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Sample records for sars coronavirus cov

  1. Bioinformatics analysis of SARS-Cov M protein provides information for vaccine development

    Institute of Scientific and Technical Information of China (English)

    LIU Wanli; LU Yun; CHEN Yinghua

    2003-01-01

    The pathogen causing severe acute respiratory syndrome (SARS) is identified to be SARS-Cov. It is urgent to know more about SARS-Cov for developing an efficient SARS vaccine to prevent this epidemic disease. In this report, the homology of SARS-Cov M protein to other members of coronavirus is illustrated, and all amino acid changes in both S and M proteins among all available SARS-Cov isolates in GenBank are described. Furthermore, one topological trans-membrane secondary structure model of M protein is proposed, which is corresponded well with the accepted topology model of M proteins of other members of coronavirus. Hydrophilic profile analysis indicated that one region (aa150~210) on the cytoplasmic domain is fairly hydrophilic, suggesting its property of antigenicity. Based on the fact that cytoplasmic domain of the M protein of some other coronavirus could induce protective activities against virus infection, this region might be one potential target for SARS vaccine development.

  2. Molecular phylogeny of coronaviruses including human SARS-CoV

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Phylogenetic tree of coronaviruses (CoVs) including the human SARS-associated virus is reconstructed from complete genomes by using our newly developed K- string composition approach. The relation of the human SARS-CoV to other coronaviruses, i.e. the rooting of the tree is suggested by choosing an appropriate outgroup. SARS-CoV makes a separate group closer but still distant from G2 (CoVs in mammalian host). The relation between different isolates of the human SARS virus is inferred by first constructing an ultrametric distance matrix from counting sequence variations in the genomes. The resulting tree is consistent with clinic relations between the SARS-CoV isolates. In addition to a larger variety of coronavirus genomes these results provide phylogenetic knowledge based on independent novel methodology as compared to recent phylogenetic studies on SARS-CoV.

  3. Understanding bat SARS-like coronaviruses for the preparation of future coronavirus outbreaks - Implications for coronavirus vaccine development.

    Science.gov (United States)

    Ng, Oi-Wing; Tan, Yee-Joo

    2017-01-02

    The severe acute respiratory syndrome coronavirus (SARS-CoV) first emerged in 2003, causing the SARS epidemic which resulted in a 10% fatality rate. The advancements in metagenomic techniques have allowed the identification of SARS-like coronaviruses (SL-CoVs) sequences that share high homology to the human SARS-CoV epidemic strains from wildlife bats, presenting concrete evidence that bats are the origin and natural reservoir of SARS-CoV. The application of reverse genetics further enabled that characterization of these bat CoVs and the prediction of their potential to cause disease in humans. The knowledge gained from such studies is valuable in the surveillance and preparation of a possible future outbreak caused by a spill-over of these bat SL-CoVs.

  4. Anti-SARS coronavirus agents: a patent review (2008 - present).

    Science.gov (United States)

    Kumar, Vathan; Jung, Young-Sik; Liang, Po-Huang

    2013-10-01

    A novel coronavirus (CoV), unlike previous typical human coronaviruses (HCoVs), was identified as causative agent for severe acute respiratory syndrome (SARS). SARS first surfaced as a pandemic in late 2002 and originated in southern China. SARS-CoV rapidly spread to > 30 countries by 2003, infecting nearly 8,000 people and causing around 800 fatalities. After 10 years of silence, a 2012 report alarmed researchers about the emergence of a new strain of CoV causing SARS-like disease. To combat SARS, scientists applied for patents on various therapeutic agents, including small-molecule inhibitors targeting the essential proteases, helicase and other proteins of the virus, natural products, approved drugs, molecules binding to the virus, neutralizing antibodies, vaccines, anti-sense RNA, siRNA and ribozyme against SARS-CoV. In this article, the patents published from 2008 to the present for the new therapeutics that could potentially be used in the prophylaxis and treatment of SARS are reviewed. The therapeutic interventions or prophylaxis discussed in this review seems to offer promising solutions to tackle SARS. Rather than being complacent about the results, we should envisage how to transform them into drug candidates that may be useful in combating SARS and related viral infections in the future.

  5. Coronaviridae and SARS-associated Coronavirus Strain HSR1

    Science.gov (United States)

    Canducci, Filippo; Pinna, Debora; Mancini, Nicasio; Carletti, Silvia; Lazzarin, Adriano; Bordignon, Claudio; Poli, Guido; Clementi, Massimo

    2004-01-01

    During the recent severe acute respiratory (SARS) outbreak, the etiologic agent was identified as a new coronavirus (CoV). We have isolated a SARS-associated CoV (SARS-CoV) strain by injecting Vero cells with a sputum specimen from an Italian patient affected by a severe pneumonia; the patient traveled from Vietnam to Italy in March 2003. Ultrastructural analysis of infected Vero cells showed the virions within cell vesicles and around the cell membrane. The full-length viral genome sequence was similar to those derived from the Hong-Kong Hotel M isolate. By using both real-time reverse transcription–polymerase chain reaction TaqMan assay and an infectivity plaque assay, we determined that approximately 360 viral genomes were required to generate a PFU. In addition, heparin (100 μg/mL) inhibited infection of Vero cells by 50%. Overall, the molecular and biologic characteristics of the strain HSR1 provide evidence that SARS-CoV forms a fourth genetic coronavirus group with distinct genomic and biologic features. PMID:15109406

  6. From SARS coronavirus to novel animal and human coronaviruses.

    Science.gov (United States)

    To, Kelvin K W; Hung, Ivan F N; Chan, Jasper F W; Yuen, Kwok-Yung

    2013-08-01

    In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) caused one of the most devastating epidemics known to the developed world. There were two important lessons from this epidemic. Firstly, coronaviruses, in addition to influenza viruses, can cause severe and rapidly spreading human infections. Secondly, bats can serve as the origin and natural animal reservoir of deadly human viruses. Since then, researchers around the world, especially those in Asia where SARS-CoV was first identified, have turned their focus to find novel coronaviruses infecting humans, bats, and other animals. Two human coronaviruses, HCoV-HKU1 and HCoV-NL63, were identified shortly after the SARS-CoV epidemic as common causes of human respiratory tract infections. In 2012, a novel human coronavirus, now called Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in the Middle East to cause fatal human infections in three continents. MERS-CoV human infection is similar to SARS-CoV in having a high fatality rate and the ability to spread from person to person which resulted in secondary cases among close contacts including healthcare workers without travel history to the Middle East. Both viruses also have close relationships with bat coronaviruses. New cases of MERS-CoV infection in humans continue to occur with the origins of the virus still unknown in many cases. A multifaceted approach is necessary to control this evolving MERS-CoV outbreak. Source identification requires detailed epidemiological studies of the infected patients and enhanced surveillance of MERS-CoV or similar coronaviruses in humans and animals. Early diagnosis of infected patients and appropriate infection control measures will limit the spread in hospitals, while social distancing strategies may be necessary to control the outbreak in communities if it remained uncontrolled as in the SARS epidemic.

  7. SARS-like WIV1-CoV poised for human emergence

    Science.gov (United States)

    Menachery, Vineet D.; Yount, Boyd L.; Sims, Amy C.; Debbink, Kari; Agnihothram, Sudhakar S.; Gralinski, Lisa E.; Graham, Rachel L.; Scobey, Trevor; Plante, Jessica A.; Royal, Scott R.; Swanstrom, Jesica; Sheahan, Timothy P.; Pickles, Raymond J.; Corti, Davide; Randell, Scott H.; Lanzavecchia, Antonio; Marasco, Wayne A.; Baric, Ralph S.

    2016-01-01

    Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations. PMID:26976607

  8. Immunological Responses against SARS-Coronavirus Infection in Humans

    Institute of Scientific and Technical Information of China (English)

    Xiaojun Xu; Xiao-Ming Gao

    2004-01-01

    Since the outbreak of a SARS epidemic last year, significant advances have been made on our understanding of the mechanisms of interaction between the SARS coronavirus (CoV) and the immune system. Strong humoral responses have been found in most patients following SARS-CoV infection, with high titers of neutralizing Abspresent in their convalescent sera. The nucleocapsid (N) and spike (S) proteins of SARS-CoV appear to be the dominant antigens recognized by serum Abs. CD4+ T cell responses against the N protein have been observed in SARS patients and an HLA-A2-restricted cytotoxic T lymphocyte epitope in the S protein has been identified.It is likely that the immune responses induced by SARS-CoV infection could also cause pathological damage to the host, especially in the case of proinflammatory cytokines. There is also evidence suggesting that SARS-CoV might be able to directly invade cells of the immune system. Our understanding on the interaction between SARS-CoV, the immune system and local tissues is essential to future diagnosis, control and treatment of this very contagious disease.

  9. Immunological Responses against SARS-Coronavirus Infection in Humans

    Institute of Scientific and Technical Information of China (English)

    XiaojunXu; Xiao-MingGao

    2004-01-01

    Since the outbreak of a SARS epidemic last year, significant advances have been made on our understanding of the mechanisms of interaction between the SARS coronavirus (CoV) and the immune system. Strong humoral responses have been found in most patients following SARS-CoV infection, with high titers of neutralizing Abs present in their convalescent sera. The nucleocapsid (N) and spike (S) proteins of SARS-CoV appear to be the dominant antigens recognized by serum Abs. CD4+ T cell responses against the N protein have been observed in SARS patients and an HLA-A2-restricted cytotoxic T lymphocyte epitope in the S protein has been identified. It is likely that the immune responses induced by SARS-CoV infection could also cause pathological damage to the host, especially in the case of proinflammatory cytokines. There is also evidence suggesting that SARS-CoV might be able to directly invade cells of the immune system. Our understanding on the interaction between SARS-CoV, the immune system and local tissues is essential to future diagnosis, control and treatment of this very contagious disease. Cellular & Molecular Immunology. 2004;1(2):119-122.

