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      Time Course and Cellular Localization of SARS-CoV Nucleoprotein and RNA in Lungs from Fatal Cases of SARS

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          Abstract

          Background

          Cellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) in the lungs of patients with SARS is important in confirming the etiological association of the virus with disease as well as in understanding the pathogenesis of the disease. To our knowledge, there have been no comprehensive studies investigating viral infection at the cellular level in humans.

          Methods and Findings

          We collected the largest series of fatal cases of SARS with autopsy material to date by merging the pathological material from two regions involved in the 2003 worldwide SARS outbreak in Hong Kong, China, and Toronto, Canada. We developed a monoclonal antibody against the SARS-CoV nucleoprotein and used it together with in situ hybridization (ISH) to analyze the autopsy lung tissues of 32 patients with SARS from Hong Kong and Toronto. We compared the results of these assays with the pulmonary pathologies and the clinical course of illness for each patient. SARS-CoV nucleoprotein and RNA were detected by immunohistochemistry and ISH, respectively, primarily in alveolar pneumocytes and, less frequently, in macrophages. Such localization was detected in four of the seven patients who died within two weeks of illness onset, and in none of the 25 patients who died later than two weeks after symptom onset.

          Conclusions

          The pulmonary alveolar epithelium is the chief target of SARS-CoV, with macrophages infected subsequently. Viral replication appears to be limited to the first two weeks after symptom onset, with little evidence of continued widespread replication after this period. If antiviral therapy is considered for future treatment, it should be focused on this two-week period of acute clinical disease.

          Abstract

          The SARS coronavirus targets primarily the pulmonary alveolar epithelium. Viral replication seems limited to the first two weeks after symptom onset and restricted to the lungs.

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          Most cited references28

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          A novel coronavirus associated with severe acute respiratory syndrome.

          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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            Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China.

            Y Guan (2003)
            A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome (SARS). SCoV-like viruses were isolated from Himalayan palm civets found in a live-animal market in Guangdong, China. Evidence of virus infection was also detected in other animals (including a raccoon dog, Nyctereutes procyonoides) and in humans working at the same market. All the animal isolates retain a 29-nucleotide sequence that is not found in most human isolates. The detection of SCoV-like viruses in small, live wild mammals in a retail market indicates a route of interspecies transmission, although the natural reservoir is not known.
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              Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome

              Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel coronavirus, but its immunopathological mechanisms have not yet been fully elucidated. We investigated changes in plasma T helper (Th) cell cytokines, inflammatory cytokines and chemokines in 20 patients diagnosed with SARS. Cytokine profile of SARS patients showed marked elevation of Th1 cytokine interferon (IFN)-γ, inflammatory cytokines interleukin (IL)-1, IL-6 and IL-12 for at least 2 weeks after disease onset, but there was no significant elevation of inflammatory cytokine tumour necrosis factor (TNF)-α, anti-inflammatory cytokine IL-10, Th1 cytokine IL-2 and Th2 cytokine IL-4. The chemokine profile demonstrated significant elevation of neutrophil chemokine IL-8, monocyte chemoattractant protein-1 (MCP-1), and Th1 chemokine IFN-γ-inducible protein-10 (IP-10). Corticosteroid reduced significantly IL-8, MCP-1 and IP-10 concentrations from 5 to 8 days after treatment (all P < 0·001). Together, the elevation of Th1 cytokine IFN-γ, inflammatory cytokines IL-1, IL-6 and IL-12 and chemokines IL-8, MCP-1 and IP-10 confirmed the activation of Th1 cell-mediated immunity and hyperinnate inflammatory response in SARS through the accumulation of monocytes/macrophages and neutrophils.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Med
                pmed
                PLoS Medicine
                Public Library of Science (San Francisco, USA )
                1549-1277
                1549-1676
                February 2006
                3 January 2006
                : 3
                : 2
                : e27
                Affiliations
                [1] 1Department of Pathology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR, China
                [2] 2Department of Pathology, University of Toronto, Toronto General Hospital, Toronto, Ontario, Canada
                [3] 3Department of Microbiology, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR, China
                [4] 4Department of Microbiology, Toronto Medical Laboratories and Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada
                Centers for Disease Control United States of America
                Author notes
                * To whom correspondence should be addressed. E-mail: nicholls@ 123456pathology.hku.hk (JMN); E-mail: mwong@ 123456pathology.hku.hk (MW)

                Competing Interests: The authors have declared that no competing interests exist.

                Author Contributions: JMN designed the study. JB analyzed the data. KHC contributed to writing the paper. JMN, MW, and JSMP are the principal investigators, with overall responsibility for the design of the study and writing of the report. SLB coordinated the autopsy organization and collection of autopsy materials of SARS deaths in Hong Kong. LLMP designed the protein expression constructs and transfection studies. LLMP and JSMP were involved in the development of the RT-PCR assay. JMN and KHC were involved in the development of the SARS-CoV IHC assay. SP was involved in the treatment of the Canadian SARS cases and the coordination of obtaining autopsy specimens from those who died. All authors were involved in the correlative interpretation of the clinical, pathological, and molecular data.

                Article
                10.1371/journal.pmed.0030027
                1324951
                16379499
                7f5c7083-0398-4506-a081-790983613039
                Copyright: © 2006 Nicholls et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 23 August 2005
                : 24 October 2005
                Categories
                Research Article
                Infectious Diseases
                Microbiology
                Virology
                Pathology
                Respiratory Medicine
                Infectious Diseases
                Lung Function
                Pathology
                Respiratory Medicine
                Travel Medicine

                Medicine
                Medicine

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