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Kwokyung Yuen - One of the best experts on this subject based on the ideXlab platform.
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the effects of temperature and relative humidity on the viability of the SARS Coronavirus
Advances in Virology, 2011Co-Authors: Kwokhung Chan, Kwokyung Yuen, J Malik S Peiris, Leo L M Poon, W H SetoAbstract:The main route of transmission of SARS CoV infection is presumed to be respiratory droplets. However the virus is also detectable in other body fluids and excreta. The stability of the virus at different temperatures and relative humidity on smooth surfaces were studied. The dried virus on smooth surfaces retained its viability for over 5 days at temperatures of 22–25°C and relative humidity of 40–50%, that is, typical air-conditioned environments. However, virus viability was rapidly lost (>3 log10) at higher temperatures and higher relative humidity (e.g., 38°C, and relative humidity of >95%). The better stability of SARS Coronavirus at low temperature and low humidity environment may facilitate its transmission in community in subtropical area (such as Hong Kong) during the spring and in air-conditioned environments. It may also explain why some Asian countries in tropical area (such as Malaysia, Indonesia or Thailand) with high temperature and high relative humidity environment did not have major community outbreaks of SARS.
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cleavage of spike protein of SARS Coronavirus by protease factor xa is associated with viral infectivity
Biochemical and Biophysical Research Communications, 2007Co-Authors: Richard Y T Kao, Dongyan Jin, Kwokyung Yuen, Shi Bo Jiang, Yusen Zhou, Guangyu Zhao, Charlotte K L Wong, Bojian ZhengAbstract:The spike (S) protein of SARS Coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection.
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SARS Coronavirus spike polypeptide dna vaccine priming with recombinant spike polypeptide from escherichia coli as booster induces high titer of neutralizing antibody against SARS Coronavirus
Vaccine, 2005Co-Authors: Patrick C Y Woo, Susanna K P Lau, Beatrice H L Wong, Hoiwah Tsoi, Kwokhung Chan, Zhiwei Chen, Linqi Zhang, Jim Kh H Chan, Lei Po Wong, Kwokyung YuenAbstract:Different forms of SARS Coronavirus (SARS-CoV) spike protein-based vaccines for generation of neutralizing antibody response against SARS-CoV were compared using a mouse model. High IgG levels were detected in mice immunized with intraperitoneal (i.p.) recombinant spike polypeptide generated by Escherichia coli (S-peptide), mice primed with intramuscular (i.m.) tPA-optimize800 DNA vaccine (tPA-S-DNA) and boosted with i.p. S-peptide, mice primed with i.m. CTLA4HingeSARS800 DNA vaccine (CTLA4-S-DNA) and boosted with i.p. S-peptide, mice primed with oral live-attenuated Salmonella typhimurium (Salmonella-S-DNA-control) and boosted with i.p. S-peptide, mice primed with oral live-attenuated S. typhimurium that contained tPA-optimize800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide, and mice primed with oral live-attenuated S. typhimurium that contained CTLA4HingeSARS800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide. No statistical significant difference was observed among the Th1/Th2 index among these six groups of mice with high IgG levels. Sera of all six mice immunized with i.p. S-peptide, i.m. DNA vaccine control and oral Salmonella-S-DNA-control showed no neutralizing antibody against SARS-CoV. Sera of the mice immunized with i.m. tPA-S-DNA, i.m. CTLA4-S-DNA, oral Salmonella-S-DNA-control boosted with i.p. S-peptide, oral Salmonella-tPA-S-DNA, oral Salmonella-tPA-S-DNA boosted with i.p S-peptide, oral Salmonella-CTLA4-S-DNA and oral Salmonella-CTLA4-S-DNA boosted with i.p. S-peptide showed neutralizing antibody titers of or=1:1280. The present observation may have major practical value, such as immunization of civet cats, since production of recombinant proteins from E. coli is far less expensive than production of recombinant proteins using eukaryotic systems.