  10. Receptor-binding domain of SARS-Cov spike protein: Soluble expression in purification and functional characterization

    Institute of Scientific and Technical Information of China (English)

    Jing Chen; Lin Miao; Jia-Ming Li; Yan-Ying Li; Qing-Yu Zhu; Chang-Lin Zhou; Hong-Qing Fang; Hui-Peng Chen

    2005-01-01

    AIM: To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability.METHODS: Three fusion tags (glutathione S-transferase,GST; thioredoxin, Trx; maltose-binding protein, MBP),which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli( BL21( DE3 ) and Rosetta-gamiB(DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay.RESULTS: RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2)positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and respectively.CONCLUSION: In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E. coli BL21(DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E. coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

  11. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence.

    Science.gov (United States)

    Menachery, Vineet D; Yount, Boyd L; Debbink, Kari; Agnihothram, Sudhakar; Gralinski, Lisa E; Plante, Jessica A; Graham, Rachel L; Scobey, Trevor; Ge, Xing-Yi; Donaldson, Eric F; Randell, Scott H; Lanzavecchia, Antonio; Marasco, Wayne A; Shi, Zhengli-Li; Baric, Ralph S

    2015-12-01

    The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations. Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein. On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Our work suggests a potential risk of SARS-CoV re-emergence from viruses currently circulating in bat populations.

  12. Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library.

    Science.gov (United States)

    Severson, William E; Shindo, Nice; Sosa, Mindy; Fletcher, Thomas; White, E Lucile; Ananthan, Subramaniam; Jonsson, Colleen B

    2007-02-01

    The authors have developed a high-throughput screen (HTS) that allows for the identification of potential inhibitors of the severe acute respiratory syndrome coronavirus (SARS CoV) from large compound libraries. The luminescent-based assay measures the inhibition of SARS CoV-induced cytopathic effect (CPE) in Vero E6 cells. The assay was validated in 96-well plates in a BSL3 containment facility. The assay is sensitive and robust, with Z values > 0.6, signal to background (S/B) > 16, and signal to noise (S/N) > 3. The assay was further validated with 2 different diversity sets of compounds against the SARS CoV. The "hit" rate for both libraries was approximately 0.01%. The validated HTS assay was then employed to screen a 100,000-compound library against SARS CoV. The hit rate for the library in a single-dose format was determined to be approximately 0.8%. Screening of the 3 libraries resulted in the identification of several novel compounds that effectively inhibited the CPE of SARS CoV in vitro-compounds which will serve as excellent lead candidates for further evaluation. At a 10-microM concentration, 3 compounds with selective indexes (SI50) of > 53 were discovered.

  13. SARS-like cluster of circulating bat coronavirus pose threat for human emergence

    Science.gov (United States)

    Menachery, Vineet D.; Yount, Boyd L.; Debbink, Kari; Agnihothram, Sudhakar; Gralinski, Lisa E.; Plante, Jessica A.; Graham, Rachel L.; Scobey, Trevor; Ge, Xing-Yi; Donaldson, Eric F.; Randell, Scott H.; Lanzavecchia, Antonio; Marasco, Wayne A.; Shi, Zhengli-Li; Baric, Ralph S.

    2016-01-01

    The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. In this study, we examine the disease potential for SARS-like CoVs currently circulating in Chinese horseshoe bat populations. Utilizing the SARS-CoV infectious clone, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild type backbone can efficiently utilize multiple ACE2 receptor orthologs, replicate efficiently in primary human airway cells, and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from CoVs utilizing the novel spike protein. Importantly, based on these findings, we synthetically rederived an infectious full length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Together, the work highlights a continued risk of SARS-CoV reemergence from viruses currently circulating in bat populations. PMID:26552008

  14. Possible SARS coronavirus transmission during cardiopulmonary resuscitation.

    Science.gov (United States)

    Christian, Michael D; Loutfy, Mona; McDonald, L Clifford; Martinez, Kennth F; Ofner, Mariana; Wong, Tom; Wallington, Tamara; Gold, Wayne L; Mederski, Barbara; Green, Karen; Low, Donald E

    2004-02-01

    Infection of healthcare workers with the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is thought to occur primarily by either contact or large respiratory droplet transmission. However, infrequent healthcare worker infections occurred despite the use of contact and droplet precautions, particularly during certain aerosol-generating medical procedures. We investigated a possible cluster of SARS-CoV infections in healthcare workers who used contact and droplet precautions during attempted cardiopulmonary resuscitation of a SARS patient. Unlike previously reported instances of transmission during aerosol-generating procedures, the index case-patient was unresponsive, and the intubation procedure was performed quickly and without difficulty. However, before intubation, the patient was ventilated with a bag-valve-mask that may have contributed to aerosolization of SARS-CoV. On the basis of the results of this investigation and previous reports of SARS transmission during aerosol-generating procedures, a systematic approach to the problem is outlined, including the use of the following: 1) administrative controls, 2) environmental engineering controls, 3) personal protective equipment, and 4) quality control.

  15. Rapid inactivation of SARS-like coronaviruses.

    Energy Technology Data Exchange (ETDEWEB)

    Kapil, Sanjay (Kansas State University, Manhattan, KS); Oberst, R. D. (Kansas State University, Manhattan, KS); Bieker, Jill Marie; Tucker, Mark David; Souza, Caroline Ann; Williams, Cecelia Victoria

    2004-03-01

    Chemical disinfection and inactivation of viruses is largely understudied, but is very important especially in the case of highly infectious viruses. The purpose of this LDRD was to determine the efficacy of the Sandia National Laboratories developed decontamination formulations against Bovine Coronavirus (BCV) as a surrogate for the coronavirus that causes Severe Acute Respiratory Syndrome (SARS) in humans. The outbreak of SARS in late 2002 resulted from a highly infectious virus that was able to survive and remain infectious for extended periods. For this study, preliminary testing with Escherichia coli MS-2 (MS-2) and Escherichia coli T4 (T4) bacteriophages was conducted to develop virucidal methodology for verifying the inactivation after treatment with the test formulations following AOAC germicidal methodologies. After the determination of various experimental parameters (i.e. exposure, concentration) of the formulations, final testing was conducted on BCV. All experiments were conducted with various organic challenges (horse serum, bovine feces, compost) for results that more accurately represent field use condition. The MS-2 and T4 were slightly more resistant than BCV and required a 2 minute exposure while BCV was completely inactivated after a 1 minute exposure. These results were also consistent for the testing conducted in the presence of the various organic challenges indicating that the test formulations are highly effective for real world application.

  16. Cell host response to infection with novel human coronavirus EMC predicts potential antivirals and important differences with SARS coronavirus.

    Science.gov (United States)

    Josset, Laurence; Menachery, Vineet D; Gralinski, Lisa E; Agnihothram, Sudhakar; Sova, Pavel; Carter, Victoria S; Yount, Boyd L; Graham, Rachel L; Baric, Ralph S; Katze, Michael G

    2013-04-30

    A novel human coronavirus (HCoV-EMC) was recently identified in the Middle East as the causative agent of a severe acute respiratory syndrome (SARS) resembling the illness caused by SARS coronavirus (SARS-CoV). Although derived from the CoV family, the two viruses are genetically distinct and do not use the same receptor. Here, we investigated whether HCoV-EMC and SARS-CoV induce similar or distinct host responses after infection of a human lung epithelial cell line. HCoV-EMC was able to replicate as efficiently as SARS-CoV in Calu-3 cells and similarly induced minimal transcriptomic changes before 12 h postinfection. Later in infection, HCoV-EMC induced a massive dysregulation of the host transcriptome, to a much greater extent than SARS-CoV. Both viruses induced a similar activation of pattern recognition receptors and the interleukin 17 (IL-17) pathway, but HCoV-EMC specifically down-regulated the expression of several genes within the antigen presentation pathway, including both type I and II major histocompatibility complex (MHC) genes. This could have an important impact on the ability of the host to mount an adaptive host response. A unique set of 207 genes was dysregulated early and permanently throughout infection with HCoV-EMC, and was used in a computational screen to predict potential antiviral compounds, including kinase inhibitors and glucocorticoids. Overall, HCoV-EMC and SARS-CoV elicit distinct host gene expression responses, which might impact in vivo pathogenesis and could orient therapeutic strategies against that emergent virus. Identification of a novel coronavirus causing fatal respiratory infection in humans raises concerns about a possible widespread outbreak of severe respiratory infection similar to the one caused by SARS-CoV. Using a human lung epithelial cell line and global transcriptomic profiling, we identified differences in the host response between HCoV-EMC and SARS-CoV. This enables rapid assessment of viral properties and the

  17. Efficacy of various disinfectants against SARS coronavirus.

    Science.gov (United States)

    Rabenau, H F; Kampf, G; Cinatl, J; Doerr, H W

    2005-10-01

    The recent severe acute respiratory syndrome (SARS) epidemic in Asia and Northern America led to broad use of various types of disinfectant in order to control the public spread of the highly contagious virus. However, only limited data were available to demonstrate their efficacy against SARS coronavirus (SARS-CoV). We therefore investigated eight disinfectants for their activity against SARS-CoV according to prEN 14476. Four hand rubs were tested at 30s (Sterillium, based on 45% iso-propanol, 30% n-propanol and 0.2% mecetronium etilsulphate; Sterillium Rub, based on 80% ethanol; Sterillium Gel, based on 85% ethanol; Sterillium Virugard, based on 95% ethanol). Three surface disinfectants were investigated at 0.5% for 30 min and 60 min (Mikrobac forte, based on benzalkonium chloride and laurylamine; Kohrsolin FF, based on benzalkonium chloride, glutaraldehyde and didecyldimonium chloride; Dismozon pur, based on magnesium monoperphthalate), and one instrument disinfectant was investigated at 4% for 15 min, 3% for 30 min and 2% for 60 min [Korsolex basic, based on glutaraldehyde and (ethylenedioxy)dimethanol]. Three types of organic load were used: 0.3% albumin, 10% fetal calf serum, and 0.3% albumin with 0.3% sheep erythrocytes. Virus titres were determined by a quantitative test (endpoint titration) in 96-well microtitre plates. With all tested preparations, SARS-CoV was inactivated to below the limit of detection (reduction factor mostly > or =4), regardless of the type of organic load. In summary, SARS-CoV can be inactivated quite easily with many commonly used disinfectants.

