Viral Diagnostics Flashcards

1
Q

How is a diagnostic approach defined?

A
  • What tests to request/complete
  • What samples to take
  • What laboratory to send
  • Interpreting the results
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2
Q

What assays exist for detection of viruses or viral proteins or nucleic acids?

A
  • Virus isolation
  • Fluorescent Antibody on tissue (FA)
  • Electron Microscopy (EM)
  • Polymerase Chain Reaction
  • Antigen Capturing ELISA (ACE)
  • Sequencing
  • Viral Microarray
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3
Q

What are Phenotypic detection methods?

A
  • Looking for the presence of the virus
  • Take small, filterable particles
    • inoculated back into susceptible hosts
    • BioAssay
  • Electron microscopy
  • In vitro replication of virus in cells
    • Tissue culture
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4
Q

What are the drawbacks of Electron microscopy?

A
  • Cost of equipment
  • technical requirements
  • lack of analytical sensitivity
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5
Q

What are the 2 categories of tissue culture?

A
  • Primary cell cultures:
    • typically have a finite life span or passage level whereas
  • Continuous cell lines:
    • Abnormal and are often transformed cell lines and can live indefinitely with proper maintenance
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6
Q

What are Primary Tissue Cultures?

A
  • Cultured cells that are derived directly from tissue (often embryonic tissue)
  • Advantages: cells have not been “modified” in any way
  • Disadvantages:
    • Mixed nature of each preparation
    • Limited lifespan
    • potential for contamination problems with other viruses
    • Many cell types are post-mitotic and will not proliferate unless transformed
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7
Q

What are Cell lines?

A
  • Specific cell types artificially maintained in the laboratory for scientific purposes
  • A population of cultured cells, of animal origin, that have undergone a change allowing the cells to grow indefinitely
  • Advantages:
    • Can grow indefinitely
    • Clonal population of cells
  • Disadvantages:
    • Not all viruses replicate well in cell lines
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8
Q

What are the steps in culturing cells?

A
  1. Homogenize tissues (primary cell culture only)
  2. Addition of culture media & antibiotics to cells
  3. Incubate
  4. Visualize
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9
Q

What are the steps to culturing viruses in 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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10
Q

What is CytoPathic Effect (CPE)?

A
  • Lytic event for the infected cell
    • cell must be infected first for CPE to occur unless Toxicity is occurring
  • CPE lacks specificity because not all viruses cause CPE during infection
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11
Q

What is Viral culture?

A
  • Propagation of the virus
  • Quantitative analysis of virus in the culture by serial dilution 1:10
    • Expressed in TCID50/ml
  • Relative amount of virus in the sample- Viral titer determined by Spearman-Karber method
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12
Q

What are the 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
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13
Q

What are Direct Assays

A
  • Use primary antibodies
  • can be either polyclonal Abs to individual antigens (epitopes of 14-18 AA) or monoclonal Abs
    *
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14
Q

What is Direct Fluorescence Assay?

A
  • Antibodies to virus proteins (labeled with a fluorescent dye)
    • Can use radiolabeled Ab - though rarely used due to health hazards
    • can use enzymatically labeled Ab (ELISA)
  • typically used to detect viral antigen in infected cells
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15
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
    • Titer corresponds to the highest dilution factor that still yields a positive reading
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16
Q

What is serodiagnosis?

A
  • Use of a serological test to determine if exposure to an antigen has occurred or not
17
Q

What actions lead to an Antibody Titer?

A
  • Animals may develop a positive antibody titer through:
    • Vaccination
    • Passive Antibody (Lactogenic Immunity, Antibody therapy)
    • Exposure to an infectious agent
      • Clinical disease insult
    • Combination9s) of the above