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Abstract

Viruses are very small and most of them can be seen only by TEM (transmission electron microscopy). TEM has therefore made a major contribution to virology, including the discovery of many viruses, the diagnosis of various viral infections and fundamental investigations of virus-host cell interactions. However, TEM has gradually been replaced by more sensitive methods, such as the PCR. In research, new imaging techniques for fluorescence light microscopy have supplanted TEM, making it possible to study live cells and dynamic interactions between viruses and the cellular machinery. Nevertheless, TEM remains essential for certain aspects of virology. It is very useful for the initial identification of unknown viral agents in particular outbreaks, and is recommended by regulatory agencies for investigation of the viral safety of biological products and/or the cells used to produce them. In research, only TEM has a resolution sufficiently high for discrimination between aggregated viral proteins and structured viral particles. Recent examples of different viral assembly models illustrate the value of TEM for improving our understanding of virus-cell interactions.

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Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome

Christian Drosten, Stephan Günther, Wolfgang Preiser … (2003)

The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. The novel coronavirus might have a role in causing SARS. Copyright 2003 Massachusetts Medical Society

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A novel coronavirus associated with severe acute respiratory syndrome.

Thomas G Ksiazek, Dean Erdman, Cynthia Goldsmith … (2003)

A worldwide outbreak of severe acute respiratory syndrome (SARS) has been associated with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiologic agent of this outbreak. We received clinical specimens from patients in seven countries and tested them, using virus-isolation techniques, electron-microscopical and histologic studies, and molecular and serologic assays, in an attempt to identify a wide range of potential pathogens. None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathological features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examination revealed ultrastructural features characteristic of coronaviruses. Immunohistochemical and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers we identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate have been used to demonstrate a virus-specific serologic response. This virus may never before have circulated in the U.S. population. A novel coronavirus is associated with this outbreak, and the evidence indicates that this virus has an etiologic role in SARS. Because of the death of Dr. Carlo Urbani, we propose that our first isolate be named the Urbani strain of SARS-associated coronavirus. Copyright 2003 Massachusetts Medical Society

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The detection of monkeypox in humans in the Western Hemisphere.

Kurt Reed, John Melski, Mary Graham … (2004)

During May and June 2003, an outbreak of febrile illness with vesiculopustular eruptions occurred among persons in the midwestern United States who had had contact with ill pet prairie dogs obtained through a common distributor. Zoonotic transmission of a bacterial or viral pathogen was suspected. We reviewed medical records, conducted interviews and examinations, and collected blood and tissue samples for analysis from 11 patients and one prairie dog. Histopathological and electron-microscopical examinations, microbiologic cultures, and molecular assays were performed to identify the etiologic agent. The initial Wisconsin cases evaluated in this outbreak occurred in five males and six females ranging in age from 3 to 43 years. All patients reported having direct contact with ill prairie dogs before experiencing a febrile illness with skin eruptions. We found immunohistochemical or ultrastructural evidence of poxvirus infection in skin-lesion tissue from four patients. Monkeypox virus was recovered in cell cultures of seven samples from patients and from the prairie dog. The virus was identified by detection of monkeypox-specific DNA sequences in tissues or isolates from six patients and the prairie dog. Epidemiologic investigation suggested that the prairie dogs had been exposed to at least one species of rodent recently imported into the United States from West Africa. Our investigation documents the isolation and identification of monkeypox virus from humans in the Western Hemisphere. Infection of humans was associated with direct contact with ill prairie dogs that were being kept or sold as pets. Copyright 2004 Massachusetts Medical Society

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PubMed ID:: 18627353

DOI:: 10.1042/BC20070173

ScienceOpen disciplines: Chemistry

Keywords: Animals,DNA, Viral,diagnostic use,ultrastructure,HIV-1,Hepacivirus,physiology,Hepatitis B,diagnosis,Hepatitis B virus,Hepatitis C,Humans,Microscopy, Electron,RNA, Viral,Virus Assembly,Virus Replication

Viral detection by electron microscopy: past, present and future.

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Atomic Force Microscopy

Most cited references 78

Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome

A novel coronavirus associated with severe acute respiratory syndrome.

The detection of monkeypox in humans in the Western Hemisphere.

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