  18. Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus.

    Science.gov (United States)

    Sevajol, Marion; Subissi, Lorenzo; Decroly, Etienne; Canard, Bruno; Imbert, Isabelle

    2014-12-19

    The successive emergence of highly pathogenic coronaviruses (CoVs) such as the Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 has stimulated a number of studies on the molecular biology. This research has provided significant new insight into functions and activities of the replication/transcription multi-protein complex. The latter directs both continuous and discontinuous RNA synthesis to replicate and transcribe the large coronavirus genome made of a single-stranded, positive-sense RNA of ∼30 kb. In this review, we summarize our current understanding of SARS-CoV enzymes involved in RNA biochemistry, such as the in vitro characterization of a highly active and processive RNA polymerase complex which can associate with methyltransferase and 3'-5' exoribonuclease activities involved in RNA capping, and RNA proofreading, respectively. The recent discoveries reveal fascinating RNA-synthesizing machinery, highlighting the unique position of coronaviruses in the RNA virus world. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Interferon-Beta 1a and SARS Coronavirus Replication

    Science.gov (United States)

    2004-02-01

    ribavirin remains uncertain because it has no activity against SARS-CoV in vitro. Molecular modeling studies suggest that rhinovirus 3Cpro inhibitors...coronavirus. Science 2003;300:1399–404. 3. Anand K, Ziebuhr J, Wadhwani P, Mesters JR, Hilgenfeld R. Coronavirus main proteinase (3CLpro) structure

  20. Coronavirus virulence genes with main focus on SARS-CoV envelope gene.

    Science.gov (United States)

    DeDiego, Marta L; Nieto-Torres, Jose L; Jimenez-Guardeño, Jose M; Regla-Nava, Jose A; Castaño-Rodriguez, Carlos; Fernandez-Delgado, Raul; Usera, Fernando; Enjuanes, Luis

    2014-12-19

    Coronavirus (CoV) infection is usually detected by cellular sensors, which trigger the activation of the innate immune system. Nevertheless, CoVs have evolved viral proteins that target different signaling pathways to counteract innate immune responses. Some CoV proteins act as antagonists of interferon (IFN) by inhibiting IFN production or signaling, aspects that are briefly addressed in this review. After CoV infection, potent cytokines relevant in controlling virus infections and priming adaptive immune responses are also generated. However, an uncontrolled induction of these proinflammatory cytokines can lead to pathogenesis and disease severity as described for SARS-CoV and MERS-CoV. The cellular pathways mediated by interferon regulatory factor (IRF)-3 and -7, activating transcription factor (ATF)-2/jun, activator protein (AP)-1, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NF-AT), are the main drivers of the inflammatory response triggered after viral infections, with NF-κB pathway the most frequently activated. Key CoV proteins involved in the regulation of these pathways and the proinflammatory immune response are revisited in this manuscript. It has been shown that the envelope (E) protein plays a variable role in CoV morphogenesis, depending on the CoV genus, being absolutely essential in some cases (genus α CoVs such as TGEV, and genus β CoVs such as MERS-CoV), but not in others (genus β CoVs such as MHV or SARS-CoV). A comprehensive accumulation of data has shown that the relatively small E protein elicits a strong influence on the interaction of SARS-CoV with the host. In fact, after infection with viruses in which this protein has been deleted, increased cellular stress and unfolded protein responses, apoptosis, and augmented host immune responses were observed. In contrast, the presence of E protein activated a pathogenic inflammatory response that may cause death in animal

  1. An Outbreak of Human Coronavirus OC43 Infection and Serological Cross-Reactivity with SARS Coronavirus

    Directory of Open Access Journals (Sweden)

    David M Patrick

    2006-01-01

    Full Text Available BACKGROUND: In summer 2003, a respiratory outbreak was investigated in British Columbia, during which nucleic acid tests and serology unexpectedly indicated reactivity for severe acute respiratory syndrome coronavirus (SARS-CoV.

  2. Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines.

    Science.gov (United States)

    Müller, Marcel A; Raj, V Stalin; Muth, Doreen; Meyer, Benjamin; Kallies, Stephan; Smits, Saskia L; Wollny, Robert; Bestebroer, Theo M; Specht, Sabine; Suliman, Tasnim; Zimmermann, Katrin; Binger, Tabea; Eckerle, Isabella; Tschapka, Marco; Zaki, Ali M; Osterhaus, Albert D M E; Fouchier, Ron A M; Haagmans, Bart L; Drosten, Christian

    2012-12-11

    A new human coronavirus (hCoV-EMC) has emerged very recently in the Middle East. The clinical presentation resembled that of the severe acute respiratory syndrome (SARS) as encountered during the epidemic in 2002/2003. In both cases, acute renal failure was observed in humans. HCoV-EMC is a member of the same virus genus as SARS-CoV but constitutes a sister species. Here we investigated whether it might utilize angiotensin-converting enzyme 2 (ACE2), the SARS-CoV receptor. Knowledge of the receptor is highly critical because the restriction of the SARS receptor to deep compartments of the human respiratory tract limited the spread of SARS. In baby hamster kidney (BHK) cells, lentiviral transduction of human ACE2 (hACE2) conferred permissiveness and replication for SARS-CoV but not for hCoV-EMC. Monkey and human kidney cells (LLC-MK2, Vero, and 769-P) and swine kidney cells were permissive for both viruses, but only SARS-CoV infection could be blocked by anti-hACE2 antibody and could be neutralized by preincubation of virus with soluble ACE2. Our data show that ACE2 is neither necessary nor sufficient for hCoV-EMC replication. Moreover, hCoV-EMC, but not SARS-CoV, replicated in cell lines from Rousettus, Rhinolophus, Pipistrellus, Myotis, and Carollia bats, representing four major chiropteran families from both suborders. As human CoV normally cannot replicate in bat cells from different families, this suggests that hCoV-EMC might use a receptor molecule that is conserved in bats, pigs, and humans, implicating a low barrier against cross-host transmission. IMPORTANCE A new human coronavirus (hCoV) emerged recently in the Middle East. The disease resembled SARS (severe acute respiratory syndrome), causing a fatal epidemic in 2002/2003. Coronaviruses have a reservoir in bats and because this novel virus is related to SARS-CoV, we investigated whether it might replicate in bat cells and use the same receptor (angiotensin-converting enzyme 2 [ACE2]). This knowledge is

  3. Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model

    Science.gov (United States)

    Cameron, Mark J.; Kelvin, Alyson A.; Leon, Alberto J.; Cameron, Cheryl M.; Ran, Longsi; Xu, Luoling; Chu, Yong-Kyu; Danesh, Ali; Fang, Yuan; Li, Qianjun; Anderson, Austin; Couch, Ronald C.; Paquette, Stephane G.; Fomukong, Ndingsa G.; Kistner, Otfried; Lauchart, Manfred; Rowe, Thomas; Harrod, Kevin S.; Jonsson, Colleen B.; Kelvin, David J.

    2012-01-01

    In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)3-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines. PMID:23029269

  4. Date of origin of the SARS coronavirus strains

    Directory of Open Access Journals (Sweden)

    Cai Lun

    2004-02-01

    Full Text Available Abstract Background A new respiratory infectious epidemic, severe acute respiratory syndrome (SARS, broke out and spread throughout the world. By now the putative pathogen of SARS has been identified as a new coronavirus, a single positive-strand RNA virus. RNA viruses commonly have a high rate of genetic mutation. It is therefore important to know the mutation rate of the SARS coronavirus as it spreads through the population. Moreover, finding a date for the last common ancestor of SARS coronavirus strains would be useful for understanding the circumstances surrounding the emergence of the SARS pandemic and the rate at which SARS coronavirus diverge. Methods We propose a mathematical model to estimate the evolution rate of the SARS coronavirus genome and the time of the last common ancestor of the sequenced SARS strains. Under some common assumptions and justifiable simplifications, a few simple equations incorporating the evolution rate (K and time of the last common ancestor of the strains (T0 can be deduced. We then implemented the least square method to estimate K and T0 from the dataset of sequences and corresponding times. Monte Carlo stimulation was employed to discuss the results. Results Based on 6 strains with accurate dates of host death, we estimated the time of the last common ancestor to be about August or September 2002, and the evolution rate to be about 0.16 base/day, that is, the SARS coronavirus would on average change a base every seven days. We validated our method by dividing the strains into two groups, which coincided with the results from comparative genomics. Conclusion The applied method is simple to implement and avoid the difficulty and subjectivity of choosing the root of phylogenetic tree. Based on 6 strains with accurate date of host death, we estimated a time of the last common ancestor, which is coincident with epidemic investigations, and an evolution rate in the same range as that reported for the HIV-1 virus.

  5. SARS and MERS: recent insights into emerging coronaviruses.

    Science.gov (United States)

    de Wit, Emmie; van Doremalen, Neeltje; Falzarano, Darryl; Munster, Vincent J

    2016-08-01

    The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 marked the second introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. The continuing introductions of MERS-CoV from dromedary camels, the subsequent travel-related viral spread, the unprecedented nosocomial outbreaks and the high case-fatality rates highlight the need for prophylactic and therapeutic measures. Scientific advancements since the 2002-2003 severe acute respiratory syndrome coronavirus (SARS-CoV) pandemic allowed for rapid progress in our understanding of the epidemiology and pathogenesis of MERS-CoV and the development of therapeutics. In this Review, we detail our present understanding of the transmission and pathogenesis of SARS-CoV and MERS-CoV, and discuss the current state of development of measures to combat emerging coronaviruses.

