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VIROLOGY > 9 – How are virus infections diagnosed? > Flashcards

9 – How are virus infections diagnosed? Flashcards

1
Q

How are diagnostic tests for viral disease used?

A
  • Clinical management of diseased animals
  • Health investigations
  • Test and removal programs
  • Exotic diseases
  • Zoonoses
  • Health certificates
  • AI/embryo transfer/blood transfusion
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2
Q

Infection does NOT equal disease. What are the 2 approaches to diagnosing viral diseases?

A
  1. Detect active infection: detect virus
  2. Detect past or subclinical infection: detect exposure or response to exposure
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3
Q

What are some things you need to consider when collecting a sample?

A
  • Alive or dead
  • Suspected pathogen, tropism, pathogenesis
  • Phase of disease
  • Virus detection or exposure
  • *Number of samples required is inversely proportional to predicted prevalence of disease/infection
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4
Q

Live animal sample collection

A
  • Nasal swabs, tracheal aspirates
  • Vesicular fluid, biopsy’s from margin of lesion
  • Feces
  • Clotted or un-clotted blood
  • Samples from unaffected animals
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5
Q

Green top tubes (heparin) for

A
  • Virus isolation from blood
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6
Q

Dead animals sample collection

A
  • Collect as soon as possible
  • Affect organs
  • Gut loops
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7
Q

Considerations for sample collection/transport

A
  • Transport medium
  • Plastic, sealed containers labeled with waterproof ink
  • Ice packs vs. frozen samples
  • *keep samples for virus isolation on ice (avoid freeze-thaw)
    o ENVELOPE viruses more susceptible
  • *keep serum samples cool OR frozen (avoid repeated freeze-thaw)
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8
Q

What should samples be accompanied by?

A
  • Case history and suspected pathogens
  • Treatment, vaccinations, numbers involved
  • List of species
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9
Q

Shipping samples

A
  • Don’t use Canada Post!
  • Use a currier service
  • *address biosecurity concerns (know the rules)
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10
Q

Sensitivity

A
  • Measure of proportion of POSITIVE test results for a viral infection in a population that is TRULY POSITIVE
  • Problem: false positives
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11
Q

Specificity

A
  • Measure or proportion of NEGATIVE test results for a viral infection in a population that is TRULY NEGATIVE
  • Problem: false negatives
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12
Q

What techniques are used to detect viruses?

A
  • Electron microscopy
  • Isolation
  • Quantitation
  • Haemagglutination
  • Capture ELISA
  • Immunological detection
  • In situ hybridization
  • PCR
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13
Q

Electron microscopy

A
  • Fast
  • Family specific (base on morphology)
  • *cheap to do: expensive to offer
  • NOT available as much anymore
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14
Q

Virus isolation in ‘experimental animals’

A
  • Egg inoculation: classical technique in diagnostic virology
  • Still used for some avian and mammalian viruses
  • *based on lesions or death of embryo
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15
Q

Virus isolation in tissue culture: a 2 step process

A
    1. Cultured cells: primary cell cultures and cell lines
    1. Assess CPE (cytopathic effect)
    1. Specific immunostaning
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16
Q

Virus quantitation (plague forming units/PFU)

A
  • Susceptible cell cultures are infected
  • Incubated for 5-7 days
  • Observe the foci of CPE
17
Q

TCID50

A
  • Tissue culture infectious dose
  • *amount where 50% have plaques
18
Q

Haemagglutination (HA)

A
  • NOT dilution of inoculum at which hemagglutination is NO longer present
  • *if agglutinated=don’t fall to bottom of well
19
Q

What are the different types of ELISA’s?

A
  • Direct assay (primary antibody conjugated to enzyme)
  • *Indirect assay: used to measure Ab (primary body detected by species SPECIFIC Ab)
  • Capture assay ‘sandwich’: used to measure antigen (2 antibodies with an antigen)
20
Q

In clinic ‘point of care’ capture ELISA (SNAP tests)

A
  • Ex. Feline leukemia virus (Ag capture and an indirect ELISA for antibody)
  • Ex. canine parvovirus
  • *lateral flow assay
21
Q

What are the 2 methods for immunological detection of viruses?

