Virus Detection Flashcards
What are some of the methods for detecting virus, virus antigens and viral genomes?
> Detection of virus or viral antigen
- In clinical samples
Election Microscopy
ELISA to detect viral antigens
Haemagglutination
- In virus infected tissues: biopsies/swabs
Immuniperoxidease assay
Immunofluorescence assay
> Viral genome detection
PCR
Hybridisation
What is good about using a EM for virus detection?
Useful when numbers of virus particles is low
Sensitivity and specificity can be improved by using virus specific Ab
Different viruses with similar morphologies can be identified
What is a classical immune EM?
Sample is mixed with Ab
Negative staining of sample
Loaded onto EM grid and visualised
What is aSolid Phase Immune EM (SPIEM)?
Grid is coated with Ab and used to capture virus particles
Virus sample is loaded onto Ab coated grid
Negative stainging etc.
What is a Haemagglutination assay?
Flu virus contains envelope glycoprotein called haemagglutinin which binds to RBC.
NO VIRUS: RBC are not agglutinated and form a tight pellet at the bottom of the well
+ VIRUS: RBC bind to virus i.e. haemagglutination occurs and virus ‘cross links’ the RBC, forms a shield.
What is the protocol for Haemagglutination?
Virus sample diluted 2 fold dilutions across starting with 1/10 dilution.
RBC added to each well including control well (C=no virus)
Incubation at room temperature for 1 hour
RBC in control well should not agglutinate but will form a buttom and bottom of well
In presence of virus, RBC will bind to each other i.e. agglutinate forming diffuse lattice that coats the well
What is immunofluorescence?
Cells from the clinical specimen fixed onto glass slide
Add virus specific
Add a labelleed Ab that binds to the virus specific Ab—>View with flourescent microscope
used commonly for resp viruses in respiratorry specimens
What is Immunoperoxidase?
Very similar but instead uses peroxidase label
What is a plaque Assay?
Determine levels of virus in tissues by titrations.
Virus spreads to adjacent cells causing damage and death.
Plaques are produced-regions with no cells which can be seen the naked eye after staining
what is the equation for virus titre (plaque assay)
Virus Titire= Number of plaques / number of replicates x dilution factor
Units PFU per ml
What are indirect methods of virus presence?
Look for signs of damage in:
Cells infected with virus in vitro:
- Cytopathic effect observed in virus infected cells
- Type of damage caused by some viruses can be used by virologists for diagnosis
Experimental infection of animals
What is a syncytium?
Fused cells containing many nuclei
What is a inclusion body?
Virus factories in nucleus or cytoplasm
What cytopathic effects are caused by picomaviruses?
Nuclear shrinking
Rounding up and detachment of cultured cells
What cytopathic effects are caused by Alphaviruses?
Proliferation of nuclear membrane
What cytopathic effects are caused by Herpesviruses?
Proliferation of nuclear membrane
Margination and breaking of chromosomes
Rounding up and detachment of cultured cells
What cytopathic effects are caused by Polyomaviruses?
Vacuoles in cytoplasm
What cytopathic effects are caused by Paramyxoviruses?
Syncytia (cell fusion)
What cytopathic effects are caused by Coronaviruses?
Syncytia
What cytopathic effects are caused by adenoviruses?
Rounding up and clustering of cells
What cytopathic effects are caused by rhabdoviruses?
Rounding up and detachment of cultured cells
What are the 5 mechanisms of damage caused by viruses?
Inhibition of host biosynthetic machinery
Toxic viral proteins
Damage to cell membranes/cytoskeleton
Lysis
Apoptosis