  6. DNA Vaccine of SARS-Cov S Gene Induces Antibody Response in Mice

    Institute of Scientific and Technical Information of China (English)

    PingZHAO; Jin-ShanKE; Zhao-LinQIN; HaoREN; Lan-JuanZHAO; Jian-GuoYU

    2004-01-01

    The spike (S) protein, a main surface antigen of SARS-coronavirus (SARS-CoV), is one of the most important antigen candidates for vaccine design. In the present study, three fragments of the truncated S protein were expressed in E.coli, and analyzed with pooled sera of convalescence phase of SARS patients.The full length S gene DNA vaccine was constructed and used to immunize BALB/c mice. The mouse serum IgG antibody against SARS-CoV was measured by ELISA with E.coli expressed truncated S protein or SARS-CoV lysate as diagnostic antigen. The results showed that all the three fragments of S protein expressed by E.coli was able to react with sera of SARS patients and the S gene DNA candidate vaccine could induce the production of specific IgG antibody against SARS-CoV efficiently in mice with seroconversion ratio of 75% after 3 times of immunization. These findings lay some foundations for further understanding the immunology of SARS-CoV and developing SARS vaccines.

  7. DNA Vaccine of SARS-Cov S Gene Induces Antibody Response in Mice

    Institute of Scientific and Technical Information of China (English)

    Ping ZHAO; Jin-Shan KE; Zhao-Lin QIN; Hao REN; Lan-Juan ZHAO; Jian-Guo YU; Jun GAO; Shi-Ying ZHU; Zhong-Tian QI

    2004-01-01

    The spike (S) protein, a main surface antigen of SARS-coronavirus (SARS-CoV), is one of the most important antigen candidates for vaccine design. In the present study, three fragments of the truncated S protein were expressed in E. Coli, and analyzed with pooled sera of convalescence phase of SARS patients.The full length S gene DNA vaccine was constructed and used to immunize BALB/c mice. The mouse serum IgG antibody against SARS-CoV was measured by ELISA with E. Coli expressed truncated S protein or SARS-CoV lysate as diagnostic antigen. The results showed that all the three fragments of S protein expressed by E. Coli was able to react with sera of SARS patients and the S gene DNA candidate vaccine could induce the production of specific IgG antibody against SARS-CoV efficiently in mice with seroconversion ratio of 75% after 3 times of immunization. These findings lay some foundations for further understanding the immunology of SARS-CoV and developing SARS vaccines.

  8. Anti-SARS virus antibody responses against human SARS-associated coronavirus and animal SARS-associated coronavirus-like virus

    Institute of Scientific and Technical Information of China (English)

    王鸣; 徐慧芳; 莫自耀; 郑伯健; 高阳; 顾菁; 秦鹏哲; 张周斌; 邹晓忠; 梁彩云; 赵宇腾; 高凯

    2004-01-01

    @@ Severe acute respiratory syndrome (SARS) is an infectious disease first recognized in November 2002 in Guangdong province, China. It was spread to many countries all over the world within a few months.1,2 By April 2003, SARS-associated coronavirus (SARS-CoV) was found to be the etiological agent.

  9. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination.

    Science.gov (United States)

    Lau, Susanna K P; Feng, Yun; Chen, Honglin; Luk, Hayes K H; Yang, Wei-Hong; Li, Kenneth S M; Zhang, Yu-Zhen; Huang, Yi; Song, Zhi-Zhong; Chow, Wang-Ngai; Fan, Rachel Y Y; Ahmed, Syed Shakeel; Yeung, Hazel C; Lam, Carol S F; Cai, Jian-Piao; Wong, Samson S Y; Chan, Jasper F W; Yuen, Kwok-Yung; Zhang, Hai-Lin; Woo, Patrick C Y

    2015-10-01

    Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS-CoV ORF8

  10. A decade after SARS: Strategies to control emerging coronaviruses

    Science.gov (United States)

    Graham, Rachel L.; Donaldson, Eric F.; Baric, Ralph S.

    2016-01-01

    Two novel coronaviruses have emerged in humans in the 21st century, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome human coronavirus (MERS-CoV), both of which cause acute respiratory distress syndrome (ARDS) and have high mortality rates. There are no clinically approved vaccines or antiviral drugs available for either of these infections; thus, a priority in the field is the development of effective therapeutic and preventive strategies that can be readily applied to new emergent strains. This review will: describe the emergence and identification of novel human coronaviruses over the last 10 years; review their key biological features, including tropism and receptor use; and summarize approaches to develop broadly effective vaccines. PMID:24217413

  11. Development of a dose-response model for SARS coronavirus.

    Science.gov (United States)

    Watanabe, Toru; Bartrand, Timothy A; Weir, Mark H; Omura, Tatsuo; Haas, Charles N

    2010-07-01

    In order to develop a dose-response model for SARS coronavirus (SARS-CoV), the pooled data sets for infection of transgenic mice susceptible to SARS-CoV and infection of mice with murine hepatitis virus strain 1, which may be a clinically relevant model of SARS, were fit to beta-Poisson and exponential models with the maximum likelihood method. The exponential model (k= 4.1 x l0(2)) could describe the dose-response relationship of the pooled data sets. The beta-Poisson model did not provide a statistically significant improvement in fit. With the exponential model, the infectivity of SARS-CoV was calculated and compared with those of other coronaviruses. The does of SARS-CoV corresponding to 10% and 50% responses (illness) were estimated at 43 and 280 PFU, respectively. Its estimated infectivity was comparable to that of HCoV-229E, known as an agent of human common cold, and also similar to those of some animal coronaviruses belonging to the same genetic group. Moreover, the exponential model was applied to the analysis of the epidemiological data of SARS outbreak that occurred at an apartment complex in Hong Kong in 2003. The estimated dose of SARS-CoV for apartment residents during the outbreak, which was back-calculated from the reported number of cases, ranged from 16 to 160 PFU/person, depending on the floor. The exponential model developed here is the sole dose-response model for SARS-CoV at the present and would enable us to understand the possibility for reemergence of SARS.

  12. A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

    Directory of Open Access Journals (Sweden)

    Andrea Balboni

    2012-01-01

    Full Text Available Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population.

  13. Development of animal models against emerging coronaviruses: From SARS to MERS coronavirus.

    Science.gov (United States)

    Sutton, Troy C; Subbarao, Kanta

    2015-05-01

    Two novel coronaviruses have emerged to cause severe disease in humans. While bats may be the primary reservoir for both viruses, SARS coronavirus (SARS-CoV) likely crossed into humans from civets in China, and MERS coronavirus (MERS-CoV) has been transmitted from camels in the Middle East. Unlike SARS-CoV that resolved within a year, continued introductions of MERS-CoV present an on-going public health threat. Animal models are needed to evaluate countermeasures against emerging viruses. With SARS-CoV, several animal species were permissive to infection. In contrast, most laboratory animals are refractory or only semi-permissive to infection with MERS-CoV. This host-range restriction is largely determined by sequence heterogeneity in the MERS-CoV receptor. We describe animal models developed to study coronaviruses, with a focus on host-range restriction at the level of the viral receptor and discuss approaches to consider in developing a model to evaluate countermeasures against MERS-CoV. Copyright © 2015. Published by Elsevier Inc.

  14. Receptor recognition and cross-species infections of SARS coronavirus.

    Science.gov (United States)

    Li, Fang

    2013-10-01

    Receptor recognition is a major determinant of the host range, cross-species infections, and pathogenesis of the severe acute respiratory syndrome coronavirus (SARS-CoV). A defined receptor-binding domain (RBD) in the SARS-CoV spike protein specifically recognizes its host receptor, angiotensin-converting enzyme 2 (ACE2). This article reviews the latest knowledge about how RBDs from different SARS-CoV strains interact with ACE2 from several animal species. Detailed research on these RBD/ACE2 interactions has established important principles on host receptor adaptations, cross-species infections, and future evolution of SARS-CoV. These principles may apply to other emerging animal viruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV). This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses". Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant.

    Science.gov (United States)

    McPherson, Clifton; Chubet, Richard; Holtz, Kathy; Honda-Okubo, Yoshikazu; Barnard, Dale; Cox, Manon; Petrovsky, Nikolai

    2016-01-01

    Given periodic outbreaks of fatal human infections caused by coronaviruses, development of an optimal coronavirus vaccine platform capable of rapid production is an ongoing priority. This chapter describes the use of an insect cell expression system for rapid production of a recombinant vaccine against severe acute respiratory syndrome coronavirus (SARS). Detailed methods are presented for expression, purification, and release testing of SARS recombinant spike protein antigen, followed by adjuvant formulation and animal testing. The methods herein described for rapid development of a highly protective SARS vaccine are equally suited to rapid development of vaccines against other fatal human coronavirus infections, e.g., the MERS coronavirus.

  16. Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research.

    Science.gov (United States)

    Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan

    2013-12-01

    The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.'' Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Identification of an epitope of SARS-coronavirus nucleocapsid protein

    Institute of Scientific and Technical Information of China (English)

    YING LIN; JIN WANG; HONG XIA WANG; HUA LIANG JIANG; JIAN HUA SHEN; YOU HUA XIE; YUAN WANG; GANG PEI; BEI FEN SHEN; JIA RUI WU; BING SUN; XU SHEN; RUI FU YANG; YI XUE LI; YONG YONG JI; YOU YU HE; MUDE SHI; WEI LU; TIE LIU SHI

    2003-01-01

    The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a majorvirion structural protein. In this study, two epitopes (N1 and N2) of the N protein of SARS-CoV werepredicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodieswere isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-inducedpolyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SARS-CoV. Furthermore, itwas confirmed that N1 peptide-specific IgG antibodies were detectable in the sera of severe acute respiratorysyndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified andN protein specific Abs were produced by peptide immunization, which will be useful for the study of SARS-CoV.

  18. Accessory proteins of SARS-CoV and other coronaviruses.

    Science.gov (United States)

    Liu, Ding Xiang; Fung, To Sing; Chong, Kelvin Kian-Long; Shukla, Aditi; Hilgenfeld, Rolf

    2014-09-01

    The huge RNA genome of SARS coronavirus comprises a number of open reading frames that code for a total of eight accessory proteins. Although none of these are essential for virus replication, some appear to have a role in virus pathogenesis. Notably, some SARS-CoV accessory proteins have been shown to modulate the interferon signaling pathways and the production of pro-inflammatory cytokines. The structural information on these proteins is also limited, with only two (p7a and p9b) having their structures determined by X-ray crystallography. This review makes an attempt to summarize the published knowledge on SARS-CoV accessory proteins, with an emphasis on their involvement in virus-host interaction. The accessory proteins of other coronaviruses are also briefly discussed. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses" (see Introduction by Hilgenfeld and Peiris (2013)). Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Coronavirus antibodies in African bat species.

