Viral Diagnostics

Question 1

How is a diagnostic approach defined?

Answer 1

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

Question 2

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

Answer 2

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

Question 3

What are Phenotypic detection methods?

Answer 3

• 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

Question 4

What are the drawbacks of Electron microscopy?

Answer 4

• Cost of equipment
• technical requirements
• lack of analytical sensitivity

Question 5

What are the 2 categories of tissue culture?

Answer 5

• 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

Question 6

What are Primary Tissue Cultures?

Answer 6

• 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

Question 7

What are Cell lines?

Answer 7

• 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

Question 8

What are the steps in culturing cells?

Answer 8

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

Question 9

What are the steps to culturing viruses in tissue culture?

Answer 9

1. Tissue to be evaluated for viruses are homogenized
2. Added to tissue culture
3. Incubate
4. Detect

Question 10

What is CytoPathic Effect (CPE)?

Answer 10

• 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

Question 11

What is Viral culture?

Answer 11

• 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

Question 12

What are the Assays for detecting antibodies?

Answer 12

• 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

Question 13

What are Direct Assays

Answer 13

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

Question 14

What is Direct Fluorescence Assay?

Answer 14

• 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

Question 15

What is a Titer?

Answer 15

• 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

Question 16

What is serodiagnosis?

Answer 16

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

Question 17

What actions lead to an Antibody Titer?

Answer 17

• 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