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Virology > Viral Assays and methods of Identification > Flashcards

Viral Assays and methods of Identification Flashcards

1
Q

What are 8 ways to diagnose viral diseases?

A

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
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2
Q

Name 3 types of viral titration

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

What is a viral plaque? How will it appear?

A

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

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4
Q

How is the titer for a plaque assay preparation calculated?

A

From # of plaques produced + dilution of the sample

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5
Q

What is ID50 and LD50?

A

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

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6
Q

What is TCID50?

A

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

Used for viruses we can’t see plaques for

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7
Q

What are the steps involved in viral isolation and identification?

A
  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
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8
Q

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

A
  1. Films and smears: inclusion bodies
  2. Biopsy or autopsy material for histo examination
  3. Electron microscopy
    • Direct
    • Immunoelectron microscopy
    • Immunogold microscopy
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9
Q

6 ways we can detect viral antigens.

A
  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)
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10
Q

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

A

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

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11
Q

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

A

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
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12
Q

Which ELISA has better sensitivity?

A

Indirect

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13
Q

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

A

Horseradish peroxidase enzyme

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14
Q

List the components of the direct versus indirect immunofluorescence

A

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
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15
Q

What sample type is used for ELISA?

A

serum or blood

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16
Q

What sample type is sent for immunofluorescence or histochemistry?

A

Tissue

17
Q

What are the components of direct versus indirect Radioimmunoassay?

A

Direct:

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

Indirect:

  • I-labeled anti-rabbit IgG
  • Rabbit antivirus Ab
  • Specimin (virus)
  • Antivirus Ab
18
Q

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

A

ELISA

19
Q

What is the basics on how a Radioimmunoprecipitate test works?

A

Virus grows in tissue culture –> labeled with radioactive substance

20
Q

Name the 3 major ways we can detect viral nucleic acid

A
  1. Nucleic acid hybridization
  2. Polymerase chain reaction (PCR; there are different types)
  3. Nucleic acid sequencing
21
Q

What are 4 nucleic acid hybridization tests?

A
  1. Dot-blot
  2. In-situ (inside cell)
  3. Southern blot (DNA viral detection)
  4. Northern blot (RNA viral detection)
22
Q

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

A
  • 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

23
Q

What is the PCR used for?

A

viral DNA or viral RNA amplification

24
Q

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

A

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

25
Q

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

A

Reverse transcriptase

26
Q

What are 3 ways we can isolate a virus?

A
  1. cell culture
  2. animal inoculation
  3. embryonated egg inoculation
27
Q

What are 7 test we use to identify viral isolates?

A
  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
28
Q

What sample type is used to do virus neutralization assays?

A

Serum

29
Q

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

A
  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
  4. highest reciprocal serum dilution resulting in reduction of virus plaque formation or TCID by 50% = virus neutralization antibody titer
30
Q

What is hemagglutination/HAI looking for?

A

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

31
Q

What does the complement fixation test look at?

A

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

32
Q

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

A

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

33
Q

What is the titer in virus neutrilization assay?

A

The last dilution that prevents plaque formation

34
Q

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

A

viral protein

labeled anti- IgG –> binds to particular bands

35
Q

What serology tests diagnose viral disease by detecting exposure?

A
  1. Virus neutralization
  2. Hemagglutination Inhibition
  3. ELISA
36
Q

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

A

Serum

Antibody

37
Q

What are limitations to serology?

A
  1. Detects exposure not when exposure occured
  2. For correlation with disease
  3. Paired sera
  4. IgM
  5. CSF
38
Q

Which of the following viruses are capable of hemagglutination?’

  1. Circoviridae
  2. Parvoviridae
  3. Papillomaviridae
  4. Polyomaviridae
  5. Adenoviridae
A

Adenoviridae

Parvoviridae

Virology (15 decks)

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