    Science.gov (United States)

    Müller, Marcel A; Paweska, Janusz T; Leman, Patricia A; Drosten, Christian; Grywna, Klaus; Kemp, Alan; Braack, Leo; Sonnenberg, Karen; Niedrig, Matthias; Swanepoel, Robert

    2007-09-01

    Asian bats have been identified as potential reservoir hosts of coronaviruses associated with severe acute respiratory syndrome (SARS-CoV). We detected antibody reactive with SARS-CoV antigen in 47 (6.7%) of 705 bat serum specimens comprising 26 species collected in Africa; thus, African bats may harbor agents related to putative group 4 CoV.

  20. Understanding the T cell immune response in SARS coronavirus infection.

    Science.gov (United States)

    Janice Oh, Hsueh-Ling; Ken-En Gan, Samuel; Bertoletti, Antonio; Tan, Yee-Joo

    2012-09-01

    The severe acute respiratory syndrome (SARS) epidemic started in late 2002 and swiftly spread across 5 continents with a mortality rate of around 10%. Although the epidemic was eventually controlled through the implementation of strict quarantine measures, there continues a need to investigate the SARS coronavirus (SARS-CoV) and develop interventions should it re-emerge. Numerous studies have shown that neutralizing antibodies against the virus can be found in patients infected with SARS-CoV within days upon the onset of illness and lasting up to several months. In contrast, there is little data on the kinetics of T cell responses during SARS-CoV infection and little is known about their role in the recovery process. However, recent studies in mice suggest the importance of T cells in viral clearance during SARS-CoV infection. Moreover, a growing number of studies have investigated the memory T cell responses in recovered SARS patients. This review covers the available literature on the emerging importance of T cell responses in SARS-CoV infection, particularly on the mapping of cytotoxic T lymphocyte (CTL) epitopes, longevity, polyfunctionality and human leukocyte antigen (HLA) association as well as their potential implications on treatment and vaccine development.

  1. Alisporivir inhibits MERS- and SARS-coronavirus replication in cell culture, but not SARS-coronavirus infection in a mouse model.

    Science.gov (United States)

    de Wilde, Adriaan H; Falzarano, Darryl; Zevenhoven-Dobbe, Jessika C; Beugeling, Corrine; Fett, Craig; Martellaro, Cynthia; Posthuma, Clara C; Feldmann, Heinz; Perlman, Stanley; Snijder, Eric J

    2017-01-15

    Currently, there is no registered treatment for infections with emerging zoonotic coronaviruses like SARS- and MERS-coronavirus. We here report that in cultured cells low-micromolar concentrations of alisporivir, a non-immunosuppressive cyclosporin A-analog, inhibit the replication of four different coronaviruses, including MERS- and SARS-coronavirus. Ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of SARS-CoV infection in a mouse model. Nevertheless, our data provide a basis to further explore the potential of Cyp inhibitors as host-directed, broad-spectrum inhibitors of coronavirus replication.

  2. [Prokaryotic expression of S2 extracellular domain of SARS coronavirus spike protein and its fusion with Hela cell membrane].

    Science.gov (United States)

    Liu, Yun; Liu, Ai-Hua; Deng, Peng; Wu, Xiang-Ling; Li, Tao; Liu, Ya-Wei; Xu, Jia; Jiang, Yong

    2009-03-01

    To construct the expression plasmid of S2 extracellular domain (S2ED) of SARS-coronavirus (SARS- Cov) spike protein (S protein) and enhanced green fluorescent protein (EGFP) to obtain the fusion protein expressed in prokaryotic cells. S2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fractions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion of S2ED with Hela cell membranes was observed with fluorescent microscope. The pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 (DE3). When incubated with Hela cells, the purified protein could not internalize through membrane fusion. The expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.

  3. Comparison between SARS CoV and MERS CoV Using Apriori Algorithm, Decision Tree, SVM

    Directory of Open Access Journals (Sweden)

    Jang Seongpil

    2016-01-01

    Full Text Available MERS (Middle East Respiratory Syndrome is a worldwide disease these days. The number of infected people is 1038(08/03/2015 in Saudi Arabia and 186(08/03/2015 in South Korea. MERS is all over the world including Europe and the fatality rate is 38.8%, East Asia and the Middle East. The MERS is also known as a cousin of SARS (Severe Acute Respiratory Syndrome because both diseases show similar symptoms such as high fever and difficulty in breathing. This is why we compared MERS with SARS. We used data of the spike glycoprotein from NCBI. As a way of analyzing the protein, apriori algorithm, decision tree, SVM were used, and particularly SVM was iterated by normal, polynomial, and sigmoid. The result came out that the MERS and the SARS are alike but also different in some way.

  4. Expression and Purification of SARS Coronavirus Membrane Protein

    Institute of Scientific and Technical Information of China (English)

    戴五星; 雷明军; 吴少庭; 陈智浩; 梁靓; 潘晖榕; 秦莉; 高士同; 袁仕善; 张仁利

    2004-01-01

    To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The re combinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropylβ-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0. 992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.

  5. Identification of myricetin and scutellarein as novel chemical inhibitors of the SARS coronavirus helicase, nsP13.

    Science.gov (United States)

    Yu, Mi-Sun; Lee, June; Lee, Jin Moo; Kim, Younggyu; Chin, Young-Won; Jee, Jun-Goo; Keum, Young-Sam; Jeong, Yong-Joo

    2012-06-15

    Severe acute respiratory syndrome (SARS) is an infectious disease with a strong potential for transmission upon close personal contact and is caused by the SARS-coronavirus (CoV). However, there are no natural or synthetic compounds currently available that can inhibit SARS-CoV. We examined the inhibitory effects of 64 purified natural compounds against the activity of SARS helicase, nsP13, and the hepatitis C virus (HCV) helicase, NS3h, by conducting fluorescence resonance energy transfer (FRET)-based double-strand (ds) DNA unwinding assay or by using a colorimetry-based ATP hydrolysis assay. While none of the compounds, examined in our study inhibited the DNA unwinding activity or ATPase activity of human HCV helicase protein, we found that myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity, but not the unwinding activity, nsP13. In addition, we observed that myricetin and scutellarein did not exhibit cytotoxicity against normal breast epithelial MCF10A cells. Our study demonstrates for the first time that selected naturally-occurring flavonoids, including myricetin and scultellarein might serve as SARS-CoV chemical inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. SARS Pathogenesis: Host Factors

    NARCIS (Netherlands)

    A. de Lang (Anna)

    2012-01-01

    textabstractWhile it is hypothesized that Sever Acute Respiratory Syndrome (SARS) in humans is caused by a disproportional immune response illustrated by inappropriate induction of inflammatory cytokines, the exact nature of the host response to SARS coronavirus (CoV) infection causing severe

  7. The SARS coronavirus nucleocapsid protein--forms and functions.

    Science.gov (United States)

    Chang, Chung-ke; Hou, Ming-Hon; Chang, Chi-Fon; Hsiao, Chwan-Deng; Huang, Tai-huang

    2014-03-01

    The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly. It is a protein with multifarious activities. In this article we will review our current understanding of the N protein structure and its interaction with nucleic acid. Highlights of the progresses include uncovering the modular organization, determining the structures of the structural domains, realizing the roles of protein disorder in protein-protein and protein-nucleic acid interactions, and visualizing the ribonucleoprotein (RNP) structure inside the virions. It was also demonstrated that N-protein binds to nucleic acid at multiple sites with a coupled-allostery manner. We propose a SARS-CoV RNP model that conforms to existing data and bears resemblance to the existing RNP structures of RNA viruses. The model highlights the critical role of modular organization and intrinsic disorder of the N protein in the formation and functions of the dynamic RNP capsid in RNA viruses. This paper forms part of a symposium in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses." Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

    NARCIS (Netherlands)

    Hamming, [No Value; Timens, W; Bulthuis, MLC; Lely, AT; Navis, GJ; van Goor, H

    2004-01-01

    Severe acute respiratory syndrome (SARS) is an acute infectious disease that spreads mainly via the respiratory route. A distinct coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Recently, a metallopeptidase named angiotensin-converting enzyme 2 (ACE2) has been identifie

  9. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

    NARCIS (Netherlands)

    Hamming, [No Value; Timens, W; Bulthuis, MLC; Lely, AT; Navis, GJ; van Goor, H

    Severe acute respiratory syndrome (SARS) is an acute infectious disease that spreads mainly via the respiratory route. A distinct coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Recently, a metallopeptidase named angiotensin-converting enzyme 2 (ACE2) has been

  10. From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.

    Science.gov (United States)

    Hilgenfeld, Rolf; Peiris, Malik

    2013-10-01

    This article introduces a series of invited papers in Antiviral Research marking the 10th anniversary of the outbreak of severe acute respiratory syndrome (SARS), caused by a novel coronavirus that emerged in southern China in late 2002. Until that time, coronaviruses had not been recognized as agents causing severe disease in humans, hence, the emergence of the SARS-CoV came as a complete surprise. Research during the past ten years has revealed the existence of a diverse pool of coronaviruses circulating among various bat species and other animals, suggesting that further introductions of highly pathogenic coronaviruses into the human population are not merely probable, but inevitable. The recent emergence of another coronavirus causing severe disease, Middle East respiratory syndrome (MERS), in humans, has made it clear that coronaviruses pose a major threat to human health, and that more research is urgently needed to elucidate their replication mechanisms, identify potential drug targets, and develop effective countermeasures. In this series, experts in many different aspects of coronavirus replication and disease will provide authoritative, up-to-date reviews of the following topics: - clinical management and infection control of SARS; - reservoir hosts of coronaviruses; - receptor recognition and cross-species transmission of SARS-CoV; - SARS-CoV evasion of innate immune responses; - structures and functions of individual coronaviral proteins; - anti-coronavirus drug discovery and development; and - the public health legacy of the SARS outbreak. Each article will be identified in the last line of its abstract as belonging to the series "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses." Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique

    Institute of Scientific and Technical Information of China (English)

    Shuai CHEN; Hua-liang JIANG; Li-li CHEN; Hai-bin LUO; Tao SUN; Jing CHEN; Fei YE; Jian-hua CAI; Jing-kang SHEN; Xu SHEN

    2005-01-01

    Aim: To characterize enzymatic activity of severe acute respiratory syndrome(SARS) coronavirus (CoV) 3C-like protease (3CLpro) and its four site-directed mutants. Methods: Based on the fluorescence resonance energy transfer (FRET)principle using 5-[(2'-aminoethyl)-amino] naphthelenesulfonic acid (EDANS) and 4-[[4-(dimethylamino) phenyl] azo] benzoic acid (Dabcyl) as the energy transfer pair, one fluorogenic substrate was designed for the evaluation of SARS-CoV 3CLpro proteolytic activity. Results: The kinetic parameters of the fluorogenic substrate have been determined as Km=404 μmol.L-1, kcat=1.08 min-1, and kcat/Km=2.7 gered activity switches, and site-directed mutagenesis analysis of SARS-CoV 3CLpro revealed that substitutions of His41, Cys145, and His163 resulted in complete loss of enzymatic activity, while replacement of Met162 with Ala caused strongly increased activity. Conclusion: This present work has provided valuable information for understanding the catalytic mechanism of SARS-CoV 3CLpro. This FRET-based assay might supply an ideal approach for the exploration SARSCoV 3CLpro putative inhibitors.

