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Chapter 7 : Imaging Viruses and Tagging Their Antigens

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Abstract:

This chapter first examines the refinements in electron microscopy (EM) that greatly expanded the understanding of the structure and replication of viruses and facilitated the application of EM to viral diagnosis. The refinements included thin sectioning, negative staining, and immunoelectron microscopy (IEM) developed for clinical diagnostic work. The use of EM in discovering the viral causes of acute gastroenteritis and infectious hepatitis are then considered. The chapter talks about the use of fluorescence microscopy for rapid viral diagnosis. It is paradoxical that IEM successfully defined the viral etiology of one of the acute nonbacterial gastroenteritis syndromes, winter vomiting disease, before basic EM defined the viral etiology of another highly prevalent gastroenteritis, acute infantile diarrhea. M. Beem and Phillip Gardner found that the respiratory syncytial virus (RSV) was inactivated by freeze-thaw specimen preparation, and specimens had to be immediately inoculated into culture for successful isolation. Further studies were performed examining the serological response to RSV. For developing countries, techniques for the rapid diagnosis of rabies, viral hepatitis, and rotavirus were encouraged. As the movement for rapid viral diagnostics gained momentum, numerous methods for antigen detection were implemented.

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7

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Figure 1

Sydney Brenner. With Robert Horne, Brenner developed negative staining to rapidly screen a large number of T-even bacteriophage fractions by EM. It became a crucial tool in the investigation and classification of viruses. Brenner continued fundamental work in molecular biology, including the triplet nature of the genetic code and the demonstration of mRNA. He won a Nobel Prize in 2002 for the development of a unique model system with which to study organ development. (Courtesy of the Salk Institute for Biological Studies.) doi:10.1128/9781555818586.ch7.f1

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 2

Robert Horne standing beside a poster showing the atomic lattice of gold after optical linear integration of an electron micrograph of gold foil. Photograph taken by Alec Bangham, 1973. With Sydney Brenner, Robert Horne developed the technique of negative staining in a study of the components of a T-even bacteriophage. This technique was to revolutionize the morphological study of all types of viruses. (Courtesy of J. Robin Harris and Edward A. Munn, , Elsevier [ ].) doi:10.1128/9781555818586.ch7.f2

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 3

“The first electron micrographs of negatively stained bacteriophages, prepared by Bob Horne.” (Courtesy of J. Robin Harris and Edward A. Munn, , Elsevier [ ].) doi:10.1128/9781555818586.ch7.f3

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 4

Adenovirus, negative stain. Negative staining allowed the demonstration of subunit construction of viruses. With nucleic acid type, the architecture revealed by negative staining served as a basis for classification of viruses. (Photo by C. K. Y. Fong. Collection of Marilyn J. August.) doi:10.1128/9781555818586.ch7.f4

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 5

TMV prepared by Robert Horne using the Horne and Pasquali-Ronchetti “Negative Staining-Carbon Film technique” showing two-dimensional paracrystalline/crystalline arrays of viruses. (Courtesy of J. Robin Harris and Edward A. Munn, , Elsevier.) doi:10.1128/9781555818586.ch7.f5

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 6

June Almeida. With training at the technical level, Almeida went on to receive a doctorate based on the body of her work. She pioneered the application of EM to clinical diagnosis, including IEM. (Courtesy of Joyce Almeida.) doi:10.1128/9781555818586.ch7.f6

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 7

John Zahorsky, a pediatrician who first described “winter vomiting disease.” He characterized the clinical characteristics of outbreaks as early as 1925. Later, the illness was called acute nonbacterial gastroenteritis. A characteristic outbreak in Norwalk, OH, in 1968, investigated by the CDC, resulted in the isolation of the Norwalk agent, soon imaged by Kapikian and colleagues. (Reprinted with permission from [ ].) doi:10.1128/9781555818586.ch7.f7

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 8

Norovirus, a cause of acute viral gastroenteritis. Originally termed Norwalk agent, it was found by Albert Kapikian and colleagues at the NIH using antibodies to aggregate the virus by IEM. Kapikian had studied in the laboratory of June Almeida, where he learned the technique. (Courtesy of the CDC, Public Health Image Library.) doi:10.1128/9781555818586.ch7.f8

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 9

Albert Kapikian. One of the members of the LID of the NIH recruited by Robert Huebner, Kapikian was to make seminal contributions to the understanding of viral gastroenteritis. He was the first to demonstrate the Norwalk agent to be a virus (norovirus), assisted in the demonstration of hepatitis A virus, and was one of the first investigators to demonstrate rotavirus associated with acute infantile diarrhea. Kapikian is shown seated (center) with Robert Chanock, seated on the left. (Courtesy of the NIH.) doi:10.1128/9781555818586.ch7.f9

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 10

Discoverers of rotavirus as the cause of acute infantile diarrhea. Ruth Bishop (left) of Melbourne, Australia, was the first to report the virus of infantile diarrhea by EM in biopsy samples of the duodenum (photo courtesy of The Royal Children’s Hospital, Melbourne). Thomas Flewett (right) of Birmingham, England, established the presence of rotavirus in stool specimens by EM (source of photo unknown). Albert Kapikian of the NIH, Bethesda, MD (not pictured here), identified the virus by using IEM and conventional EM (see Figure 9 ). doi:10.1128/9781555818586.ch7.f10

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 11

Rotavirus in stool. This negative-stained preparation shows the characteristic wheel-like appearance of rotavirus. The name was derived from the Latin , for “wheel.” (Collection of Marilyn J. August.) doi:10.1128/9781555818586.ch7.f11

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 12

Albert Coons, the originator of the FA staining technique. The technique identified antigens in tissues using antibodies tagged with compounds which would fluoresce under illumination by ultraviolet light. It allowed the development of rapid viral diagnosis. (Courtesy of the Albert Lasker Foundation.) doi:10.1128/9781555818586.ch7.f12

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 13

Clinical specimen diagnosed as RSV by immunofluorescence. Rapid viral diagnosis was revolutionized with the implementation of immunofluorescence staining techniques. A sample collected on a swab from the nasopharynx was processed in the laboratory, and harvested cells were used to prepare a smear for staining. This is a direct immunofluorescent stain for RSV showing typical apple-green staining in the cytoplasm of infected cells against a background of negative cells counterstained with Evans blue. (Courtesy of Indiana Pathology Images.) doi:10.1128/9781555818586.ch7.f13

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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Figure 14

Phillip Gardner. With Joyce McQuillin, Gardner pioneered the use of fluorescent-antibody staining techniques for the rapid diagnosis of viral infections. He also played a crucial role in the creation of a group for the advancement of rapid viral diagnosis in Europe and encouraged a similar group in North America. He is shown here reviewing the book he coauthored with Joyce McQuillin. (Courtesy of Dick Madeley and June Almeida.) doi:10.1128/9781555818586.ch7.f14

Citation: Booss J, August M. 2013. Imaging Viruses and Tagging Their Antigens, p 197-248. In To Catch a Virus. ASM Press, Washington, DC. doi: 10.1128/9781555818586.ch7
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