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detection of severe acute respiratory syndrome SARS Coronavirus nucleocapsid protein in SARS patients by enzyme linked immunosorbent assay
Journal of Clinical Microbiology, 2004Co-Authors: Susanna K P Lau, Patrick C Y Woo, Beatrice H L Wong, Hoiwah Tsoi, Gibson K S Woo, Rosana W S Poon, Kwokhung Chan, William I Wei, J S Peiris, Kwokyung YuenAbstract:We report the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of severe acute respiratory syndrome (SARS) Coronavirus (CoV) nucleocapsid protein. The assay was carried out with hyperimmune polyclonal nucleocapsid-specific antibodies from guinea pigs and rabbits immunized with recombinant His6-tagged SARS CoV nucleocapsid protein. The assay was used for the detection of SARS CoV nucleocapsid protein in nasopharyngeal aspirate, urine, and fecal samples collected from patients with confirmed SARS between days 2 and 33 after the onset of illness. The ELISA was capable of detecting this protein in SARS CoV cell culture lysates at 15 50% tissue culture infective doses/ml but did not produce positive signals when tested with cell culture lysates of human Coronaviruses OC43 and 229E. When tested with 120 nasopharyngeal aspirate, 100 urine, and 100 fecal specimens from hospitalized patients without SARS, the assay was shown to have high specificities—96.7, 99, and 96%, respectively. In an evaluation of clinical specimens from SARS patients, 34 (52%) of 66 nasopharyngeal aspirate samples from 50 patients, 5 (5%) of 94 urine samples from 94 patients, and 36 (55%) of 65 fecal samples from 65 patients tested positive for SARS CoV nucleocapsid protein. Nucleocapsid protein could be detected from days 6 to 24 in nasopharyngeal aspirate specimens, from days 11 to 31 in urine specimens, and from days 8 to 32 in fecal specimens after the onset of illness. Moreover, the protein could be detected in 25 (83%) of 30 nasopharyngeal aspirate specimens obtained from days 11 to 15 and in all 7 fecal specimens obtained from days 21 to 32. Since the present ELISA is more convenient and economical than reverse transcription-PCR, it may serve as an alternative tool for the early diagnosis of SARS CoV infection in laboratories with limited resources and expertise and for mass screening for the reservoir of SARS CoV. Further studies on serial clinical specimens should reveal the duration of nucleocapsid protein shedding and may reveal a higher detection rate in SARS patients.
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rapid detection of the severe acute respiratory syndrome SARS Coronavirus by a loop mediated isothermal amplification assay
Clinical Chemistry, 2004Co-Authors: Leo L M Poon, Kwokhung Chan, Kwokyung Yuen, Yi Guan, Cynthia S W Leung, Masato Tashiro, Bonnie W Y Wong, Jsm PeirisAbstract:Severe acute respiratory syndrome (SARS) is a newly emerging disease that first emerged in Guangdong Province, China in November 2002 (1). The SARS Coronavirus (SARS-CoV) was found to be the etiology of the disease (2)(3)(4). Subsequent surveillance studies have indicated that this virus is of animal origin and have suggested that the source of the disease is still circulating in this geographic region (5). Indeed, the potential risk of reemergence of SARS is further highlighted by a recent confirmed SARS case in January 2004 (6). Therefore, the establishment of a rapid SARS diagnostic method is a high priority for control of the disease. Currently, there are two major diagnostic approaches for SARS. Detection of antibodies against SARS-CoV is a sensitive and specific diagnostic approach, but serconversion can be detected only around day 10 of illness (7). In contrast, PCR-based tests have been shown to be useful for early SARS diagnosis (8). Quantitative PCR approaches are a powerful tool for identifying SARS-CoV early after disease onset (4)(9)(10)(11). However, because of the requirements for sophisticated instrumentation and expensive reagents, these rapid molecular tests might not be the method of choice in basic clinical settings in developing countries or in field situations. It is therefore critical to develop simple and economical molecular tests for the above scenarios. The invention of loop-mediated isothermal amplification (LAMP) has opened up a new horizon for molecular diagnosis (12). This method depends on autocycling strand displacement DNA synthesis performed by a Bst DNA polymerase, and a detailed amplification mechanism has been described elsewhere (12). The reaction relies on recognition of the DNA target by six independent sequences, making this kind of assay highly specific. This method is rapid and has a DNA amplification efficiency equivalent to that of …
Kwokhung Chan - One of the best experts on this subject based on the ideXlab platform.
