Viral Diagnostics: Application for Vet Med Flashcards
Selective Assays for detection of viruses or viral proteins or nucleic acids
virus isolation
fluorescent antibody on tissue
electron microscopy
Polymerase Chain Reactions
Antigen Capturing ELISA
Sequencing
Viral micorarray
Veterinary DIagnostics:
What are they good for
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
Veterinary Diagnostics:
Develop a differential list
- 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
Example of Viral DIagnoisi:
Canine Parvovirus
- History and Clinical signs
- In feces:
- viral antigens
- hemafflutination assays
- ELISA - snap test
- Virus morphology
- electron microscopy
- Virus
- isolation / tissue culture
- viral antigens
- 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
Classical Diagnostic Problem Solving Process
ask the right question and set your goasl on this case
Sample collection
Sample preparation
Sample submission
Processing
Amplification
Detection
Analysis / interpretaiton
Specific Diagnostic Approaches:
Phenotypic Detection Method
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
Intracerebral inoculation
pasteur used intracerebral inoculation of dogs with tissues form rabies suspect dogs to diagnose canine rabies
Virus Culturing
Woodruff and goodpasture showed that cowpox and some other viruses could be grown in the tisseus of chick embryos
Tissue Culture Categories:
Primary Tissue Cultures
Typically have a finite life span or passage level
Tissue Culture Categories:
Continuous cell lines
are, by definition, abnormal and are often transformed cell lines and can live indefinitely with proper maintenance
Primary Tissue Cultures:
Pros, Cons,
- 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
Cell Lines:
Pros, Cons
- 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
Steps in culturing cells
- Homogenize tissues
- addition of culture media and antibiotics to cells
- Incubate
- Visualize
Steps in culturing vituses on tissue culture
- TIssue to be evaluated for viruses are homogenized
- Added to tissue culture
- Incubate
- Detect
Cytopathic Effect (CPE)
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
Viral Culture
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
Assays for detecting Antibodies
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
Direct Antibody-based test
Use only primary antibodies
They can be either polyclonal Abs to individual antigens or monoclonal Abs.
Monoclonal Antibody
Continuous cultures of fused cells secreting antibody of predefined specificity
Direct FA
antibodies to virus proteins labeled with a fluorescent dye
What is a Titer
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
Serodiagnosis
use of a serological test to determine if exposure to an antigen has occurred or not
What “actions” lead to an antibody titer
- 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
What “actions” cause titer variation?
pathogen
degree of exposure
Maternal antibody
Vaccine type
Single vs. multiple dose exposure
Boost of exposed animals
Cross reaction
What “actions” negatively impact titers
- Animals may fail to develop a positive antibody titer due to:
- improper vaccine handling
- Poor nutrition
- Antigenic stimulation
- Immune suppression
- Maternal interference
- Stress
Improving serology testing restults:
Golden Rule
serodiagnostics is you must have acute and convalescent serum samples for a truly meaningful interpretation
Improving serology testing results:
Silver Rule
serodiagnostics is that sometimes what you have is better than nothing at all
Improving Serology testing results:
Other rules of thumb
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
Hemagglutination of RBCs
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
Hemagglutination:
Advantages / Disadvantages
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
Specific Diagnostic Approaches:
Phenotypic detection methods:
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
Fluorescent Antibody Testing
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
Case Study:
Clinical Signs in cat:
anorexia
weight loss
icterus
pale mucosal membrane
Dyspnea
Outcome: sudden death
Feline infections peritonitis (FIP)
coronavirus by FE direct assay
Inclusion Bodies:
accumulation of viral components in cytoplasm of infected cells
Syncytia or giant cells
due to fusion protein in some viruses causing cells to fuse
