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Chapter 5 : Electron Microscopy and Immunoelectron Microscopy
Authors:
Raymond Tellier,
John Nishikawa,
Martin Petric
Content Type: Reference
Publication Year:
2009
Category: Clinical Microbiology; Viruses and Viral Pathogenesis
Book DOI:
10.1128/9781555815974
Chapter DOI:
10.1128/9781555815974.ch5
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Electron Microscopy and Immunoelectron Microscopy, Page 1 of 2
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Abstract:
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.
Citation:
Tellier R, Nishikawa J, Petric M.
2009.
Electron Microscopy and Immunoelectron Microscopy,
p 64-76.
In
Specter S, Hodinka R, Young S, Wiedbrauk D (ed),
Clinical Virology Manual, Fourth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815974.ch5
Figures
Electron Microscopy and Immunoelectron Microscopy
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Citation:
Tellier R, Nishikawa J, Petric M.
2009.
Electron Microscopy and Immunoelectron Microscopy,
p 64-76.
In
Specter S, Hodinka R, Young S, Wiedbrauk D (ed),
Clinical Virology Manual, Fourth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815974.ch5
/content/10.1128/9781555815974.ch05.ch05fig01
FIGURE 1
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.)
10.1128/9781555815974/ch05fig1_thmb.gif
10.1128/9781555815974/ch05fig1.gif
FIGURE 1
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.)
Citation:
Tellier R, Nishikawa J, Petric M.
2009.
Electron Microscopy and Immunoelectron Microscopy,
p 64-76.
In
Specter S, Hodinka R, Young S, Wiedbrauk D (ed),
Clinical Virology Manual, Fourth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815974.ch5
/content/10.1128/9781555815974.ch05.ch05fig02
FIGURE 2
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.
10.1128/9781555815974/ch05fig2_thmb.gif
10.1128/9781555815974/ch05fig2.gif
FIGURE 2
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.
Citation:
Tellier R, Nishikawa J, Petric M.
2009.
Electron Microscopy and Immunoelectron Microscopy,
p 64-76.
In
Specter S, Hodinka R, Young S, Wiedbrauk D (ed),
Clinical Virology Manual, Fourth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815974.ch5
/content/10.1128/9781555815974.ch05.ch05fig03
FIGURE 3
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.
10.1128/9781555815974/ch05fig3_thmb.gif
10.1128/9781555815974/ch05fig3.gif
FIGURE 3
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.
Citation:
Tellier R, Nishikawa J, Petric M.
2009.
Electron Microscopy and Immunoelectron Microscopy,
p 64-76.
In
Specter S, Hodinka R, Young S, Wiedbrauk D (ed),
Clinical Virology Manual, Fourth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815974.ch5
/content/10.1128/9781555815974.ch05.ch05fig04
FIGURE 4
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.
10.1128/9781555815974/ch05fig4_thmb.gif
10.1128/9781555815974/ch05fig4.gif
FIGURE 4
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.
Citation:
Tellier R, Nishikawa J, Petric M.
2009.
Electron Microscopy and Immunoelectron Microscopy,
p 64-76.
In
Specter S, Hodinka R, Young S, Wiedbrauk D (ed),
Clinical Virology Manual, Fourth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815974.ch5
/content/10.1128/9781555815974.ch05.ch05fig05
FIGURE 5
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.)
10.1128/9781555815974/ch05fig5_thmb.gif
10.1128/9781555815974/ch05fig5.gif
FIGURE 5
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.)
Citation:
Tellier R, Nishikawa J, Petric M.
2009.
Electron Microscopy and Immunoelectron Microscopy,
p 64-76.
In
Specter S, Hodinka R, Young S, Wiedbrauk D (ed),
Clinical Virology Manual, Fourth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815974.ch5
/content/10.1128/9781555815974.ch05.ch05fig06
FIGURE 6
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.
10.1128/9781555815974/ch05fig6_thmb.gif
10.1128/9781555815974/ch05fig6.gif
FIGURE 6
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.
Citation:
Tellier R, Nishikawa J, Petric M.
2009.
