Thursday, August 18, 2011

Electron Microscopy for Viruses

Virus diagnosis by electron microscopy relies on the detection and identification of viruses on the basis of their characteristic morphology. A major advantage of virus diagnosis by EM is the ability to visualize the virus. By identifying the virus directly, it is possible to perform an examination without a preconceived concept of the aetiological agent, in contrast with those assays which require a specific viral probe. Speed is another advantage of EM as the specimen can be processed within minutes of receipt and thus EM can be used as a rapid diagnostic method. On the other hand, the main disadvantage of EM is its inability to examine multiple specimens coincidentally. Secondly, there must be a minimum number of virus particles present (around 106 virus particles per ml for detection) Some viruses such as SRSV may give a non-distinct morphological appearance which may make detection very difficult. Finally, EM is a very expensive service to provide and requires highly skilled personnel. EM has found a particular niche in the detection of fastidious gastroenteritis viruses such as rota, adeno, astro, Norwalk, and Caliciviruses. It is also used for the rapid diagnosis of herpesvirus infection. It is occasionally used for the diagnosis of human papillomavirus infections and infections by members of the poxvirus family. In addition EM may be used to confirm the results of virus isolation by cell culture such as for parainfluenza viruses.
There are two types of EM methods;- direct or immunoelectron microscopy (IEM). With direct methods, negative staining is normally used which requires little special equipment, in contrast to thin sectioning techniques. The specimens may be used directly or the virus particles may be concentrated before negative staining. Several methods are available for concentration, including differential centrifugation, ammonium persulphate precipitation, and agar diffusion method. Fomvar coated copper grids are used. Immunoelectron microscopy is a means of increasing the sensitivity and specificity of EM and is particularly useful in the following situations;-
1. The number of virus particles present is small.
2. Many different viruses have different morphology e.g. herpesviruses and picornaviruses. IEM may identify the virus
3. In an outbreak situation where the pathogens responsible has been identified so that it may be useful to go back to look at the negative specimens again with IEM.
There are 2 types of IEM, simple IEM, where the specimen is incubated with specific antibody before staining in the hope that the antibody will agglutinate the specimen, and solid phase IEM (SPIEM), where the copy grid is coated with specific antibody which is used to capture virus particles from the specimen.

Electron micrographs of viruses commonly found in stool specimens from patients suffering from gastroenteritis. From left to right: rotavirus, adenovirus, astroviruses

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