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      Chicken single-chain variable fragments against the SARS-CoV spike protein

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          The major concern for severe acute respiratory syndrome (SARS), caused by the SARS-associated coronavirus (SARS-CoV), is the lack of diagnostic and therapeutic agents. Using a phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the SARS-CoV spike protein were characterized. Ten truncated spike protein gene fragments were expressed in Escherichia coli cells. Following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scFv) antibody libraries were established with short or long linkers to contain 5 × 10 7 and 9 × 10 6 transformants, respectively. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were demonstrated by Coomassie blue staining, and verified by western blot analysis. In a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scFv antibodies reacted significantly with SARS-CoV-infected Vero cells, and that those two specific scFv antibodies recognized the same region of the spike protein spanning amino acid residues 750–1000. In conclusion, the results suggest that the chicken scFv phage display system can be a potential model for mass production of high-affinity antibodies against the SARS-CoV spike protein.

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          Most cited references 29

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

          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.

            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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              Characterization of a novel coronavirus associated with severe acute respiratory syndrome.

               P Rota (2003)
              In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.

                Author and article information

                J Virol Methods
                J. Virol. Methods
                Journal of Virological Methods
                Elsevier B.V.
                23 July 2007
                December 2007
                23 July 2007
                : 146
                : 1
                : 104-111
                [a ]Graduate Institute of Pharmaceutical Science, Taipei Medical University, Taipei, Taiwan
                [b ]Graduate Institute of Cell and Molecular Biology, Taipei Medical University, Taipei, Taiwan
                [c ]Department of Neurology, Taipei Medical University, Taipei, Taiwan
                [d ]National Institute of Cancer Research, National Health Research Institutes, Taipei, Taiwan
                [e ]Applied Bioscience Division, Taiwan Sugar Research Institute, Tainan, Taiwan
                [f ]School of Medical Laboratory Science and Biotechnology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan
                [g ]Department of Laboratory Medicine, Mackay Memorial Hospital, Taipei, Taiwan
                [h ]Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan
                [i ]Department of Neurosurgery, Taipei Municipal Wan Fang Hospital, Taipei, Taiwan
                Author notes
                [* ]Corresponding author. Tel.: +886 2 27361661x3325; fax: +886 2 27324510. yangyuan@ 123456tmu.edu.tw
                Copyright © 2007 Elsevier B.V. All rights reserved.

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