Virology Flashcards
Describe the electron micrograph of a herpes virus
- Nucleocapsid surrounds viral DNA and an outer envelope
- Symmetry= icosahedral
What is viral antigen detection?
- Frequent test used for virus detection
- Blood screened for surface antigens in immunoassay test
Describe the HBV test
- HB antigens present in serum of chronic hepatitis B carrier
- Excess protein assembles into small spheres or tubules, which are not infectious
- Material aggregated by specific antibody to HBs antigen (immune EM)
Why is there HBV carriers?
Excess protein assembles into small spheres or tubules, which are not infectious- a liver cell infected by hepatitis B virus manufactures a huge excess of HBsAg which enters the blood making this a highly sensitive marker of hepatitis B infection
What test is better than electron microscopy?
ELISA= Enzyme-Linked Immuno Assay
- means of detecting antigen (or antibody) that is widely used in diagnostic virology and other work.
- EIA is carried out on liquid samples so blood serum or plasma can be used directly in tests, or (for other viruses) extracts may be prepared from respiratory secretions or faeces for testing
Describe the stages of an ELISA test
- Antibody= specific antibody is require to react with the antigen of interest in the sample cup- can be used as coating to trap antigen from solution
- Enzyme-Labelled Antibody= labelled antibody against antigen or first antibody will be bound to the antigen hence enzyme-label can be detected
- Enzyme substrate= chromogen that changes colour/ light-emitting reagent.
How are the results from the ELISA calculated/ automated?
- testing large numbers of samples efficient.
- Many laboratories use closed system Random Access analysers, identical to those in biochemistry, for enzyme-linked immunoassays for antigen or for antibody.
- These assays are now sensitive enough to detect 0.5 international units of HBs antigen in 1 ml plasma/serum - important for excluding infected blood or tissue donations
How may immunofluorescence be used?
- In some centres immunofluorescence is still used to reveal viral antigens in cell samples.
- This can easily be done for individual samples or small numbers at a time, by hand. This is useful in small labs which don’t have the sample throughput to carry out molecular tests for viruses
- For instance epithelial cells fixed on a glass slide can be stained with fluorescein (FITC)-labelled antibody to a specific virus, and viewed in a special light microscope with UV illumination to detect the fluorescence emitted.
When may immunofluorescence be used?
-Direct immunofluorescence can be applied to cells from epithelial or mucosal sites, and is used for rapid direct detection of any respiratory virus, such as influenza, or of herpes simplex or varicella-zoster in cells from base of skin blister.
-Getting an accurate diagnosis can influence specific antiviral treatment, management of contacts, and infection control decisions.
-Immunofluorescence tests have the advantage of being rapid to carry out on a small number of samples but may be less sensitive than molecular tests, which are now tending to replImmunofluorescence is also used for rapid identification of viruses causing cytopathic effects in cell cultures, by fixing some cells from the tube culture on a slide. Type-specific monoclonal antibodies are also used, commonly to differentiate HSV type1 from HSV type 2 in cells. (The type-specific antigens are on surface glycoproteins)ace them.
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What is Respiratory syncytial virus (RSV)?
RSV is the most important respiratory pathogen in infancy. One in every hundred babies born is admitted to hospital in the first year of life with serious RSV infection, usually bronchiolitis.
As RSV spreads it may induce adjacent cells to fuse into a syncytium (hence the name of this virus). Human metapneumovirus (hMPV) is another paramyxovirus like RSV that causes respiratory infections that are more severe in young children, the elderly and the immunosuppressed. Its importance has become more widely understood with the introduction of molecular diagnostic tests
What is sensitivity and specificity?
Sensitivity= how many of the true positives are found
no. true positives/ no. true positives + false negatives
Specificity= how many of the true negatives are identified
no. true negative/ no. true negative + false positive
Describe nucleic acid detection
- When sufficient nucleic acid is present, separation of fragments by size on gel electrophoresis is possible.
- Polymerase chain reaction (PCR)
When can gel electrophoresis be used?
Children with gastroenteritis due to rotavirus infection shed large numbers of virus particles in stools, as seen in the EM image earlier. Rotavirus has 11 RNA genes, and these are easily extracted and detected by electrophoresis in a polyacrylamide gel (PAGE). Note that because so much rotavirus was shed in these stools there has been no need to amplify a fragment of its genome by PCR in this case - the entire genome has simply been extracted and detected.
What does PCR require?
- A known (usually short) DNA sequence of interest
- synthetic oligonucleotide sequences (“primers”)
- deoxyribonucleotide triphosphates (dNTPs)
- heat-stable DNA polymerase (eg “Taq”)
- buffer with MgCl, etc
- sample with DNA to be tested
- positive and negative control material
- a thermal cycler
How does PCR work?
The microprocessor-controlled thermal cycler repeats a chosen number of cycles of denaturation, hybridisation, and DNA synthesis (eg 30 to 40 cycles).
Eventually, a vastly-amplified amount of double-stranded DNA copied from the original sample is present in the small reaction tube.
The chosen stretch of DNA that has been amplified must then be detected by electrophoresis, or by a hybridisation probe, or an EIA for a label incorporated into the new DNA.