A
  • Immunofluorescence
  • Immunohistochemistry (seeing the virus in the LESION)
22
Q

In situ hybridization

A
  • Detecting genetic material in the LESION
    o Labeled probe to detect and localized specific RNA or DNA in a tissue or chromosome
23
Q

What is PCR?

A
  • Very sensitive method of detecting DNA
  • *need to do reverse transcriptase-PCR to turn DNA to RNA for RNA viruses
24
Q

What should be the generic features of the amplified sequence?

A
  • Should be UNIQUE to group of agents you are testing for
  • Should be SHARED by all members of the group of agents you are testing for
25
Q

Multiplex PCR

A
  • Multiple primer pairs in ‘one tube’
  • Allows detection of multiple pathogens in one reaction
  • *commonly applied by commercial labs providing diagnostic testing in vet med
26
Q

“real time” PCR

A
  • More virus there is=smaller value b/c fewer cycles are need to amplify detectable virus
27
Q

What does the PCR NOT tell you?

A
  • If it is ALIVE or not
28
Q

What is used to detect exposure to viruses?

A
  • *SEROLOGY
  • Agar gel immunodiffusion test (AGID)
  • Virus neutralization
  • Haemagglutination inhibition
  • Indirect ELISA
29
Q

What is an (agar gel immunodiffusion test) AGID test?

A
  • Add antigen to center well:
    o Antigen diffuses out
  • Add test serum and positive control serum to ALTERNATING peripheral wells
  • *observe precipitin lines for positive or negative results
    o Negative=no line
    o Positive=line
  • *Cogins test: equine infectious anemia virus
30
Q

What is virus neutralization test?

A
  • Serially dilated serum
  • Add EQUAL amounts of virus (100 plaque forming units) to each tube
  • Infect cultured cells
    o Wait 5-7 days
    o Look for plaques: figure out the titre
  • *intra- and inter-lab variability: NEVER ASSUME RESULTS AMONG LABS ARE THE SAME (even from day to day)
    o Ex. older cell cultures don’t grow viruses as well
31
Q

What is a titre?

A
  • *Last DILUTION that can prevent plaque formation is titre
32
Q

What factors can lead to variation in titers within a lab and among labs?

A
  • Type of cells used
  • Age or passage level of cells
  • ‘standard’ amount of virus added
  • Isolation or strain of virus used
33
Q

What is Haemagglutination inhibition (HAI) test? How is it performed?

A
  • Hea to inactive complement in serum
  • Adsorb to RBC then remove
  • Dilute
  • Add virus
  • Incubate
  • Add RBCs
  • Incubate
  • *looking for the ‘cut-off’ (titre): measuring last well at which haemagglutination occurs
  • Ex. used for parvo
34
Q

What is indirect ELISA?

A
  • Easier to do and STANDARIZED
  • Faster (done in hours)
  • *results reported in optical density (OD)
    o Based on comparing test results to a standard curve derived from dilutions of positive serum
  • *don’t get a TITRE
    o Just do it with one dilution of serum, NOT MULTIPLE dilutions
35
Q

What are the limitations of serology?

A
  • Detects exposure and NOT WHEN exposure occurred
  • Limited use in vaccinated animals
36
Q

Virus neutralization (VN) vs. ELISA: what do each measure?

A
  • VN: FUNCTIONAL Ab response to external proteins
  • ELISA: depending on antigen preparation, can measure response to INTERNAL PROTEINS
    o Good for indicating exposure, but maybe NOT protective immune response
37
Q

What needs to be done to correlation the serology results with disease?

A
  • Paired sera (
  • IgM: used to approximate timing of infection (occurs first) (Ex. WNV in horses)
  • CSF
  • *COORDINATED EFFORT!

VIROLOGY (12 decks)

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