  12. Dissection of SARS Coronavirus Spike Protein into Discrete Folded Fragments

    Institute of Scientific and Technical Information of China (English)

    LI Shuang; CAI Zhen; CHEN Yong; LIN Zhanglin

    2006-01-01

    The spike protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) mediates cell fusion by binding to target cell surface receptors. This paper reports a simple method for dissecting the viral protein and for searching for foldable fragments in a random but systematic manner. The method involves digestion by DNase I to generate a pool of short DNA segments, followed by an additional step of reassembly of these segments to produce a library of DNA fragments with random ends but controllable lengths. To rapidly screen for discrete folded polypeptide fragments, the reassembled gene fragments were further cloned into a vector as N-terminal fusions to a folding reporter gene which was a variant of green fluorescent protein. Two foldable fragments were identified for the SARS-CoV spike protein, which coincide with various anti-SARS peptides derived from the hepated repeat (HR) region 2 of the spike protein. The method should be applicable to other viral proteins to isolate antigen or vaccine candidates, thus providing an alternative to the full-length proteins (subunits) or linear short peptides.

  13. Development of chemical inhibitors of the SARS coronavirus: viral helicase as a potential target.

    Science.gov (United States)

    Keum, Young-Sam; Jeong, Yong-Joo

    2012-11-15

    Severe acute respiratory syndrome (SARS) was the first pandemic in the 21st century to claim more than 700 lives worldwide. However, effective anti-SARS vaccines or medications are currently unavailable despite being desperately needed to adequately prepare for a possible SARS outbreak. SARS is caused by a novel coronavirus, and one of its components, a viral helicase, is emerging as a promising target for the development of chemical SARS inhibitors. In the following review, we describe the characterization, family classification, and kinetic movement mechanisms of the SARS coronavirus (SCV) helicase-nsP13. We also discuss the recent progress in the identification of novel chemical inhibitors of nsP13 in the context of our recent discovery of the strong inhibition of the SARS helicase by natural flavonoids, myricetin and scutellarein. These compounds will serve as important resources for the future development of anti-SARS medications. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines

    NARCIS (Netherlands)

    M.A. Müller (Marcel); V.S. Raj (V. Stalin); D. Muth; B. Meyer (Bernhard); S. Kallies (Stephan); S.L. Smits (Saskia); R. Wollny (Robert); T.M. Bestebroer (Theo); S. Specht (Sabine); T. Suliman (Tasnim); K. Zimmermann (Kathrin); T. Binger (Tabea); I. Eckerle; M. Tschapka (Marco); A.M. Zaki (Ali); A.D.M.E. Osterhaus (Albert); R.A.M. Fouchier (Ron); B.L. Haagmans (Bart); C. Drosten (Christian)

    2012-01-01

    textabstractA new human coronavirus (hCoV-EMC) has emerged very recently in the Middle East. The clinical presentation resembled that of the severe acute respiratory syndrome (SARS) as encountered during the epidemic in 2002/2003. In both cases, acute renal failure was observed in humans. HCoV-EMC i

  15. A simple and rapid approach for screening of SARS-coronavirus genotypes: an evaluation study

    Directory of Open Access Journals (Sweden)

    Jin Yongjie

    2005-10-01

    Full Text Available Abstract Background The Severe Acute Respiratory Syndrome (SARS was a newly emerged infectious disease which caused a global epidemic in 2002–2003. Sequence analysis of SARS-coronavirus isolates revealed that specific genotypes predominated at different periods of the epidemic. This information can be used as a footprint for tracing the epidemiology of infections and monitor viral evolution. However, direct sequencing analysis of a large number of clinical samples is cumbersome and time consuming. We present here a simple and rapid assay for the screening of SARS-coronavirus genotypes based on the use of fluorogenic oligonucleotide probes for allelic discrimination. Methods Thirty SARS patients were recruited. Allelic discrimination assays were developed based on the use of fluorogenic oligonucleotide probes (TaqMan. Genotyping of the SARS-coronavirus isolates obtained from these patients were carried out by the allelic discrimination assays and confirmed by direct sequencing. Results Genotyping based on the allelic discrimination assays were fully concordant with direct sequencing. All of the 30 SARS-coronavirus genotypes studied were characteristic of genotypes previously documented to be associated with the latter part of the epidemic. Seven of the isolates contained a previously reported major deletion but in patients not epidemiologically related to the previously studied cohort. Conclusion We have developed a simple and accurate method for the characterization and screening of SARS-coronavirus genotypes. It is a promising tool for the study of epidemiological relationships between documented cases during an outbreak.

  16. Peptide Mimicrying Between SARS Coronavirus Spike Protein and Human Proteins Reacts with SARS Patient Serum

    Directory of Open Access Journals (Sweden)

    K.-Y. Hwa

    2008-01-01

    Full Text Available Molecular mimicry, defined as similar structures shared by molecules from dissimilar genes or proteins, is a general strategy used by pathogens to infect host cells. Severe acute respiratory syndrome (SARS is a new human respiratory infectious disease caused by SARS coronavirus (SARS-CoV. The spike (S protein of SARS-CoV plays an important role in the virus entry into a cell. In this study, eleven synthetic peptides from the S protein were selected based on its sequence homology with human proteins. Two of the peptides D07 (residues 927–937 and D08 (residues 942–951 were recognized by the sera of SARS patients. Murine hyperimmune sera against these peptides bound to proteins of human lung epithelial cells A549. Another peptide D10 (residues 490–502 stimulated A549 to proliferate and secrete IL-8. The present results suggest that the selected S protein regions, which share sequence homology with human proteins, may play important roles in SARS-CoV infection.

  17. SARS CORONAVIRUS ENRICHED IN LYMPHOCYTES:AN EARLY DETECTION OF SARS%检测淋巴细胞内的冠状病毒RNA可作为SARS的早期诊断指标

    Institute of Scientific and Technical Information of China (English)

    王海滨; 毛远丽; 鞠连才

    2004-01-01

    Because of the highly contagious nature of the SARS-Coronavirus (CoV),a rapid and reliable diagnostic test is urgently needed for making a definite diagnosis at early phase so that infected individuals can be isolated while avoiding blanket quarantines and the unnecessary burden on the medical care system.Unfortanantly,current kits/procedures had only less than 20 % positive detecting rate with serum or other samples of real SARS patients.Due to the compositions varies with times and individuals,the septum and lung lavage are impossible to serve as a standardized sample source for the comparison or monitoring of viral load.Therefore,the currently available kits/procedures should be improved and a good sample source should be identified.[Methods] Viral RNA in two hundred microlitter plasma or one million lymphocytes from SARS patients were extracted with Trizol,and then entire RNA was used as a template in a modified procedure of RT-PCR.CoV viral load was quantified in 45 patients at different phases of SARS infection and compared between the paired samples of plasma and lymphocytes obtained from 5 patients who had recovered from SARS for 2 months and lived in a normal life.[Results] Our methods detected CoV at a level of a few copies with 60-80% positive detection rate for plasma from SARS patients within 1 to 7 days after fever,which was much more sensitive than other current available method(positive rate at 8%~20%).The mean viral copies in 17 acute phase patients(1 to 7 days after fever)was 8,951,while that in 18 sub-acute phase patients(13 to 36 days after fever)was 98 and that in 10 recovered patients(79 to 91 days after fever)was 88.Importantly,the levels of CoV in lymphocytes of 5 patients who had recovered from SARS for 2 months were 2 to 3 orders of magnitude higher than that in the plasma.[Conclusions] The early and definite diagnosis of SARS can be achieved by our modified RT-PCR.CoV load peaked during the acute phase and rapidly dropped in sub

  18. Prevalence and phylogeny of coronaviruses in wild birds from the Bering Strait area (Beringia.

    Directory of Open Access Journals (Sweden)

    Shaman Muradrasoli

    Full Text Available Coronaviruses (CoVs can cause mild to severe disease in humans and animals, their host range and environmental spread seem to have been largely underestimated, and they are currently being investigated for their potential medical relevance. Infectious bronchitis virus (IBV belongs to gamma-coronaviruses and causes a costly respiratory viral disease in chickens. The role of wild birds in the epidemiology of IBV is poorly understood. In the present study, we examined 1,002 cloacal and faecal samples collected from 26 wild bird species in the Beringia area for the presence of CoVs, and then we performed statistical and phylogenetic analyses. We detected diverse CoVs by RT-PCR in wild birds in the Beringia area. Sequence analysis showed that the detected viruses are gamma-coronaviruses related to IBV. These findings suggest that wild birds are able to carry gamma-coronaviruses asymptomatically. We concluded that CoVs are widespread among wild birds in Beringia, and their geographic spread and frequency is higher than previously realised. Thus, Avian CoV can be efficiently disseminated over large distances and could be a genetic reservoir for future emerging pathogenic CoVs. Considering the great animal health and economic impact of IBV as well as the recent emergence of novel coronaviruses such as SARS-coronavirus, it is important to investigate the role of wildlife reservoirs in CoV infection biology and epidemiology.

