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Virology > Diagnosis of Viral Infections L4 > Flashcards

Diagnosis of Viral Infections L4 Flashcards

1
Q

When would laboratory conformation be needed with viral diseases?

A
  1. For specific diagnosis of zoonotic disease.
  2. When a specific diagnosis is required to optimise clinical management (diarrhoea in a large group)
  3. When it is necessary to certify an animal free of particular infections (export)
  4. To prevent transmission of pathogens (AI, embryo transfer, blood transfusion)
  5. To facilitate disease eradication programs.
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2
Q

What are non-specific diagnostic tests?

A

Haematology/biochemistry/cytology, post-mortem.

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3
Q

What information do non-specific diagnostic tests give you?

A

Components/ signs of the pathogen. Multiple pieces to the puzzle but not one direct answer.

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4
Q

What information does specific diagnostic tests give you?

A

Detection of specific pathogen or detection of specific antibodies.

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5
Q

What does the presence of inclusion bodies suggest?

A

Viral infection

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6
Q

What are the two types of inclusion bodies?

A

Intracytoplasmic (e.g. Rabies virus)
Intranuclear (e.g. Herpesviruses)
Both (e.g. paramyxoviruses)

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7
Q

What are inclusion bodies?

A

Viral structural components within the cell. Can represent packed virus particles in an almost crystalline array.

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8
Q

What are 3 diagnostic test approaches?

A
  1. Detection of whole pathogen.
  2. Detection of pathogen’s components.
  3. Detection of specific antibodies.
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9
Q

Does the virus have to be alive to isolate it?

A

Yes- temperature, time, media and containment are important.

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10
Q

How many passages might it take for the virus to show cytopathic effects (CPE)?

A

Three passages (3 weeks)

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11
Q

Do all viruses produce a cytopathic effect?

A

No- can be difficult to recognize viral growth when isolating it.

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12
Q

For virus isolation, the sample must be maintained ___.

A

Cold - NOT frozen.

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13
Q

For successful virus isolation, samples need to be…

A

Collected at the early stages of disease.
Collected into a viral transport medium.
Transported to the lab ASAO.
Maintained cold.
Processed ASAP or stored at -80C.

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14
Q

What does electron microscopy allow?

A

Direct visualization of viruses. (Very expensive)

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15
Q

True or False:

Electron microscopy requires a lot of skill - but can give rapid results.

A

True

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16
Q

Electron microscopy is/is not very sensitive?

A

IS NOT

Need high concentration of viruses in sample.

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17
Q

Can electron microscopy differentiate between members of the same virus family?

A

Nope.

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18
Q

What are 2 ways of detecting pathogen’s nucleic acids?

A
  1. PCR (traditional/real-time)

2. In-situ hybridisation

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19
Q

What is PCR?

A

Polymerase chain reaction - repeated cycles of heating and cooling to make many copies of DNA.

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20
Q

What region of an antibody binds specific antigens?

A

The Fab Region at the antigen binding site.

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21
Q

What does it mean that an antibody is conjugated?

A

The Fc region can be linked/conjugated to an enzyme or a fluorescent molecule. If you add substrate and the enzyme is present, it will bind causing a change - typically colour change.

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22
Q

What is an antigen?

A

A portion of a virus.

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23
Q

What does an antibody do?

A

Specifically binds foreign antigens.

24
Q

Does conjugation of antibodes and enzymes occur naturally?

A

No- it is made artificially in the lab.

25
Q

How does the colour change of an enzyme attached to an antibody work in in situ hybridization?

A
  1. Piece of tissue on a slide with a virus.
  2. Add RNA (reagent)
  3. DNA probe binds to the tissue- complimentary to virus sequence.
  4. Add antibody- recognizes the antigen on the virus and binds.
  5. Add substrate- colour change due to enzyme conjugated to antibody
26
Q

What happens if there is no virus present in the tissue sample of an in-situ hybridization test?

A
  1. Add RNA (reagent)
  2. Doesn’t bind because no complimentary DNA sequence.
  3. Add antibody - nothing to bind to.
  4. Wash slide
  5. Add substrate - no colour change because antibody with enzyme was washed away due to absence of viral antigen.
27
Q

What is the difference between in situ hybridization and immunohistochemistry?

A

In situ hybridization uses DNA probes.

Immunohistochemistry uses labelled antibodies.

28
Q

What does the antibody bind to in a immunohistochemistry test?

A

Viral proteins embedded in the cell surface.

29
Q

What is a labelled antibody?

A

It has an enzyme attached to it that can allow a change that we can see to conclude that the virus is present.

30
Q

True or False:
You can have a primary antibody that isn’t labelled and add a secondary antibody that binds to the primary antibody that is labelled.

