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
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
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
4
Q
What are the drawbacks of Electron microscopy?
A
- Cost of equipment
- technical requirements
- lack of analytical sensitivity
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
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
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
8
Q
What are the steps in culturing cells?
A
- Homogenize tissues (primary cell culture only)
- Addition of culture media & antibiotics to cells
- Incubate
- Visualize
9
Q
What are the steps to culturing viruses in tissue culture?
A
- Tissue to be evaluated for viruses are homogenized
- Added to tissue culture
- Incubate
- Detect
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
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
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
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
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