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the effects of temperature and relative humidity on the viability of the SARS Coronavirus
Advances in Virology, 2011Co-Authors: Kwokhung Chan, Kwokyung Yuen, J Malik S Peiris, Leo L M Poon, W H SetoAbstract:The main route of transmission of SARS CoV infection is presumed to be respiratory droplets. However the virus is also detectable in other body fluids and excreta. The stability of the virus at different temperatures and relative humidity on smooth surfaces were studied. The dried virus on smooth surfaces retained its viability for over 5 days at temperatures of 22–25°C and relative humidity of 40–50%, that is, typical air-conditioned environments. However, virus viability was rapidly lost (>3 log10) at higher temperatures and higher relative humidity (e.g., 38°C, and relative humidity of >95%). The better stability of SARS Coronavirus at low temperature and low humidity environment may facilitate its transmission in community in subtropical area (such as Hong Kong) during the spring and in air-conditioned environments. It may also explain why some Asian countries in tropical area (such as Malaysia, Indonesia or Thailand) with high temperature and high relative humidity environment did not have major community outbreaks of SARS.
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SARS Coronavirus spike polypeptide dna vaccine priming with recombinant spike polypeptide from escherichia coli as booster induces high titer of neutralizing antibody against SARS Coronavirus
Vaccine, 2005Co-Authors: Patrick C Y Woo, Susanna K P Lau, Beatrice H L Wong, Hoiwah Tsoi, Kwokhung Chan, Zhiwei Chen, Linqi Zhang, Jim Kh H Chan, Lei Po Wong, Kwokyung YuenAbstract:Different forms of SARS Coronavirus (SARS-CoV) spike protein-based vaccines for generation of neutralizing antibody response against SARS-CoV were compared using a mouse model. High IgG levels were detected in mice immunized with intraperitoneal (i.p.) recombinant spike polypeptide generated by Escherichia coli (S-peptide), mice primed with intramuscular (i.m.) tPA-optimize800 DNA vaccine (tPA-S-DNA) and boosted with i.p. S-peptide, mice primed with i.m. CTLA4HingeSARS800 DNA vaccine (CTLA4-S-DNA) and boosted with i.p. S-peptide, mice primed with oral live-attenuated Salmonella typhimurium (Salmonella-S-DNA-control) and boosted with i.p. S-peptide, mice primed with oral live-attenuated S. typhimurium that contained tPA-optimize800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide, and mice primed with oral live-attenuated S. typhimurium that contained CTLA4HingeSARS800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide. No statistical significant difference was observed among the Th1/Th2 index among these six groups of mice with high IgG levels. Sera of all six mice immunized with i.p. S-peptide, i.m. DNA vaccine control and oral Salmonella-S-DNA-control showed no neutralizing antibody against SARS-CoV. Sera of the mice immunized with i.m. tPA-S-DNA, i.m. CTLA4-S-DNA, oral Salmonella-S-DNA-control boosted with i.p. S-peptide, oral Salmonella-tPA-S-DNA, oral Salmonella-tPA-S-DNA boosted with i.p S-peptide, oral Salmonella-CTLA4-S-DNA and oral Salmonella-CTLA4-S-DNA boosted with i.p. S-peptide showed neutralizing antibody titers of or=1:1280. The present observation may have major practical value, such as immunization of civet cats, since production of recombinant proteins from E. coli is far less expensive than production of recombinant proteins using eukaryotic systems.
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in vitro susceptibility of 10 clinical isolates of SARS Coronavirus to selected antiviral compounds
Journal of Clinical Virology, 2004Co-Authors: Feng Chen, Kwokhung Chan, Y Jiang, Richard Y T Kao, K W Fan, Vincent C C Cheng, W H W Tsui, Ivan Hung, T S W Lee, Yi GuanAbstract:Effective antiviral agents are urgently needed to combat the possible return of severe acute respiratory syndrome (SARS). Commercial antiviral agents and pure chemical compounds extracted from traditional Chinese medicinal herbs were screened against 10 clinical isolates of SARS Coronavirus by neutralisation tests with confirmation by plaque reduction assays. Interferon-beta-1a, leukocytic interferon-alpha, ribavirin, lopinavir, rimantadine, baicalin and glycyrrhizin showed antiviral activity. The two interferons were only active if the cell lines were pre-incubated with the drugs 16 h before viral inoculation. Results were confirmed by plaque reduction assays. Antiviral activity varied with the use of different cell lines. Checkerboard assays for synergy were performed showing combinations of interferon beta-1a or leukocytic interferon-alpha with ribavirin are synergistic. Since the clinical and toxicity profiles of these agents are well known, they should be considered either singly or in combination for prophylaxis or treatment of SARS in randomised placebo controlled trials in future epidemics.