Electron Microscopy and Immunoelectron Microscopy,
p 64-76.
In
Specter S, Hodinka R, Young S, Wiedbrauk D (ed),
Clinical Virology Manual, Fourth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555815974.ch5
References
/content/book/10.1128/9781555815974.ch05
1.
Almeida, J.,, B. Cinader, and, A. Howatson. 1963. The structure of antigen antibody complexes. J. Exp. Med. 118: 327– 340.
2.
Anderson, N.,, and F. W. Doane. 1972. Agar diffusion method for negative staining of microbial suspensions in salt solutions. Appl. Microbiol. 24: 495– 496.
3.
Anderson, N.,, and F. W. Doane. 1973. Specific identification of enteroviruses by immuno-electron microscopy using a serum-in-agar diffusion method. Can. J. Microbiol. 19: 585– 589.
4.
Bayer, M. E.,, B. S. Blumberg, and, B. Werner. 1968. Particles associated with Australia antigen in the sera of patients with leukemia, Down’s syndrome and hepatitis. Nature 218: 1057– 1059.
5.
Beesley, J. E.,, and M. P. Betts. 1985. Virus diagnosis: a novel use for the protein A-gold probe. Med. Lab. Sci. 42: 161– 165.
6.
Best, J. M.,, J. E. Banatvala,, J. D. Almeida, and, A. P. Waterson. 1967. Morphological characteristics of rubella virus. Lancet ii: 237– 239.
7.
Brenner, S.,, and R. W. Horne. 1959. A negative stain method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34: 103– 110.
8.
Carlson, J. A.,, P. J. Middleton,, M. T. Szymanski,, J. Huber, and, M. Petric. 1978. Fatal rotavirus gastroenteritis: an analysis of 21 cases. Am. J. Dis. Child. 132: 477– 479.
9.
Doane, F. W.,, N. Anderson,, A. Zbitnew, and, A. J. Rhodes. 1969. Application of electron microscopy to the diagnosis of virus infections. Can. Med. Assoc. J. 100: 1043– 1049.
10.
Doane, F. W.,, and N. Anderson. 1987. Electron Microscopy in Diagnostic Virology: a Practical Guide and Atlas. Cambridge University Press, New York, NY.
11.
Duckmanton, L.,, B. Luan,, J. Devenish,, R. Tellier, and, M. Petric. 1997. Characterization of torovirus from human fecal specimens. Virology 239: 158– 168.
12.
Edwards, E. A.,, W. A. Valters,, L. G. Boehm, and, M. J. Rosenbaum. 1975. Visualization by immune electron microscopy of viruses associated with acute respiratory diseases. J. Immunol. Methods 8: 159– 167.
13.
Feinstone, S. M.,, A. Z. Kapikian, and, R. H. Purcell. 1973. Hepatitis A: detection by immune electron microscopy of a virus like antigen associated with acute illness. Science 182: 1026– 1028.
14.
Geoghegan, W. D.,, and G. A. Ackerman. 1977. Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin, and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application. J. Histochem. Cytochem. 25: 1187– 1200.
15.
Gerna, G.,, A. Sarasini,, B. S. Coulson,, M. Parea,, M. Torsellini,, E. Arbustini, and, M. Battaglia. 1988. Comparative sensitivities of solid phase immune electron microscopy and enzyme linked immunosorbent assay for serotyping human rotavirus strains with neutralizing monoclonal antibodies. J. Clin. Microbiol. 26: 1383– 1387.
16.
Gregory, D. W.,, and B. J. S. Pirie. 1973. Wetting agents for biological electron microscopy. I. General considerations and negative staining. J. Microsc. 99: 261– 265.
17.
Hammond, G.,, P. R. Hazelton,, I. Chuang, and, B. Klisko. 1981. Improved detection of viruses by electron microscopy after direct ultracentrifuge preparation of specimens. J. Clin. Microbiol. 14: 210– 221.
18.
Hayat, M. A.,, and S. E. Miller. 1990. Negative Staining. McGraw Hill, New York, NY.
19.
Hazelton, P. R.,, and H. R. Gelderblom. 2003. Electron microscopy for rapid diagnosis of infectious agents in emergent situations. Emerg. Infect. Dis. 9: 294– 303.