  19. MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment

    NARCIS (Netherlands)

    A.H. de Wilde (Adriaan); V.S. Raj (Stalin); D. Oudshoorn (Diede); T.M. Bestebroer (Theo); S. van Nieuwkoop (Stefan); R. Limpens (Ronald); C.C. Posthuma (Clara); Y. van der Meer (Yvonne); M. Bárcena (Montserrat); B.L. Haagmans (Bart); E.J. Snijder (Eric); B.G. van den Hoogen (Bernadette)

    2013-01-01

    textabstractCoronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle Ea

  20. Influence of hydrophobic and electrostatic residues on SARS-coronavirus S2 protein stability: insights into mechanisms of general viral fusion and inhibitor design.

    Science.gov (United States)

    Aydin, Halil; Al-Khooly, Dina; Lee, Jeffrey E

    2014-05-01

    Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS-coronavirus (SARS-CoV). SARS-CoV entry is facilitated by the spike protein (S), which consists of an N-terminal domain (S1) responsible for cellular attachment and a C-terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS-CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site-directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH-independent retroviral fusion proteins, SARS-CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS-CoV S2 analysis showed that specific hydrophobic positions at the C-terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C-terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C-terminal hydrophobic residues led us to identify a 42-residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation=0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections. © 2014 The Protein Society.

  1. Transmission of SARS and MERS coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination.

    Science.gov (United States)

    Otter, J A; Donskey, C; Yezli, S; Douthwaite, S; Goldenberg, S D; Weber, D J

    2016-03-01

    Viruses with pandemic potential including H1N1, H5N1, and H5N7 influenza viruses, and severe acute respiratory syndrome (SARS)/Middle East respiratory syndrome (MERS) coronaviruses (CoV) have emerged in recent years. SARS-CoV, MERS-CoV, and influenza virus can survive on surfaces for extended periods, sometimes up to months. Factors influencing the survival of these viruses on surfaces include: strain variation, titre, surface type, suspending medium, mode of deposition, temperature and relative humidity, and the method used to determine the viability of the virus. Environmental sampling has identified contamination in field-settings with SARS-CoV and influenza virus, although the frequent use of molecular detection methods may not necessarily represent the presence of viable virus. The importance of indirect contact transmission (involving contamination of inanimate surfaces) is uncertain compared with other transmission routes, principally direct contact transmission (independent of surface contamination), droplet, and airborne routes. However, influenza virus and SARS-CoV may be shed into the environment and be transferred from environmental surfaces to hands of patients and healthcare providers. Emerging data suggest that MERS-CoV also shares these properties. Once contaminated from the environment, hands can then initiate self-inoculation of mucous membranes of the nose, eyes or mouth. Mathematical and animal models, and intervention studies suggest that contact transmission is the most important route in some scenarios. Infection prevention and control implications include the need for hand hygiene and personal protective equipment to minimize self-contamination and to protect against inoculation of mucosal surfaces and the respiratory tract, and enhanced surface cleaning and disinfection in healthcare settings.

  2. Genome sequence variation analysis of two SARS coronavirus isolates after passage in Vero cell culture

    Institute of Scientific and Technical Information of China (English)

    JIN Weiwu; LI Ning; HU Liangxiang; DU Zhenglin; GAO Qiang; GAO Hong; NING Ye; FENG Jidong; ZHANG Jiansan; YIN Weidong

    2004-01-01

    SARS coronavirus is an RNA virus whose replication is error-prone, which provides possibility for escape of host defenses, and even leads to evolution of new viral strains during the passage or the transmission. Lots of variations have been detected among different SARS-CoV strains. And a study on these variations is helpful for development of efficient vaccine. Moreover, the test of nucleic acid characterization and genetic stability of SARS-CoV is important in the research of inactivated vaccine. The whole genome sequences of two SARS coronavirus strains after passage in Vero cell culture were determined and were compared with those of early passages, respectively. Results showed that both SARS coronavirus strains have high genetic stability, although nearly 10 generations were passed. Four nucleotide variations were observed between the second passage and the 11th passage of Sino1 strain for identification of SARS inactivated vaccine. Moreover, only one nucleotide was different between the third passage and the 10th passage of Sino3 strain for SARS inactivated vaccine. Therefore, this study suggested it was possible to develop inactivated vaccine against SARS-CoV in the future.

  3. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds.

    Science.gov (United States)

    Báez-Santos, Yahira M; St John, Sarah E; Mesecar, Andrew D

    2015-03-01

    Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of

  4. CLONING SEGMENT SPIKE PROTEIN GENE OF SARS-COV AND ITS EXPRESSION IN ESCHERICHIA COLI

    Institute of Scientific and Technical Information of China (English)

    刘中华; 许文波; 毛乃颖; 张燕; 朱贞; 崔爱利; 杨建国; 胡海涛

    2004-01-01

    Objective Expressing and purifying the segment of SARS-CoV spike protein in E.Coli. Methods The target gene was obtained by RT-PCR. The PCR product was cloned into pEGM- T Easy Vector, sequencing and double restriction digestion ( BamHⅠ,PstⅠ) were performed. The target gene was subcloned into PQE30 expression vector. The gene was expressed in the E.coli strain M15 cells induced by IPTG. The protein was purified with a nickel HiTrap chelating metal affinity column. Results The recombinant expression plasmid was successfully constructed and the protein was well expressed in E. coli strain M15 cells. The ideal pure protein was obtained by purification. Western blotting analysis suggested the protein could act with the convalescent sera of lab confirmed SARS patients. Conclusion The segment of SARS-CoV spike protein was well expressed and purified, and can be applied in diagnosis and immunological research of SARS.

  5. SARS coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon.

    Science.gov (United States)

    Totura, Allison L; Baric, Ralph S

    2012-06-01

    SARS-CoV is a pathogenic coronavirus that emerged from a zoonotic reservoir, leading to global dissemination of the virus. The association SARS-CoV with aberrant cytokine, chemokine, and Interferon Stimulated Gene (ISG) responses in patients provided evidence that SARS-CoV pathogenesis is at least partially controlled by innate immune signaling. Utilizing models for SARS-CoV infection, key components of innate immune signaling pathways have been identified as protective factors against SARS-CoV disease, including STAT1 and MyD88. Gene transcription signatures unique to SARS-CoV disease states have been identified, but host factors that regulate exacerbated disease phenotypes still remain largely undetermined. SARS-CoV encodes several proteins that modulate innate immune signaling through the antagonism of the induction of Interferon and by avoidance of ISG effector functions. Copyright © 2012. Published by Elsevier B.V.

  6. Interaction of SARS and MERS Coronaviruses with the Antiviral Interferon Response.

    Science.gov (United States)

    Kindler, E; Thiel, V; Weber, F

    2016-01-01

    Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) are the most severe coronavirus (CoV)-associated diseases in humans. The causative agents, SARS-CoV and MERS-CoV, are of zoonotic origin but may be transmitted to humans, causing severe and often fatal respiratory disease in their new host. The two coronaviruses are thought to encode an unusually large number of factors that allow them to thrive and replicate in the presence of efficient host defense mechanisms, especially the antiviral interferon system. Here, we review the recent progress in our understanding of the strategies that highly pathogenic coronaviruses employ to escape, dampen, or block the antiviral interferon response in human cells. © 2016 Elsevier Inc. All rights reserved.

  7. Coronavirus 3CLpro proteinase cleavage sites: Possible relevance to SARS virus pathology

    Directory of Open Access Journals (Sweden)

    Blom Nikolaj

    2004-06-01

    Full Text Available Abstract Background Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS, efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR, transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. Conclusions Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

  8. SARS-unique fold in the Rousettus bat coronavirus HKU9.

    Science.gov (United States)

    Hammond, Robert G; Tan, Xuan; Johnson, Margaret A

    2017-09-01

    The coronavirus nonstructural protein 3 (nsp3) is a multifunctional protein that comprises multiple structural domains. This protein assists viral polyprotein cleavage, host immune interference, and may play other roles in genome replication or transcription. Here, we report the solution NMR structure of a protein from the "SARS-unique region" of the bat coronavirus HKU9. The protein contains a frataxin fold or double-wing motif, which is an α + β fold that is associated with protein/protein interactions, DNA binding, and metal ion binding. High structural similarity to the human severe acute respiratory syndrome (SARS) coronavirus nsp3 is present. A possible functional site that is conserved among some betacoronaviruses has been identified using bioinformatics and biochemical analyses. This structure provides strong experimental support for the recent proposal advanced by us and others that the "SARS-unique" region is not unique to the human SARS virus, but is conserved among several different phylogenetic groups of coronaviruses and provides essential functions. © 2017 The Protein Society.

  9. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins.

    Science.gov (United States)

    Wang, Sheng-Fan; Tseng, Sung-Pin; Yen, Chia-Hung; Yang, Jyh-Yuan; Tsao, Ching-Han; Shen, Chun-Wei; Chen, Kuan-Hsuan; Liu, Fu-Tong; Liu, Wu-Tse; Chen, Yi-Ming Arthur; Huang, Jason C

    2014-08-22

    The severe acute respiratory syndrome coronavirus (SARS-CoV) still carries the potential for reemergence, therefore efforts are being made to create a vaccine as a prophylactic strategy for control and prevention. Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Molecular Advances in Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV)

    Institute of Scientific and Technical Information of China (English)

    Ken Yan Ching Chow; Chung Chau Hon; Raymond Kin Hi Hui; Raymond Tsz Yeung Wong; Chi Wai Yip; Fanya Zeng; Frederick Chi Ching Leung

    2003-01-01

    The sudden outbreak of severe acute respiratory syndrome (SARS) in 2002 prompted the establishment of a global scientific network subsuming most of the traditional rivalries in the competitive field of virology. Within months of the SARS outbreak, collaborative work revealed the identity of the disastrous pathogen as SARS-associated coronavirus (SARS-CoV). However, although the rapid identification of the agent represented an important breakthrough, our understanding of the deadly virus remains limited. Detailed biological knowledge is crucial for the development of effective countermeasures, diagnostic tests, vaccines and antiviral drugs against the SARS-CoV. This article reviews the present state of molecular knowledge about SARS-CoV, from the aspects of comparative genomics, molecular biology of viral genes, evolution, and epidemiology, and describes the diagnostic tests and the anti-viral drugs derived so far based on the available molecular information.

