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      Human airway epithelial cell culture to identify new respiratory viruses: Coronavirus NL63 as a model


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          Propagation of new human respiratory virus pathogens in established cell lines is hampered by a lack of predictability regarding cell line permissivity and by availability of suitable antibody reagents to detect infection in cell lines that do not exhibit significant cytopathic effect. Recently, molecular methods have been used to amplify and identify novel nucleic acid sequences directly from clinical samples, but these methods may be hampered by the quantity of virus present in respiratory secretions at different time points following the onset of infection. Human airway epithelial (HAE) cultures, which effectively mimic the human bronchial environment, allow for cultivation of a wide variety of human respiratory viral pathogens. The goal of the experiments described here was to determine if propagation and identification of a human respiratory virus may be achieved through inoculation of HAE cultures followed by whole transcriptome amplification (WTA) and sequence analysis. To establish proof-of-principle human coronavirus NL63 (HCoV-NL63) was evaluated, and the first visualization of HCoV-NL63 virus by transmission electron microscopy (TEM) is reported. Initial propagation of human respiratory secretions onto HAE cultures followed by TEM and WTA of culture supernatant may be a useful approach for visualization and detection of new human respiratory pathogens that have eluded identification by traditional approaches.

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          • Record: found
          • Abstract: found
          • Article: not found

          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
            • Record: found
            • Abstract: found
            • Article: not found

            A newly discovered human pneumovirus isolated from young children with respiratory tract disease

            From 28 young children in the Netherlands, we isolated a paramyxovirus that was identified as a tentative new member of the Metapneumovirus genus based on virological data, sequence homology and gene constellation. Previously, avian pneumovirus was the sole member of this recently assigned genus, hence the provisional name for the newly discovered virus: human metapneumovirus. The clinical symptoms of the children from whom the virus was isolated were similar to those caused by human respiratory syncytial virus infection, ranging from upper respiratory tract disease to severe bronchiolitis and pneumonia. Serological studies showed that by the age of five years, virtually all children in the Netherlands have been exposed to human metapneumovirus and that the virus has been circulating in humans for at least 50 years.
              • Record: found
              • Abstract: found
              • Article: not found

              Cloning of a human parvovirus by molecular screening of respiratory tract samples.

              The identification of new virus species is a key issue for the study of infectious disease but is technically very difficult. We developed a system for large-scale molecular virus screening of clinical samples based on host DNA depletion, random PCR amplification, large-scale sequencing, and bioinformatics. The technology was applied to pooled human respiratory tract samples. The first experiments detected seven human virus species without the use of any specific reagent. Among the detected viruses were one coronavirus and one parvovirus, both of which were at that time uncharacterized. The parvovirus, provisionally named human bocavirus, was in a retrospective clinical study detected in 17 additional patients and associated with lower respiratory tract infections in children. The molecular virus screening procedure provides a general culture-independent solution to the problem of detecting unknown virus species in single or pooled samples. We suggest that a systematic exploration of the viruses that infect humans, "the human virome," can be initiated.

                Author and article information

                J Virol Methods
                J. Virol. Methods
                Journal of Virological Methods
                Elsevier B.V.
                5 December 2008
                March 2009
                5 December 2008
                : 156
                : 1
                : 19-26
                [a ]Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, 2160 South First Avenue, Maywood, IL, United States
                [b ]Department of Pathology, George Washington University School of Medicine, Washington, DC, United States
                [c ]Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, United States
                [d ]Department of Pediatrics and Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL, United States
                Author notes
                [* ]Corresponding author at: Department of Microbiology and Immunology, Loyola University Medical center, 2160 South First Avenue, Building 105, Maywood, IL 60153, United States. Tel.: +1 708 216 6910; fax: +1 708 216 9574. sbaker1@ 123456lumc.edu
                Copyright © 2008 Elsevier B.V. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                : 22 July 2008
                : 7 October 2008
                : 13 October 2008

                Microbiology & Virology
                hcov-nl63, human coronavirus nl63,wta, whole transcriptome amplification,sars-cov, severe acute respiratory syndrome coronavirus,cpe, cytopathic effect,tem, transmission electron microscopy,hae, human airway epithelium,hae culture,coronavirus,hcov-nl63,tem,whole transcriptome amplification


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