viral antigens.

Answer 9

1. Enzyme linked Immunosorbent assay
2. Radioimmunoassay (RIA)
3. Immunofluorescence (IFA)
4. Immunoperoxidase staining (ELISA with tissue) = histochemistry
5. Radioimmunoprecipitation (RIP)
6. Western blotting (Immunoblotting)

Question 10

What do we use to see the Ag-Ab reaction in diagnostic tests that look for antigen?

Answer 10

Substrate that will interact with the enzyme attached to the detection antibody

Question 11

What would an indirect ELISA be better at doing than the direct ELISA? What is the difference in components used in each?

Answer 11

Indirect ELISA amplifies the signal seen much better than the direct ELISA.
Direct:
- Substrate
- Enzyme linked antivirus Ab: detection Ab
- Specimen (antigen)
- Antivirus antibody: capture Ab

Indirect:

• Substrate
• Enzyme labeled avidin
• Biotin labeled antibody: detection Ab
• specimen (antigen)
• Antivirus antibody: capture Ab

Question 12

Which ELISA has better sensitivity?

Answer 12

Indirect

Question 13

What enzyme tag is attached to Ab with the immunohistochemistry to combine with a substrate to make color?

Answer 13

Horseradish peroxidase enzyme

Question 14

List the components of the direct versus indirect immunofluorescence

Answer 14

Direct:
- Fluorescein labeled anti-IgG –>binds to viral antigen fixed in cells

Indirect:

• Fluorescein labeled goat anti-rabbit Ab
• Antivirus Ab (rabbit)
• viral antigen fixed in cells

Question 15

What sample type is used for ELISA?

Answer 15

serum or blood

Question 16

What sample type is sent for immunofluorescence or histochemistry?

Answer 16

Tissue

Question 17

What are the components of direct versus indirect Radioimmunoassay?

Answer 17

Direct:

• I labeled antivirus Ab (IgG
• Specimen (antigen)
• Antivirus Ab

Indirect:

• I-labeled anti-rabbit IgG
• Rabbit antivirus Ab
• Specimin (virus)
• Antivirus Ab

Question 18

If there is an enzyme tag in the viral test you are using, what test is this considered?

Answer 18

ELISA

Question 19

What is the basics on how a Radioimmunoprecipitate test works?

Answer 19

Virus grows in tissue culture –> labeled with radioactive substance

Question 20

Name the 3 major ways we can detect viral nucleic acid

Answer 20

1. Nucleic acid hybridization
2. Polymerase chain reaction (PCR; there are different types)
3. Nucleic acid sequencing

Question 21

What are 4 nucleic acid hybridization tests?

Answer 21

1. Dot-blot
2. In-situ (inside cell)
3. Southern blot (DNA viral detection)
4. Northern blot (RNA viral detection)

Question 22

What is used in nucleic acid hybridization tests to visibly show what we are looking for and allow them to work?

Answer 22

• Single stranded DNA or RNA
• Probes with a complementary base sequence to the NA we are looking for.

Needs a single stranded radiolabeled or enzyme labeled NA probe + substrate to make it visible

Question 23

What is the PCR used for?

Answer 23

viral DNA or viral RNA amplification

Question 24

What is the heat resistant polymerase from Thermus aquaticus used in PCR? What is its purpose?

Answer 24

Taq polymerase

Extend primers b/w two fixed points on a DNA molecule. Label two parts on DNA and we can read everything in between

Question 25

What do we have to use with PCR if we want to look at RNA?

Answer 25

Reverse transcriptase

Question 26

What are 3 ways we can isolate a virus?

Answer 26

1. cell culture 2. animal inoculation 3. embryonated egg inoculation

Question 27

What are 7 test we use to identify viral isolates?

Answer 27

1. Virus neutralization 2. Hemagglutination and hemagglutination inhibition 3. Complement fixation 4. Immunodiffusion 5. Restriction endonuclease mapping 6. Radiolabeling of proteins and Immunoprecipitation 7. Western Blot Analysis

Question 28

What sample type is used to do virus neutralization assays?

Answer 28

Serum

Question 29

What are the major steps to virus neutralization and what are we looking for in our results?

Answer 29

1. use 100 PFU or TCID50 of particular virus 2. incubate with serial dilutions of test serum 3. virus serum mixture absorbed onto appropriate cells 4 Plaques from unneutrilized virus population is counted 5. highest reciprocal serum dilution resulting in reduction of virus plaque formation or TCID by 50% = virus neutralization antibody titer

Question 30

What is hemagglutination/HAI looking for?

Answer 30

Hemagglutination: virus HAI: viral Ab --> add virus and no agglutination because Ab are there

Question 31

What does the complement fixation test look at?

Answer 31

Complement added to mixture of Ag-Ab --> fixed No Ag-Ab --> complement remains free Addition of sheep RBC coated with Ab against RBC (Ag-Ab) No lysis --> positive: complement is fixed to the patient's RBC therefore, it can't bind to the sheep RBC Lysis --> negative: complement is free because no fixation to patient's cells

Question 32

What sample type is used for Immunodiffusion? What is a positive result?

Answer 32

patient's serum is used containing Ab and positive control of Ag and negative control are put on gel agar Positive = precipitation line b/w patient's serum well and positive Ag well

Question 33

What is the titer in virus neutrilization assay?

Answer 33

The last dilution that prevents plaque formation

Question 34

What is the western blot looking for? What does it use to detect the item of interest?

Answer 34

viral protein | labeled anti- IgG --> binds to particular bands

Question 35

What serology tests diagnose viral disease by detecting exposure?

Answer 35

1. Virus neutralization 2. Hemagglutination Inhibition 3. ELISA

Question 36

What sample types do serology look at and what are they looking for?

Answer 36

Serum | Antibody

Question 37

What are limitations to serology?

Answer 37

1. Detects exposure not when exposure occured 2. For correlation with disease 3. Paired sera 4. IgM 5. CSF

Question 38

Which of the following viruses are capable of hemagglutination?' 1. Circoviridae 2. Parvoviridae 3. Papillomaviridae 4. Polyomaviridae 5. Adenoviridae

Answer 38

Adenoviridae | Parvoviridae