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detection of severe acute respiratory syndrome SARS Coronavirus nucleocapsid protein in SARS patients by enzyme linked immunosorbent assay
Journal of Clinical Microbiology, 2004Co-Authors: Susanna K P Lau, Patrick C Y Woo, Beatrice H L Wong, Hoiwah Tsoi, Gibson K S Woo, Rosana W S Poon, Kwokhung Chan, William I Wei, J S Peiris, Kwokyung YuenAbstract:We report the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of severe acute respiratory syndrome (SARS) Coronavirus (CoV) nucleocapsid protein. The assay was carried out with hyperimmune polyclonal nucleocapsid-specific antibodies from guinea pigs and rabbits immunized with recombinant His6-tagged SARS CoV nucleocapsid protein. The assay was used for the detection of SARS CoV nucleocapsid protein in nasopharyngeal aspirate, urine, and fecal samples collected from patients with confirmed SARS between days 2 and 33 after the onset of illness. The ELISA was capable of detecting this protein in SARS CoV cell culture lysates at 15 50% tissue culture infective doses/ml but did not produce positive signals when tested with cell culture lysates of human Coronaviruses OC43 and 229E. When tested with 120 nasopharyngeal aspirate, 100 urine, and 100 fecal specimens from hospitalized patients without SARS, the assay was shown to have high specificities—96.7, 99, and 96%, respectively. In an evaluation of clinical specimens from SARS patients, 34 (52%) of 66 nasopharyngeal aspirate samples from 50 patients, 5 (5%) of 94 urine samples from 94 patients, and 36 (55%) of 65 fecal samples from 65 patients tested positive for SARS CoV nucleocapsid protein. Nucleocapsid protein could be detected from days 6 to 24 in nasopharyngeal aspirate specimens, from days 11 to 31 in urine specimens, and from days 8 to 32 in fecal specimens after the onset of illness. Moreover, the protein could be detected in 25 (83%) of 30 nasopharyngeal aspirate specimens obtained from days 11 to 15 and in all 7 fecal specimens obtained from days 21 to 32. Since the present ELISA is more convenient and economical than reverse transcription-PCR, it may serve as an alternative tool for the early diagnosis of SARS CoV infection in laboratories with limited resources and expertise and for mass screening for the reservoir of SARS CoV. Further studies on serial clinical specimens should reveal the duration of nucleocapsid protein shedding and may reveal a higher detection rate in SARS patients.
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rapid detection of the severe acute respiratory syndrome SARS Coronavirus by a loop mediated isothermal amplification assay
Clinical Chemistry, 2004Co-Authors: Leo L M Poon, Kwokhung Chan, Kwokyung Yuen, Yi Guan, Cynthia S W Leung, Masato Tashiro, Bonnie W Y Wong, Jsm PeirisAbstract:Severe acute respiratory syndrome (SARS) is a newly emerging disease that first emerged in Guangdong Province, China in November 2002 (1). The SARS Coronavirus (SARS-CoV) was found to be the etiology of the disease (2)(3)(4). Subsequent surveillance studies have indicated that this virus is of animal origin and have suggested that the source of the disease is still circulating in this geographic region (5). Indeed, the potential risk of reemergence of SARS is further highlighted by a recent confirmed SARS case in January 2004 (6). Therefore, the establishment of a rapid SARS diagnostic method is a high priority for control of the disease. Currently, there are two major diagnostic approaches for SARS. Detection of antibodies against SARS-CoV is a sensitive and specific diagnostic approach, but serconversion can be detected only around day 10 of illness (7). In contrast, PCR-based tests have been shown to be useful for early SARS diagnosis (8). Quantitative PCR approaches are a powerful tool for identifying SARS-CoV early after disease onset (4)(9)(10)(11). However, because of the requirements for sophisticated instrumentation and expensive reagents, these rapid molecular tests might not be the method of choice in basic clinical settings in developing countries or in field situations. It is therefore critical to develop simple and economical molecular tests for the above scenarios. The invention of loop-mediated isothermal amplification (LAMP) has opened up a new horizon for molecular diagnosis (12). This method depends on autocycling strand displacement DNA synthesis performed by a Bst DNA polymerase, and a detailed amplification mechanism has been described elsewhere (12). The reaction relies on recognition of the DNA target by six independent sequences, making this kind of assay highly specific. This method is rapid and has a DNA amplification efficiency equivalent to that of …
Eric J Snijder - One of the best experts on this subject based on the ideXlab platform.
