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Veterinary Virology DMP851 > Viral Diagnostics: Application for Vet Med > Flashcards

Viral Diagnostics: Application for Vet Med Flashcards

1
Q

Selective Assays for detection of viruses or viral proteins or nucleic acids

A

virus isolation
fluorescent antibody on tissue
electron microscopy
Polymerase Chain Reactions
Antigen Capturing ELISA
Sequencing
Viral micorarray

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2
Q

Veterinary DIagnostics:

What are they good for

A

Identifying infection and disease

Applied to health programs

Disease eradication programs

Public health and economic impact

The most important: to help you succeed in your practice

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3
Q

Veterinary Diagnostics:

Develop a differential list

A
  • Rule in/out
  • Define diagnostic approach
    • what test to request / complete
    • What sample to take
      • follow aseptic techniques always during sample collection
    • What laboratory to send
    • Interpreting the results
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4
Q

Example of Viral DIagnoisi:

Canine Parvovirus

A
  • History and Clinical signs
  • In feces:
    • viral antigens
      • hemafflutination assays
      • ELISA - snap test
    • Virus morphology
      • electron microscopy
    • Virus
      • isolation / tissue culture
  • In blood / serology:
    • blood profile
    • Hemagglutination inhibition
    • ELISA
    • serum neutralization assay
  • By PCR:
    • in feces, blood or tissue

Focus on what these tests demonstrate, advantages/disadvantages, applicability to assist you in managing a case, cost vs. need, requirements, type of sample available to test

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5
Q

Classical Diagnostic Problem Solving Process

A

ask the right question and set your goasl on this case

Sample collection

Sample preparation

Sample submission

Processing

Amplification

Detection

Analysis / interpretaiton

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6
Q

Specific Diagnostic Approaches:

Phenotypic Detection Method

A

Phenotype = the entire physical, biochemical and physiological make-up of a virus

Looking for the presense of the virus

Small, filterable particles:

inoclated back into susceptible hosts

Bioassay

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7
Q

Intracerebral inoculation

A

pasteur used intracerebral inoculation of dogs with tissues form rabies suspect dogs to diagnose canine rabies

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8
Q

Virus Culturing

A

Woodruff and goodpasture showed that cowpox and some other viruses could be grown in the tisseus of chick embryos

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9
Q

Tissue Culture Categories:

Primary Tissue Cultures

A

Typically have a finite life span or passage level

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10
Q

Tissue Culture Categories:

Continuous cell lines

A

are, by definition, abnormal and are often transformed cell lines and can live indefinitely with proper maintenance

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11
Q

Primary Tissue Cultures:

Pros, Cons,

A
  • Cultured cells that are derived directly form tissues
  • Pros:
    • the cells have not been “modified” in any way
  • Cons:
    • mixed nature of each preparation
    • Limit lifespan of the culture
    • Potential contamination problems with other viruses
    • Remember many cell types are post-mitotic and will not proliferate unless transformed
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12
Q

Cell Lines:

Pros, Cons

A
  • Specific cell types artificially maintained in the laboratory for scientific purposes
  • Population of cultured cells, of animla origin, that have undergone a change allowing the cells to grow indefinitely
  • Pros:
    • can grow indefinitely
    • Clonal polulation of cells
  • Cons:
    • cell lines are that not all viruses replicate well in cell lines
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13
Q

Steps in culturing cells

A
  1. Homogenize tissues
  2. addition of culture media and antibiotics to cells
  3. Incubate
  4. Visualize
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14
Q

Steps in culturing vituses on tissue culture

A
  1. TIssue to be evaluated for viruses are homogenized
  2. Added to tissue culture
  3. Incubate
  4. Detect
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15
Q

Cytopathic Effect (CPE)

A

is a lytic event for the infected cell; the cell must be infected first for CPE to occur unless Toxicity is occuring

Lacks specificity because no all viruses cause CPE during infection

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16
Q

Viral Culture

A

propagation of the virus

Quantitative analysis of virus in the culture by serial dilution 1:10

Relative amount of virus in the sample - Viral titer determined by : Spearman-Karber method

17
Q

Assays for detecting Antibodies

A

ELISA - enzyme-linked immunosorbent assay

VN - virus neutralization

HI - Heamagglutination Inhibition assay

IFA - Indirect Fluorescent assay

CF - complement fixation

WB - western blotting

AGID - Agar Gel Immunodiffusion Assay

18
Q

Direct Antibody-based test

A

Use only primary antibodies

They can be either polyclonal Abs to individual antigens or monoclonal Abs.

19
Q

Monoclonal Antibody

A

Continuous cultures of fused cells secreting antibody of predefined specificity

20
Q

Direct FA

A

antibodies to virus proteins labeled with a fluorescent dye

21
Q

What is a Titer

A

an expression of concentration or level of antibodies or viruses

Titer testing uses serial dilutions to obtain semi-quantitative information from a series of positive / negative results

The titer corresponds to the highest dilution factor that still yields a positive reading

22
Q

Serodiagnosis

A

use of a serological test to determine if exposure to an antigen has occurred or not

23
Q

What “actions” lead to an antibody titer

A
  • Animals may develop a positive antibody titer through the following
    • vaccination
    • passive antibody
    • Exposure to an infectious agent
      • clinical disease insult
      • Sub-clinical disease insult
    • Combination of the above
24
Q

What “actions” cause titer variation?

A

pathogen

degree of exposure

Maternal antibody

Vaccine type

Single vs. multiple dose exposure

Boost of exposed animals

Cross reaction

25
Q

What “actions” negatively impact titers

A
  • Animals may fail to develop a positive antibody titer due to:
    • improper vaccine handling
    • Poor nutrition
    • Antigenic stimulation
    • Immune suppression
    • Maternal interference
    • Stress
26
Q

Improving serology testing restults:

Golden Rule

A

serodiagnostics is you must have acute and convalescent serum samples for a truly meaningful interpretation

27
Q

Improving serology testing results:

Silver Rule

A

serodiagnostics is that sometimes what you have is better than nothing at all

28
Q

Improving Serology testing results:

Other rules of thumb

A

spin down serum prior to shipping, use serum separator tubes

Ship cool, not frozen on ice, by overnight courier

Avoid hemolyzed samples

Proper container - sterile plastic snap tubes

Proper amount of serum

29
Q

Hemagglutination of RBCs

A

1941

McLelland and Hare showed that human influenza virus would agglutinate red blood cells

The first application of this technique was in the titration of myxoviruses

30
Q

Hemagglutination:

Advantages / Disadvantages

A

relatively easy to complete

Relatively inexpensive

Many different variables

Not virus specific

Used more to finalize viral characteristics.

Remember: its a lab finding and does not occur in vivo

31
Q

Specific Diagnostic Approaches:

Phenotypic detection methods:

A

THe entire physical, biochemical and physiological make-up of a virus

Looking for the presence of the virus

Looking for the host’s response to infection

32
Q

Fluorescent Antibody Testing

A

can be completed in tissue culture to indentify the presence of infectious virus from samples collected from the suspect animal

Can also be completed directly on tissues

33
Q

Case Study:

Clinical Signs in cat:

anorexia

weight loss

icterus

pale mucosal membrane

Dyspnea

Outcome: sudden death

A

Feline infections peritonitis (FIP)

coronavirus by FE direct assay

34
Q

Inclusion Bodies:

A

accumulation of viral components in cytoplasm of infected cells

35
Q

Syncytia or giant cells

A

due to fusion protein in some viruses causing cells to fuse

Veterinary Virology DMP851 (12 decks)

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