Quantification of Viruses Flashcards
TCID50 =
% Infection above 50% - 50%
% Infection above 50% - % Infection below 50%
Viral quantification test that is most common
Biological Quanitfication Tests
Tissue Culture Infectivity Dose50
Tissue culture infectious dose which will infect 50% of the cell monolayers challenged with the defined medium
Quantitative Polymerase Chain Reaction (qPCR)
Utilizes polymerase chain reaction chemistry to amplify viral DNA or RNA to produce high enough concentrations for detection and quantification by fluorescence
Methods of quantification based on antigen concentration
Hemagglutination Assay
ELISA
High Performance Liquid Chromatography
Single Radial Immunodiffusion
Titer (PFU/mL) =
Average Plaque Count x Reciprocal of the Dilution Selected
High Performance Liquid Chromatography (HPLC)
Concentration of specific viral antigens may be quantified through UV analysis of fractions generated
Embryo Lethal Dose50
Dilution of virus that will kill 50% of embryos
Paralytic Dose50
Dilution of virus that will cause paralysis in 50% of case subjects
Transformation Assay
Quantitative determination of titers of oncogenic viruses - transformed cells lose contact inhibition and become heaped upone one another
Quantal Assay
Measures the presence or absence of infection
Hemagglutination Assay
Asseses virus quantification on antigen concentration. Virus interaction with red blood cells causes clumping
Endpoint
Virus dilution that affects 50% of the test subjects
Methods of quantification based on gene expression
Quantitative Polymerase Chain Reaction
Virus Counter 2100
Specialized version of flow cytometry developed specifically for use with nanometer scale particles - Sample stained with two dyes (one stains nucleic acid the other protein) and is anaylzed as they flow through a laser beam
Virus Titer
Lowest concentration of virus that still infects cells
Examples of Biological Quantification Tests
Plaque Assay
Pock Assay
Endpoint Titration Methods
Plaque
Circular zone of necrotic cells surrounded by viable cells in a monolayer
When is a quantal assay used?
Certain viruses that do not form plaques or for determining the virulence of a virus in animals or eggs
Multiplicity of Infection (MOI)
Average number of virus particles infecting each cell
Single Radial Immunodiffusion (SRID)
Radial diffusion of purified viral antigens (standards) and viral particles through agarose gel seeded with polyclonal antisera against a viral antigen
Most direct method to determine the concentration of virus particles in a sample
Transmission Electron Microscopy (TEM)
Most accurate quantitative biological assay
Monolayer plaque assay
When crystal violet is used in a monolayer plaque assay, viable cells stain what color
Blue
Quantification tests that depend on a virus particle initiating a successful replication cycle
Biological Viral Quantification Tests
Virus Quantification
Counts the number of viruses in a specific volume to determine the virus concentration
Monolayer Plaque Assay
Measures the number of virus particles capable of forming plaques per unit volume
Two types of viral quantification tests
Biological
Physical
Pock Assay
Necrotic area on chorioallantoic membrane (CAM) of embryonated egg
Lethal Dose50
Dilution of the virus that will kill 50% of case subjects
Methods of direct quantification
TEM
Virus Counter 2100
Examples of Physical Quantification Tests
Electron Microscopic Particle Counts
Hemagglutination
Immunological Assay
Quantitative PCR Assay
Flow Cytometry