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the SARS Coronavirus nsp7 nsp8 complex is a unique multimeric rna polymerase capable of both de novo initiation and primer extension
Nucleic Acids Research, 2012Co-Authors: Aartjan Te J W Velthuis, Sjoerd H E Van Den Worm, Eric J SnijderAbstract:Uniquely among RNA viruses, replication of the ~30-kb SARS-Coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities. The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this 'improved' recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, ~20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.
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the rna polymerase activity of SARS Coronavirus nsp12 is primer dependent
Nucleic Acids Research, 2010Co-Authors: Aartjan Te J W Velthuis, Jamie J Arnold, Craig E Cameron, Sjoerd H E Van Den Worm, Eric J SnijderAbstract:An RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit of the RNA-synthesizing machinery of all positive-strand RNA viruses. Usually, RdRp domains are readily identifiable by comparative sequence analysis, but biochemical confirmation and characterization can be hampered by intrinsic protein properties and technical complications. It is presumed that replication and transcription of the approximately 30-kb severe acute respiratory syndrome (SARS) Coronavirus (SARS-CoV) RNA genome are catalyzed by an RdRp domain in the C-terminal part of nonstructural protein 12 (nsp12), one of 16 replicase subunits. However, thus far full-length nsp12 has proven refractory to expression in bacterial systems, which has hindered both the biochemical characterization of Coronavirus RNA synthesis and RdRp-targeted antiviral drug design. Here, we describe a combined strategy involving bacterial expression of an nsp12 fusion protein and its in vivo cleavage to generate and purify stable SARS-CoV nsp12 (106 kDa) with a natural N-terminus and C-terminal hexahistidine tag. This recombinant protein possesses robust in vitro RdRp activity, as well as a significant DNA-dependent activity that may facilitate future inhibitor studies. The SARS-CoV nsp12 is primer dependent on both homo- and heteropolymeric templates, supporting the likeliness of a close enzymatic collaboration with the intriguing RNA primase activity that was recently proposed for Coronavirus nsp8.
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SARS Coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum
PLOS Biology, 2008Co-Authors: Kevin Knoops, Sjoerd H E Van Den Worm, Marjolein Kikkert, Jessika C Zevenhovendobbe, Yvonne Van Der Meer, Abraham J Koster, Mieke A Mommaas, Eric J SnijderAbstract:Positive-strand RNA viruses, a large group including human pathogens such as SARS-Coronavirus (SARS-CoV), replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs) were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of Coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200–300 nm), and “vesicle packets” apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this “replication network” will aid to further dissect the early stages of the Coronavirus life cycle and its virus-host interactions.
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SARS Coronavirus replication transcription complexes are membrane protected and need a host factor for activity in vitro
PLOS Pathogens, 2008Co-Authors: Martijn J Van Hemert, Sjoerd H E Van Den Worm, Kevin Knoops, Mieke A Mommaas, Alexander E Gorbalenya, Eric J SnijderAbstract:SARS-Coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.
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the SARS Coronavirus plnc domain of nsp3 as a replication transcription scaffolding protein
Virus Research, 2008Co-Authors: Isabelle Imbert, Eric J Snijder, Maria Dimitrova, Jeanclaude Guillemot, Patrick Lecine, Bruno CanardAbstract:Abstract Many genetic and mechanistic features distinguish the Coronavirus replication machinery from that encoded by most other RNA viruses. The Coronavirus replication/transcription complex is an assembly of viral and, most probably, cellular proteins that mediate the synthesis of both the unusually large (∼30 kb) RNA genome and an extensive set of subgenomic mRNAs. The viral components of the complex are encoded by the giant replicase gene, which is expressed in the form of two polyproteins (pp1a and pp1ab) that are processed into 16 cleavage products (nonstructural proteins 1–16). Using the combination of yeast two-hybrid screening and GST pull-down assays, we have now analyzed all potential interactions between SARS-Coronavirus nonstructural proteins, which may contribute to the structure and/or function of the viral replication/transcription complex. We demonstrate the existence of a complex network of interactions involving all 16 nonstructural proteins. Our results both confirmed previously described associations and identified novel heterodimerizations. The interaction map thus provides a sum of the interactions that may occur at some point during Coronavirus RNA synthesis and provides a framework for future research.
