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Identification of enterically transmitted hepatitis virus particles by solid phase immune electron microscopy
C D Humphrey1, E H Cook, D W Bradley
1Ultrastructure Activity, Center for Infectious Diseases Centers for Disease Control, Atlanta, Georgia 30333.
Abstract:
Small 'featureless' viruses (less than 50 nm) are difficult to identify by routine immune electron microscopy techniques, particularly when they are mixed with debris from stool or cell culture extracts. A combination of conventional immune electron microscopy (IEM) and solid phase IEM (SPIEM) methodologies was used to identify hepatitis A virus (HAV) in stool and cell culture extracts and non-A non-B hepatitis (hepatitis E) in stool extracts. Compared with conventional IEM, the modified SPIEM method resulted in a significant increase in the number of particles observed. Several small aggregates, each containing 2-20 particles, were observed scattered randomly within most grid squares. Similar results were seen with stool extracts from hepatitis E (HEV) infections. The SPIEM method is a simple, highly sensitive specific assay that facilitates rapid identification of enteric hepatitis viruses. Several experiments were done to characterize the effects of altered physical environment within the assay and to evaluate potential modifications.
Insights
Identifying small enteric hepatitis viruses like hepatitis A virus (HAV) and hepatitis E virus (HEV) is challenging. Solid phase immune electron microscopy (SPIEM) significantly improves detection sensitivity and speed for these viruses in clinical samples.
Area of Science:
- Virology
- Immunology
- Microscopy
Background:
- Small viruses (<50 nm), such as hepatitis A virus (HAV) and hepatitis E virus (HEV), are difficult to detect using standard immune electron microscopy (IEM).
- Routine IEM methods struggle with identifying these viruses in complex biological matrices like stool and cell culture extracts due to interference from debris.
Purpose of the Study:
- To evaluate the efficacy of a modified solid phase immune electron microscopy (SPIEM) technique for enhanced detection of small enteric hepatitis viruses.
- To compare the sensitivity of SPIEM against conventional IEM for identifying hepatitis A virus (HAV) and hepatitis E virus (HEV).
Main Methods:
- Utilized a combination of conventional immune electron microscopy (IEM) and a modified solid phase immune electron microscopy (SPIEM) methodology.
- Applied the techniques to identify HAV in stool and cell culture extracts, and HEV in stool extracts.
- Investigated the effects of altered physical environments and potential modifications within the SPIEM assay.
Main Results:
- The modified SPIEM method demonstrated a significant increase in the number of viral particles observed compared to conventional IEM.
- SPIEM facilitated the visualization of small aggregates (2-20 particles) of HAV and HEV, which were not readily apparent with standard IEM.
- The technique proved effective for both hepatitis A virus (HAV) and hepatitis E virus (HEV) detection in relevant clinical samples.
Conclusions:
- Solid phase immune electron microscopy (SPIEM) is a highly sensitive and specific assay for the rapid identification of enteric hepatitis viruses.
- The SPIEM method offers a significant improvement over conventional IEM for detecting small, 'featureless' viruses in challenging sample types.
- SPIEM facilitates faster and more accurate diagnosis of hepatitis A and E infections.