virology Flashcards
Ways to visualize virus/bacteria/parasite
light/electron microscope
ways to detect protein
ELISA, IFA, IHC
Ways to detect DNA or RNA
PCR, NGS
electron microscopy advantages/disadvantages
advantages:
* allows visualization of virus
* rapid
* detects viruses that cannot be isolated
* live virus not required
disadvantages:
* expensive
* need high numbers of virus
* specialized training
virus isolation
grow virus in cell culture
Advantages
* Sensitive
* Allows for further characterization of the virus
* Allows for identification of unknown viruses
Disadvantages
* No longer widely available
* Slow, time consuming
* Requires specialized training
* Costly to perform
antibody-antigen based tests
detect viral protein
ELISA
Immunofluorescence (IFA)
Immunohistochemistry (IHC)
Enzyme-linked immunosorbent assay (ELISA)
Antigen based (“antigen capture”)
* Conjugated primary or secondary antibody
* Substrate gives a color reaction
* SNAP tests
* detects viral proteins
Antibody based ELISA
* Dilutions allow for detecting antibody titers
* A second antibody can detect IgM, IgA or IgG
Immunofluorescent antibody tests (IFA)
- Cell culture or tissues
- Antibody has a fluorescent tag and is read with a fluorescence microscope
- Requires high viral titer
Immunohistochemistry
Uses paraffin-embedded tissues mounted on glass slides
Sections are incubated with specific antibodies tagged with a substrate
Substrate gives a color reaction
Allows detection of the antigen in the lesion or in specific cells
antibody based tests advantages/disadvantages
Advantages
* Rapid
* Sensitive
* Easy to use
* Can detect different serotypes
Disadvantages
* Not available for all organisms
* Can be difficult to interpret
types of Genomic Detection (DNA or RNA)
PCR
In-situ hybridization
Next generation sequencing
Real time PCR
more DNA= more fluorescence sooner (earlier cycle)
actively replicating= more viral DNA/RNA= detected at an earlier threshold cycle
In-situ hybridization
Allows for visualization of the virus in the tissue or at the site of infection
Allows for determination of which virus is responsible for pathologic lesions
Next Generation Sequencing
Whole genome sequencing
Detection of unknown viruses
DNA-based tests advantages/disadvantages
Advantages
* Rapid
* Sensitive
* Specific
* Relatively inexpensive
* Potentially applicable to all viruses
Disadvantages
* Sensitive – detecting DNA does not necessarily indicate causation
* Risk of DNA contamination (PCR, NGS)
* Have to know what you’re looking for (PCR)
serology
Measurement of antigen-antibody interactions
Looks for antibodies to determine exposure/disease/immunity
Many different tests available
* ELISA
* Immunodiffusion
* Virus neutralization
antibody titers
Definition: The highest dilution (lowest concentration) of serum need to detectably interact with the antigen
* Gives you the relative concentration of antibody
Method: Create a series of standard dilutions of the original serum samples and test against the antigen of interest
* Not all tests produce titers
Immunodiffusion
- Antigen is loaded at the center of the gel
- Test serum is loaded on the periphery
- A charge is added and antibodies move towards the antigen and precipitate creating a band
Virus Neutralization
Using antibodies in serum to inhibit virus replication
The** titer is the inverse of the highest dilution needed to neutralize the virus**
Highly specific
Slow or expensive because it relies on inhibition of virus growth in cell culture