A

True

31
Q

Instead of an enzyme, there is a fluorescent molecule attached to the antibody. What is this diagnostic test called?

A

Immunofluorescence

32
Q

Immunohistochemistry and immunofluorescence are the same principal, they’re just _____ to different molecules.

A

Conjugated

33
Q

What microscope do you have to use to gather the results for immunofluorescence and immunohistochemistry?

A

Immunofluorescence- fluorescence microscope as red light.

Immunohistochemistry- normal microscope

34
Q

What does the ELISA test look for?

A

Viral proteins.

35
Q

A well is coated with capturing antibodies - specifically recognizes one virus. The clinical sample is added to the well and then it is washed. If the virus is present it will bind to the capturing antibody, if there is no antibody then everything will be washed away. A labelled antibody is then added. If the virus is present it will bind, if not it will be washed away. Finally, a substrate is added that binds to the labelled antibody if it is present results in a colour change. What is this test called?

A

Antigen Detection ELISA test

36
Q

What test involves a peatry dish with holes cut into the agarose.

A

Agar gel immunodiffusion.

37
Q

What are the holes in the agar gel immunodiffusion filled with?

A 
Known antiserum (antibody) - in the centre. 
Control antigen (virus) - in 3 
Test samples we are looking for antigen - 3 (alternating with control)

OR

Also used to detect viral antibodies:
Known antigen (virus)- in the centre.
Control antibody - in 3 holes (every other)
Test samples - in 3 (every other hole)

38
Q

How does agar gel immunodiffusion work?

A

The virus migrates through the agarose. At some point the virus/antigens and the antibodies meet. They then bind together and get stuck because they’re too big. This generates a white line between the antibody hole and the virus hole in the agar.

39
Q

How do you know if the agar gel immunodiffusion is positive?

A

You have to look to see if there a continuous line between the control and the antibody/virus and antibody. If they’re the same then the sample is positive. If the line is different or absent then the sample is negative.

40
Q

True or False?

A single serum sample positive for antibody to a virus tells you nothing about the timing of infection.

A

TRUE

41
Q

What does a positive titre test tell you?

A

That the animal has been exposed at some point in its life - could be infection or vaccine and the timing is unknown from the titre.

42
Q

If you want to link detection of antibody to timing what do you have to look for?

A

At least a four fold rise in tire between acute and convalescent samples - this indicates recent infection. Test 2-4 weeks after first sample.

43
Q

When are single serum samples useful for detection of anti-viral antibodies?

A
  1. Detection of IgM- this antibody is only generated very early in infections.
  2. Persistent infections (FIV)- once the animal if infected, they’re infected for life. Still doesn’t tell you the timing.
  3. Detection of antibodies for prevalence studies.
  4. Monitoring for absence of specific diseases (export testing)
44
Q

The plate is covered with the antigen (virus) and the clinical sample is added. If antibodies are present, will bind to virus and it stays when the plate is washed. A secondary labelled antibody that is directed against the primary antibody is added and binds to the primary antibody if still present (bound to virus). Substrate is added > colour change. What test is this?

A

Antibody direct ELISA test.

45
Q

True or False:

Antibody direct ELISA tests are species specific.

A

TRUE

46
Q

The plate is covered with the antigen (virus) and the clinical sample is added along with a reagent (labelled antibody) that recognizes the virus. The reagent competes with the antibody in the sample. If negative - the reagent can bind to the virus and when substrate is added there is a colour change. What is this test called?

A

Antibody blocking/competition ELISA.

47
Q

If an antibody blocking/competition ELISA test DOES have a colour change then the test is positive or negative?

A

NEGATIVE

48
Q

If an antibody/competition ELISA test DOESN’T have a colour change then the test is positive or negative?

A

POSITIVE

49
Q

If an antibody direct ELISA test DOES have a colour change then the test is positive or negative?

A

POSITIVE

50
Q

If an antibody direct ELISA test DOESN’T have a colour change then the test is positive or negative?

A

NEGATIVE

51
Q

Can antibody blocking ELISA test be used for similar viruses across species?

A

Yes - for example canine corona virus

52
Q

Can antibody direct ELISA test be used for similar viruses across species?

A

NO

53
Q

A reciprocal of the highest dilution of the serum which completely inhibits a virus-specific effect is a…?

A

Serum Titre

54
Q

If a serum dilution is 1/16, what is the titre?

A

16

55
Q

What two tests involve mixing serum from the animal with the virus and you look to see if this has inhibited the specific virus effects?

A
  1. Virus neutralisation (VNT) - production of CPE in cell culture.
  2. Hemagglutination inhibition (HI)- hemagglutination of RBCs by the virus.
56
Q

What is an acute vs convalescence serum test?

A

Acute is the initial serum blood test.

Convalescence is the second serum blood sample.

Virology (4 decks)

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