Patrick C Y Woo - One of the best experts on this subject based on the ideXlab platform.
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suppression of innate antiviral response by severe acute respiratory syndrome Coronavirus m protein is mediated through the first transmembrane domain
Cellular & Molecular Immunology, 2014Co-Authors: Kam-leung Siu, Kinhang Kok, Patrick C Y Woo, Chiping Chan, Dongyan JinAbstract:Coronaviruses have developed various measures to evade innate immunity. We have previously shown that severe acute respiratory syndrome (SARS) Coronavirus M protein suppresses type I interferon (IFN) production by impeding the formation of functional TRAF3-containing complex. In this study, we demonstrate that the IFN-antagonizing activity is specific to SARS Coronavirus M protein and is mediated through its first transmembrane domain (TM1) located at the N terminus. M protein from human Coronavirus HKU1 does not inhibit IFN production. Whereas N-linked glycosylation of SARS Coronavirus M protein has no influence on IFN antagonism, TM1 is indispensable for the suppression of IFN production. TM1 targets SARS Coronavirus M protein and heterologous proteins to the Golgi apparatus, yet Golgi localization is required but not sufficient for IFN antagonism. Mechanistically, TM1 is capable of binding with RIG-I, TRAF3, TBK1 and IKKe, and preventing the interaction of TRAF3 with its downstream effectors. Our work defines the molecular architecture of SARS Coronavirus M protein required for suppression of innate antiviral response.
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SARS Coronavirus spike polypeptide dna vaccine priming with recombinant spike polypeptide from escherichia coli as booster induces high titer of neutralizing antibody against SARS Coronavirus
Vaccine, 2005Co-Authors: Patrick C Y Woo, Susanna K P Lau, Beatrice H L Wong, Hoiwah Tsoi, Kwokhung Chan, Zhiwei Chen, Linqi Zhang, Jim Kh H Chan, Lei Po Wong, Kwokyung YuenAbstract:Different forms of SARS Coronavirus (SARS-CoV) spike protein-based vaccines for generation of neutralizing antibody response against SARS-CoV were compared using a mouse model. High IgG levels were detected in mice immunized with intraperitoneal (i.p.) recombinant spike polypeptide generated by Escherichia coli (S-peptide), mice primed with intramuscular (i.m.) tPA-optimize800 DNA vaccine (tPA-S-DNA) and boosted with i.p. S-peptide, mice primed with i.m. CTLA4HingeSARS800 DNA vaccine (CTLA4-S-DNA) and boosted with i.p. S-peptide, mice primed with oral live-attenuated Salmonella typhimurium (Salmonella-S-DNA-control) and boosted with i.p. S-peptide, mice primed with oral live-attenuated S. typhimurium that contained tPA-optimize800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide, and mice primed with oral live-attenuated S. typhimurium that contained CTLA4HingeSARS800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide. No statistical significant difference was observed among the Th1/Th2 index among these six groups of mice with high IgG levels. Sera of all six mice immunized with i.p. S-peptide, i.m. DNA vaccine control and oral Salmonella-S-DNA-control showed no neutralizing antibody against SARS-CoV. Sera of the mice immunized with i.m. tPA-S-DNA, i.m. CTLA4-S-DNA, oral Salmonella-S-DNA-control boosted with i.p. S-peptide, oral Salmonella-tPA-S-DNA, oral Salmonella-tPA-S-DNA boosted with i.p S-peptide, oral Salmonella-CTLA4-S-DNA and oral Salmonella-CTLA4-S-DNA boosted with i.p. S-peptide showed neutralizing antibody titers of or=1:1280. The present observation may have major practical value, such as immunization of civet cats, since production of recombinant proteins from E. coli is far less expensive than production of recombinant proteins using eukaryotic systems.
