Virus detection Flashcards
Electron microscopy
- Shows the morphology of viruses
- Can be used for characterisation and identification
- Can be performed on specimens directly or viruses concentrated
from: - Faeces (rotavirus, calicivirus)
- Vesicle fluid (herpes simplex)
- Skin scrapings (Papillomavirus)
- Fast – can be done in a few minutes
- Excellent method for detecting rotaviruses, adenoviruses,
astroviruses, caliciviruses
Cons of EM
- Expensive
- Need specialise equipment
- Requires skilled personnel
- Low sensitivity
- Need concentrated virus samples (106
particles/ml)
Immune EM
- Sensitivity and specificity can be improved by using virus specific antibodies
- Different viruses with similar morphologies can be identified
*useful when virus particle number is low
− Classical Immune EM
* Sample is mixed with antibody
* Negative staining of sample
* Loaded onto EM grid and visualised
− Solid phase Immune EM (SPIEM)
* Grid is coated with antibody and used to capture virus particles
* Virus sample is loaded onto antibody coated grid
* Negative staining etc.
ELISA
Add sample containing Ag -> Wash well -> Add antibody that recognises the Ag (detecting Ab) -> Wash well -> Add conjugate labelled secondary Ab
(anti IgG peroxidase) -> Wash well -> Add substrate -> either coloured or colourless liquid
Haemagglutination assay
Some viruses contain proteins that can bind to RBC - adenoviridae, paramyxoviridae
Ifluenza virus contains an envelope glycoprotein called haemagglutinin that binds to erythrocytes
How does HA work?
If there is no virus present, RBC are not agglutinated and form a tight pellet at the well bottom
If virus is present, it binds to RBC, haemagglutination occurs and virus ‘cross-links’ the RBC, forms a shield
Haemagglutination protocol
*Virus sample diluted in 2 fold dilutions across plate starting with 1/10 dilution
*Red blood cells (RBC) added to each well including control well (C = no virus)
* Incubation at room temperature for 1 hour
Immunofluorescence
Cells from the clinical specimen
(i.e. nasopharyngeal aspirate) fixed onto glass slide -> Add virus specific antibody -> Add a labelled antibody (fluorescent
dye) that binds to the virus specific
antibody -> view with fluorescent microscope
Immunoperoxidase is also similar to this but a peroxidase label is used
Virus quantification: plaque assay
To determine levels of virus (titre) in tissues by titration
* Virus spreads to adjacent cells causing damage and death
* Plaques are produced – regions with no cells, which can be seen with the naked eye after staining
* Virologists equivalent of bacterial colonies
* Used to quantify viruses
* virus titre – exact number of plaques
* LD50 – dilution of virus that kills 50% of cells
Plaque assay
- Monolayers of cultured cells are incubated with serial dilution of virus to allow adsorption to cells
- After removal of the inoculum, the cells are overlayed with semi solid media (agar) which limits the spread of virus to
neighbouring cells - Hence, each infectious particle produces a circular zone of infected cells which damage or kill the cells resulting in a plaque
- Only viruses that cause visible damage can be assayed in this way
- Stain with crystal violet (stains living cells) to enable counting the visible plaques
Intepretation of plaque assay
Virus Titre = Number of plaques (10) /number of replicates (4) x dilution factor
Viral genome detection
PCR
−Nucleic acid purification(RNA or DNA)
−Reverse transcription for RNA virus
- Using virus specific primers to amplify a specific target
- Repeated cycles of denaturation, annealing and elongation
−Agarose gel electrophoresis to visualise PCR products
Viral Cytopathic Effect (CPE) observed in vitro
- Grow cells in vitro and infect with viruses taken from clinical
samples - Some viruses kill the cells they infect and as more cells are infected
these changes can be visualised with a light microscope - Easier to see this than the viruses themselves (too small)
- Range from massive damage to no visible damage (non-cytopathic)
- If CPE is not easily visible, can look for viral antigens in cells by immunofluorescence or immunoperoxidase
Preparation of primary cell culture
Tissue fragments (cut with scissors/scalpel) -> Treated with trypsin or collagenase -> Cell suspension -> Addition of liquid media (Eagle’s + animal serum)
-> Incubated in petri dish or tissue culture flask -> Cells attach to solid surface and start dividing
Primary cell lines
Prepared directly from tissue
Subcultured 1-2 times
Technically more difficult
Supports wide range of viruses
Used when the state of cell differentiation is important
Difficult to obtain a reliable supply
Expensive
Example: monkey kidney
