Application of transmission electron microscopy to the clinical study of viral and bacterial infections: present and future

Abstract

Transmission electron microscopy has had a profound impact on our knowledge and understanding of viruses and bacteria. The 1000-fold improvement in resolution provided by electron microscopy (EM) has allowed visualization of viruses, the existence of which had previously only been suspected as the causative agents of transmissible infectious disease. Viruses are grouped into families based on their morphology. Viruses from different families look different and these morphological variances are the basis for identification of viruses by EM. Electron microscopy initially came to prominence in diagnostic microbiology in the late 1960s when it was used in the rapid diagnosis of smallpox, by differentiating, on a morphological basis, poxviruses from the less problematic herpesviruses in skin lesions. Subsequently, the technique was employed in the diagnosis of other viral infections, such as hepatitis B and parvovirus B19. Electron microscopy has led to the discovery of many new viruses, most notably the various viruses associated with gastroenteritis, for which it remained the principal diagnostic method until fairly recent times. Development of molecular techniques, which offer greater sensitivity and often the capacity to easily process large numbers of samples, has replaced EM in many areas of diagnostic virology. Hence the role of EM in clinical virology is evolving with less emphasis on diagnosis and more on research, although this is likely only to be undertaken in specialist centres. However, EM still offers tremendous advantages to the microbiologist, both in the speed of diagnosis and the potential for detecting, by a single test, any viral pathogen or even multiple pathogens present within a sample. There is continuing use of EM for the investigation of new and emerging agents, such as SARS and human monkeypox virus. Furthermore, EM forms a vital part of the national emergency response programme of many countries and will provide a frontline diagnostic service in the event of a bioterrorism incident, particularly in the scenario of a deliberate release of smallpox virus. In the field of bacteriology, EM is of little use diagnostically, although some bacterial pathogens can be identified in biopsy material processed for EM examination. Electron microscopy has been used, however, to elucidate the structure and function of many bacterial features, such as flagellae, fimbriae and spores and in the study of bacteriophages. The combined use of EM and gold-labelled antibodies provides a powerful tool for the ultrastructural localisation of bacterial and viral antigens.

Fig. 1

Metal shadowed Vaccinia virus—this was the original method of producing contrast in microorganisms before the advent of negative staining. Vaccinia is about 250×200 nm in size.

Fig. 2

A preparation of plasmid DNA—low-angle metal shadowing is still used to visualise nucleic acids under the electron microscope.

Fig. 3

Thin section of a cultured cell containing replicating adenoviruses. Note crystalline arrays of virus assembling within the cell nucleus. Adenoviruses are about 80 nm in diameter.

Fig. 4

Monkeypox virus negatively stained with PTA—this is a large brick-shaped virus covered in ill-defined surface filaments. Monkeypox is an orthopox virus that normally infects certain rodents, but sometimes may infect humans. Monkeypox is about 250×200 nm in diameter.

Fig. 5

Preparation of a herpesvirus from a skin lesion negatively stained with PTA. The virion is surrounded by a limiting lipid bi-layer (‘fried egg’ appearance). This envelope is structurally and functionally an integral part of the virion. The virion appears to have a hexagonal outline (icosahedral morphology) and is covered with 152 tubular capsomeres. The members of this group of viruses all look identical, but each member of the group produces different symptoms. The internal virion of all herpes group viruses is about 100 nm in diameter and the complete virus with envelope is 150–180 nm.

Fig. 6

Parvovirus B19 particles negatively stained with PTA. These small round featureless viruses have been aggregated into a clump by specific antibody. Parvoviruses are 21–26 nm in diameter.

Fig. 7

Thin section from a small intestinal biopsy showing Whipples disease bacteria (Tropheryma whippelii) in the lamina propria of an infected patient. Bacteria replicate extracellularly, but are phagocytosed by macrophages and after digestion are reduced to intracellular myelin figures.

Fig. 8

A negatively stained (PTA) preparation of Helicobacter pylori showing a terminal bunch of sheathed flagellar filaments. The flagellar sheath is thought to be acid resistant, an important adaptation to life in the gastric environment. H. pylori is 2–6 μm in length and 0.5 μm wide.

Fig. 9

A negatively stained (PTA) preparation of a bacteriophage showing its icosahedral head and contractile tail. The phage head is about 100 nm in diameter.

Fig. 10

Paramyxovirus negatively stained with PTA. The nucleic acid (RNA) is protected by proteins (nucleocapsid), which give the filament a ‘herring bone’ appearance. This nucleocapsid is contained inside a membrane envelope that features various surface proteins and appears as a fringed envelope. In this micrograph, the virus membrane has ruptured and the nucleocapsid is spilling out. The nucleocapsid is 15–20 nm wide.

Fig. 11

Two parapoxviruses negatively stained with PTA. This is a large ‘sausage-shaped’ virus with a distinctive surface filament that appears to wrap around the virion. The virus illustrated is a sheep pathogen (orf virus) that can sometimes infect the skin of humans. Orf virus is about 250×150 nm in size.

Fig. 12

Negatively stained (PTA) spheres and tubules of hepatitis B surface antigen and Dane particles (infective virus) from the blood of an infected individual. The Dane particle is 40–45 nm in diameter and the surface antigen 18–22 nm wide.

Fig. 13

Norovirus negatively stained with PTA. This small virus is from the stool specimen of a patient with ‘winter vomiting disease’. Note the fuzzy or ragged-edged morphology of this virus and the presence of some empty virions. Norovirus is 30–35 nm in diameter.

Fig. 14

A negatively stained (PTA) preparation of rotaviruses from a diarrhoeal sample of a child. Note the spokes between the two shells of this virus, giving it a ‘wheel-like’ appearance. Rotaviruses are 70–75 nm in diameter.

Fig. 15

A negatively stained (PTA) preparation of two adenoviruses from a diarrhoeal sample of a child. Note the hexagonal outline (icosahedral shape) and spherical capsomeres making up the shell of this virion. Adenoviruses are around 80 nm in diameter.

Fig. 16

Negatively stained (PTA) preparation showing a group of astroviruses from a diarrhoeal sample from a childhood case of gastroenteritis. Note the surface star that is characteristic of this virus. Astroviruses are 27–28 nm in diameter.

Fig. 17

Negatively stained (PTA) preparation showing a group of sapoviruses from a diarrhoeal sample from a childhood case of gastroenteritis. Note surface cups that are characteristic of this virus and various orientations consistent with icosahedral symmetry. Sapoviruses are 33–35 nm in diameter.

Fig. 18

Negatively stained (PTA) preparation of polyomaviruses from a urine sample of a bone marrow transplant patient with symptoms of bloody urine. Polyomaviruses are 40–45 nm in diameter.

Fig. 19

A negatively stained (PTA) preparation of the SARS virus showing the characteristic surface ‘petals’ of this newly recognised coronavirus. The SARS virus is about 100 nm in diameter.

Fig. 20

A negatively stained (PTA) preparation of Ebola virus, a cause of haemorrhagic fever. Ebola virus is about 80 nm wide, but very variable in length (600+nm).