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detection of severe acute respiratory syndrome SARS Coronavirus nucleocapsid protein in SARS patients by enzyme linked immunosorbent assay
Journal of Clinical Microbiology, 2004Co-Authors: Susanna K P Lau, Patrick C Y Woo, Beatrice H L Wong, Hoiwah Tsoi, Gibson K S Woo, Rosana W S Poon, Kwokhung Chan, William I Wei, J S Peiris, Kwokyung YuenAbstract:We report the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of severe acute respiratory syndrome (SARS) Coronavirus (CoV) nucleocapsid protein. The assay was carried out with hyperimmune polyclonal nucleocapsid-specific antibodies from guinea pigs and rabbits immunized with recombinant His6-tagged SARS CoV nucleocapsid protein. The assay was used for the detection of SARS CoV nucleocapsid protein in nasopharyngeal aspirate, urine, and fecal samples collected from patients with confirmed SARS between days 2 and 33 after the onset of illness. The ELISA was capable of detecting this protein in SARS CoV cell culture lysates at 15 50% tissue culture infective doses/ml but did not produce positive signals when tested with cell culture lysates of human Coronaviruses OC43 and 229E. When tested with 120 nasopharyngeal aspirate, 100 urine, and 100 fecal specimens from hospitalized patients without SARS, the assay was shown to have high specificities—96.7, 99, and 96%, respectively. In an evaluation of clinical specimens from SARS patients, 34 (52%) of 66 nasopharyngeal aspirate samples from 50 patients, 5 (5%) of 94 urine samples from 94 patients, and 36 (55%) of 65 fecal samples from 65 patients tested positive for SARS CoV nucleocapsid protein. Nucleocapsid protein could be detected from days 6 to 24 in nasopharyngeal aspirate specimens, from days 11 to 31 in urine specimens, and from days 8 to 32 in fecal specimens after the onset of illness. Moreover, the protein could be detected in 25 (83%) of 30 nasopharyngeal aspirate specimens obtained from days 11 to 15 and in all 7 fecal specimens obtained from days 21 to 32. Since the present ELISA is more convenient and economical than reverse transcription-PCR, it may serve as an alternative tool for the early diagnosis of SARS CoV infection in laboratories with limited resources and expertise and for mass screening for the reservoir of SARS CoV. Further studies on serial clinical specimens should reveal the duration of nucleocapsid protein shedding and may reveal a higher detection rate in SARS patients.
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detection of specific antibodies to severe acute respiratory syndrome SARS Coronavirus nucleocapsid protein for serodiagnosis of SARS Coronavirus pneumonia
Journal of Clinical Microbiology, 2004Co-Authors: Patrick C Y Woo, Susanna K P Lau, Beatrice H L Wong, Hoiwah Tsoi, Kwokhung Chan, Ami M Y Fung, Victoria K P Tam, Jsm Peiris, Kwokyung YuenAbstract:We report the evaluation of recombinant severe acute respiratory syndrome (SARS) Coronavirus (SARS-CoV) nucleocapsid protein enzyme-linked immunosorbent assay (ELISA)-based antibody tests for serodiagnosis of SARS-CoV pneumonia and compare the sensitivities and specificities of this ELISA for detection of immunoglobulin G (IgG), IgM, IgA, and their combinations with serum samples from 149 healthy blood donors who donated blood 3 years ago as controls and 106 SARS-CoV pneumonia patients in Hong Kong. The specificities of the ELISA for IgG, IgM, and IgA detection were 95.3, 96.6, and 96.6%, respectively, with corresponding sensitivities of 94.3, 59.4, and 60.4%, respectively. The present ELISA appears to be a sensitive test for serodiagnosis of SARS-CoV pneumonia, is much more economical and less labor-intensive than the indirect immunofluorescence assay, and does not require cultivation of SARS-CoV.
You Xin Jin - One of the best experts on this subject based on the ideXlab platform.
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inhibition of genes expression of SARS Coronavirus by synthetic small interfering rnas
Cell Research, 2005Co-Authors: Yi Shi, De Hua Yang, Jie Xiong, Jie Jia, Bing Huang, You Xin JinAbstract:RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNA-mediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression. Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS Coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0 approximately 60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5'end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS Coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.
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inhibition of genes expression of SARS Coronavirus by synthetic small interfering rnas
Cell Research, 2005Co-Authors: Yi Shi, De Hua Yang, Jie Xiong, Jie Jia, Bing Huang, You Xin JinAbstract:RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNA-mediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression. Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS Coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0 similar to 60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS Coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.
