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Chapter 5 : Electron Microscopy and Immunoelectron Microscopy
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In clinical virology, electron microscopy (EM) has achieved a role equivalent to that of conventional light microscopy in clinical microbiology. EM allows for the rapid detection of the virus in a clinical specimen, at least at the level of the family into which it is classified, with a very high degree of specificity. Immunoelectron microscopy (IEM) arose from the combination of EM with the immunospecific interaction of viruses with their respective antibodies. Several methods such as negative staining methods, direct-application method and water drop method will concern only the direct visualization of viruses after negative staining. EM was instrumental in the identification of hendraviruses in cell cultures when this virus first emerged. It has a well-established potential for the rapid differentiation of varicella-zoster virus from poxviruses in skin lesions. With developments of enzyme immunoassays and molecular approaches such as polymerase chain reaction (PCR) and reverse transcription-PCR, EM is now mainly used in reference laboratories or laboratories in tertiary health care centers.
Gastroenteritis viruses detected in stool specimens by EM using the direct-application method. (A) Calicivirus; (B) Norwalk-like virus; (C) astrovirus; (D) small round virus; (E) adenovirus; (F) coronavirus; (G) torovirus-like particles. Bars, 100 nm. (Reprinted from Petric and Tellier, 2003, with permission.)
Microorganisms diagnosed by EM using the direct-application method. (A) Reovirus; (B) rotavirus. Note differences in arrangement of capsomeres between rotavirus and reovirus. Arrows indicate single capsid particles. (C) Campylobacter. Note sinusoidal appearance with bipolar flagella. Bars, 100 nm.
Viruses seen by EM in lesion specimens from skin or mucous membrane secretions. (A) Herpes group virus from herpes, varicella, or shingles; (B) papovavirus from respiratory tract secretions of an immunocompromised patient; (C) molluscipoxvirus from molluscum contagiosum; (D) parapoxvirus from orf lesions. Bars, 100 nm.
Agents seen in EM examination of cell cultures showing CPE. (A) Respiratory syncytial virus. Virus particles are generally intact with well-defined fringe of spikes. (B) Parainfluenza virus. Typically, most particles are broken, with the nucleocapsid visible as herringbone rods. (C) Influenza virus. Note well-defined spike proteins. (D) Mycoplasma hyorrhinis from a contaminated cell culture. (E) Foamy agent seen in contaminated cell cultures. (F) Rubella virus from infected cell culture. The preparation was treated with glutaraldehyde to stabilize the viruses. Bars, 100 nm.
IEM of viruses. (A) Human torovirus-like particles reacted with patient convalescent-phase serum. (B) Astrovirus from a stool specimen reacted with antiserum produced in guinea pig. (C) Serum from an HBV-infected patient reacted with reference antiserum to hepatitis B surface antigen. Note intact hepatitis B virions (arrow) and 22-nm-diameter surface antigen spheres. (D) Rotavirus reacted with reference antibody and protein A labeled with colloidal gold. Note association of gold granules with the virus particles. Bars, 100 nm. (Panel D reprinted from Hopley and Doane, 1985, with permission.)
Examples of emerging and newly described viruses. (A) Severe acure respiratory syndrome coronavirus from inoculated cell culture. (B) Human metapneumovirus (hMPV). Left panel: hMPV in a nasopharyngeal sample directly examined by EM. The finding of virions by direct sample examination suggests a high viral load. Right panel: hMPV grown in cell culture (R-Mix). Bars, 100 nm.