There are four approaches to confirming a viral infection in the laboratory. Namely:
- serology: demonstrating an antibody response in a patient's serum
- direct detection of viral antigens in a clinical sample
- virus culture
- viral nucleic acid detection
1. Antibody assays:
An acute or recent infection may be confirmed by demonstrating the presence of specific IgM in a single serum sample, or showing a sero-conversion or rise in titre of specific IgG in paired sera.
In general, the presence of IgG and the absence of IgM, is indicative of past infection or immunity.
These days, antibody assays are usually tested by means of the enzyme-linked immuno-assay (ELISA) technique.
2. Direct demonstration of virus
(a) Electron Microscopy
Viruses are very small and cannot be visualized by light microscopy. Historically the electron microscope was very useful in defining the morphology of many human viruses. However, it is not a tool that is routinely used to identify viruses in a diagnostic setting. This is because viruses are usually present in very small numbers in clinical specimens and other contaminating material tends to obscure their presence.
(b) Demonstration of virus-infected cells in clinical samples by labelled antibodies.
This technique is commonly used to identify the causative agent in a patient with a respiratory infection, caused by viruses such as RSV, Influenza or Adenovirus.
Infected cells synthesize and express viral proteins (antigens). The presence of these can be detected using specific mono-clonal or poly-clonal antibodies labelled with fluorescene (a green dye). The antibody binds to the cells if they express the corresponding antigen. The cells can then be visualized by examination under a fluorescent microscope. Positive cells fluoresce a bright green colour.
The limitation of the test is that you have to know what virus you are looking for.
The advantage is that one can get a very rapid answer as to which virus is causing the problem.
Viruses can only replicate in living cells. Therefore to culture them in vitro one must provide them with living cells. In the past it was common to use laboratory animals, or chick embryos to grow viruses, but these have largely been replaced by the use of cell monolayers.
The clinical sample is inoculated into a test tube containing a glass cover slip on which a cell monolayer is growing. Replicating viruses change the appearance of the cells to induce a cytopathic effect. Different viruses cause different types of cytopathic effects. Only some medically important viruses can be cultured.
Immunofluorescence: Another way to identify a virus growing in a cell culture is to add fluorosceine labelled monoclonal antibodies to likely viruses to the cell sheet and examine under a fluorescent microscope.
4. Molecular techniques
Nucleic acid amplification techniques such as polymerase chain reaction (PCR) can be used to detect viral genomes in clinical material. The same technique can be used to detect any DNA sequence (viral, bacterial or other). To detect RNA, an initial reverse transcription step is performed (converts RNA into cDNA). After this, PCR can be performed. Molecular assays are very sensitive (able to detect only a few viruses in a clinical sample.) They can also be used to measure the amount of virus (viral load) in a patient's sample.