20.
Hebert, T. T. 1963. Precipitation of plant viruses by polyethylene glycol. Phytopathology 53: 362.
21.
Hopley, J. F.,, and F. W. Doane. 1985. Development of a sensitive protein A-gold immunoelectron microscopy method for detecting viral antigens in fluid specimens. J. Virol. Methods 12: 135– 147.
22.
Howell, D. N.,, C. M. Payne,, S. E. Miller, and, J. D. Shelburne. 1998. Special techniques in diagnostic electron microscopy. Hum. Pathol. 29: 1339– 1346.
23.
Johnson, R. P. C.,, and D. W. Gregory. 1993. Viruses accumulate spontaneously near droplet surfaces: a method to concentrate viruses for electron microscopy. J. Microsc. 171: 125– 136.
24.
Kapikian, A. Z.,, R. G. Wyatt,, R. Dolin,, T. S. Thornhill,, A. R. Kalica, and, R. M. Chanock. 1972. Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. J. Virol. 10: 1075– 1081.
25.
Kapikian, A. Z.,, S. M. Feinstone,, R. H. Purcell,, R. G. Wyatt,, T. S. Thornhill,, A. R. Kalica, and, R. M. Chanock. 1975. Detection and identification by immune electron microscopy of fastidious agents associated with respiratory illness, acute non-bacterial gastroenteritis and hepatitis A. Perspect. Virol. 9: 9– 47.
26.
Lecatsas, G.,, and E. G. Boes. 1980. Urinary virus excretion in pregnancy. So. Afr. Med. J. 57: 988– 990.
27.
Lee, F. K.,, A. J. Nahmias, and, S. Stagno. 1978. Rapid diagnosis of cytomegalovirus infection in infants by electron microscopy. N. Engl. J. Med. 299: 1266– 1270.
28.
MacRae, J.,, and M. Srivastava. 1998. Detection of viruses by electron microscopy: an efficient approach. J. Virol. Methods 72: 105– 108.
29.
Middleton, P. J.,, M. T. Szymanski, and, M. Petric. 1977. Viruses associated with acute gastroenteritis in young children. Am. J. Dis. Child. 131: 733– 737.
30.
Muller, G.,, M. Bruns,, L. Martinez Peralta, and, F. Lehmann-Grube. 1983. Lymphocytic choriomeningitis virus. IV. Electron microscopic investigation of the virion. Arch. Virol. 74: 229.
31.
Murray, K.,, P. Selleck,, P. Hooper, et al. 1995. A morbillivirus that caused fatal disease in horses and humans. Science 268: 94– 97.
32.
Peiris, J. S.,, S. T. Lai,, L. L. Poon,, Y. Guan,, L. Y. Yam,, W. Lim,, J. Nicholls,, W. K. Yee,, W. W. Yan,, M. T. Cheung,, V. C. Cheng,, K. H. Chan,, D. N. Tsang,, R. W. Yung,, T. K. Ng, and, K. Y. Yuen. 2003. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361: 1319– 1325.
33.
Petric, M.,, and R. Tellier. 2003. Rotaviruses, caliciviruses, astroviruses and other diarrheic viruses, p. 1439–1451. In P. R. Murray,, E. J. Baron,, J. H. Jorgensen,, M. A. Pfaller, and, R. H. Yolken (ed.), Manual of Clinical Microbiology, 8th ed. ASM Press, Washington, DC.
34.
Petrovicova, A.,, and A. S. Juck. 1977. Serotyping of coxsackieviruses by immune electron microscopy. Acta Virol. 21: 165– 167.
35.
Stannard, L. M.,, M. Lennon,, M. Hodgkiss, and, H. Smuts. 1982. An electron microscopic demonstration of immune complexes of hepatitis B e-antigen using colloidal gold as a marker. J. Med. Virol. 9: 165– 175.
36.
van Rooyen, C. E.,, and M. A. Scott. 1948. Smallpox diagnosis with special reference to electron microscopy. Can. J. Pub. Health 39: 467– 477.