  11. Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination.

    Directory of Open Access Journals (Sweden)

    Sjoerd H E van den Worm

    Full Text Available Severe acute respiratory syndrome (SARS is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV. Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs. In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E. Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.

  12. Reverse Genetics of SARS-Related Coronavirus Using Vaccinia Virus-Based Recombination

    Science.gov (United States)

    Zevenhoven, Jessika C.; Weber, Friedemann; Züst, Roland; Kuri, Thomas; Dijkman, Ronald; Chang, Guohui; Siddell, Stuart G.; Snijder, Eric J.; Thiel, Volker; Davidson, Andrew D.

    2012-01-01

    Severe acute respiratory syndrome (SARS) is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV) that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime) as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV). Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs). In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E). Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs. PMID:22412934

  13. Design and application of 60mer oligonucleotide microarray in SARS coronavirus detection

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The 60mer oligonucleotide microarray was designed and applied to detecting of SARS (severe acute respiratory syndrome) coronavirus. Thirty 60mer specific oligos were designed to cover the whole genome of the first submitted coronavirus strain, according to the sequence of TOR2 (GENEBANK Accession: AY274119). These primers were synthesized and printed into a microarray with 12×12 spots. RNAs were extracted from the throat swab and gargling fluid of SARS patients and reverse-transcripted into the double strand cDNAs. The cDNAs were prepared as restricted cDNA fragments by the restriction display (RD) technique and labeled by PCR with the Cy5-universal primer. The labeled samples were then applied to the oligo microarray for hybridization. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The scanning result showed that samples of SARS patients were hybridized with multiple SARS probes on the microarray, and there is no signal on the negative and blank controls. These results indicate that the genome of SARS coronavirus can be detected in parallel by the 60mer oligonucleotide microarray, which can improve the positive ratio of the diagnosis. The oligo microarray can also be used for monitoring the behavior of the virus genes in different stages of the disease status.

  14. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.

    Science.gov (United States)

    Ge, Xing-Yi; Li, Jia-Lu; Yang, Xing-Lou; Chmura, Aleksei A; Zhu, Guangjian; Epstein, Jonathan H; Mazet, Jonna K; Hu, Ben; Zhang, Wei; Peng, Cheng; Zhang, Yu-Ji; Luo, Chu-Ming; Tan, Bing; Wang, Ning; Zhu, Yan; Crameri, Gary; Zhang, Shu-Yi; Wang, Lin-Fa; Daszak, Peter; Shi, Zheng-Li

    2013-11-28

    The 2002-3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history. An ongoing outbreak of Middle East respiratory syndrome coronavirus suggests that this group of viruses remains a key threat and that their distribution is wider than previously recognized. Although bats have been suggested to be the natural reservoirs of both viruses, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa, but none is considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2). Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

  15. The role of epidermal growth factor receptor (EGFR) signaling in SARS coronavirus-induced pulmonary fibrosis.

    Science.gov (United States)

    Venkataraman, Thiagarajan; Frieman, Matthew B

    2017-07-01

    Many survivors of the 2003 outbreak of severe acute respiratory syndrome (SARS) developed residual pulmonary fibrosis with increased severity seen in older patients. Autopsies of patients that died from SARS also showed fibrosis to varying extents. Pulmonary fibrosis can be occasionally seen as a consequence to several respiratory viral infections but is much more common after a SARS coronavirus (SARS-CoV) infection. Given the threat of future outbreaks of severe coronavirus disease, including Middle East respiratory syndrome (MERS), it is important to understand the mechanisms responsible for pulmonary fibrosis, so as to support the development of therapeutic countermeasures and mitigate sequelae of infection. In this article, we summarize pulmonary fibrotic changes observed after a SARS-CoV infection, discuss the extent to which other respiratory viruses induce fibrosis, describe available animal models to study the development of SARS-CoV induced fibrosis and review evidence that pulmonary fibrosis is caused by a hyperactive host response to lung injury mediated by epidermal growth factor receptor (EGFR) signaling. We summarize work from our group and others indicating that inhibiting EGFR signaling may prevent an excessive fibrotic response to SARS-CoV and other respiratory viral infections and propose directions for future research. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. The Role of Severe Acute Respiratory Syndrome (SARS)-Coronavirus Accessory Proteins in Virus Pathogenesis

    Science.gov (United States)

    McBride, Ruth; Fielding, Burtram C.

    2012-01-01

    A respiratory disease caused by a novel coronavirus, termed the severe acute respiratory syndrome coronavirus (SARS-CoV), was first reported in China in late 2002. The subsequent efficient human-to-human transmission of this virus eventually affected more than 30 countries worldwide, resulting in a mortality rate of ~10% of infected individuals. The spread of the virus was ultimately controlled by isolation of infected individuals and there has been no infections reported since April 2004. However, the natural reservoir of the virus was never identified and it is not known if this virus will re-emerge and, therefore, research on this virus continues. The SARS-CoV genome is about 30 kb in length and is predicted to contain 14 functional open reading frames (ORFs). The genome encodes for proteins that are homologous to known coronavirus proteins, such as the replicase proteins (ORFs 1a and 1b) and the four major structural proteins: nucleocapsid (N), spike (S), membrane (M) and envelope (E). SARS-CoV also encodes for eight unique proteins, called accessory proteins, with no known homologues. This review will summarize the current knowledge on SARS-CoV accessory proteins and will include: (i) expression and processing; (ii) the effects on cellular processes; and (iii) functional studies. PMID:23202509

  17. Differential stepwise evolution of SARS coronavirus functional proteins in different host species

    Directory of Open Access Journals (Sweden)

    Tang Xianchun

    2009-03-01

    Full Text Available Abstract Background SARS coronavirus (SARS-CoV was identified as the etiological agent of SARS, and extensive investigations indicated that it originated from an animal source (probably bats and was recently introduced into the human population via wildlife animals from wet markets in southern China. Previous studies revealed that the spike (S protein of SARS had experienced adaptive evolution, but whether other functional proteins of SARS have undergone adaptive evolution is not known. Results We employed several methods to investigate selective pressure among different SARS-CoV groups representing different epidemic periods and hosts. Our results suggest that most functional proteins of SARS-CoV have experienced a stepwise adaptive evolutionary pathway. Similar to previous studies, the spike protein underwent strong positive selection in the early and middle phases, and became stabilized in the late phase. In addition, the replicase experienced positive selection only in human patients, whereas assembly proteins experienced positive selection mainly in the middle and late phases. No positive selection was found in any proteins of bat SARS-like-CoV. Furthermore, specific amino acid sites that may be the targets of positive selection in each group are identified. Conclusion This extensive evolutionary analysis revealed the stepwise evolution of different functional proteins of SARS-CoVs at different epidemic stages and different hosts. These results support the hypothesis that SARS-CoV originated from bats and that the spill over into civets and humans were more recent events.

  18. Discovery of Anti-SARS Coronavirus Drug Based on Molecular Docking and Database Screening

    Institute of Scientific and Technical Information of China (English)

    CHEN,Hai-Feng(陈海峰); YAO,Jian-Hua(姚建华); SUN,Jing(孙晶); LI,Qiang(李强); LI,Feng(李丰); FAN,Bo-Tao(范波涛); YUAN,Shen-Gang(袁身刚)

    2004-01-01

    The active site of 3CL proteinase (3CLpro) for coronavirus was identified by comparing the crystal structures of human and porcine coronavirus. The inhibitor of the main protein of rhinovirus (Ag7088) could bind with 3CLpro of human coronavirus, then it was selected as the reference for molecular docking and database screening. The ligands from two databases were used to search potential lead structures with molecular docking. Several structures from natural products and ACD-SC databases were found to have lower binding free energy with 3CLpro than that of Ag7088. These structures have similar hydrophobicity to Ag7088. They have complementary electrostatic potential and hydrogen bond acceptor and donor with 3CLpro, showing that the strategy of anti-SARS drug design based on molecular docking and database screening is feasible.

  19. Protection from SARS coronavirus conferred by live measles vaccine expressing the spike glycoprotein.

    Science.gov (United States)

    Escriou, Nicolas; Callendret, Benoît; Lorin, Valérie; Combredet, Chantal; Marianneau, Philippe; Février, Michèle; Tangy, Frédéric

    2014-03-01

    The recent identification of a novel human coronavirus responsible of a SARS-like illness in the Middle-East a decade after the SARS pandemic, demonstrates that reemergence of a SARS-like coronavirus from an animal reservoir remains a credible threat. Because SARS is contracted by aerosolized contamination of the respiratory tract, a vaccine inducing mucosal long-term protection would be an asset to control new epidemics. To this aim, we generated live attenuated recombinant measles vaccine (MV) candidates expressing either the membrane-anchored SARS-CoV spike (S) protein or its secreted soluble ectodomain (Ssol). In mice susceptible to measles virus, recombinant MV expressing the anchored full-length S induced the highest titers of neutralizing antibodies and fully protected immunized animals from intranasal infectious challenge with SARS-CoV. As compared to immunization with adjuvanted recombinant Ssol protein, recombinant MV induced stronger and Th1-biased responses, a hallmark of live attenuated viruses and a highly desirable feature for an antiviral vaccine. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. The SARS-unique domain (SUD of SARS coronavirus contains two macrodomains that bind G-quadruplexes.

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    Jinzhi Tan

    2009-05-01

    Full Text Available Since the outbreak of severe acute respiratory syndrome (SARS in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV, the non-structural proteins (Nsps, have been determined. However, within the large Nsp3 (1922 amino-acid residues, the structure and function of the so-called SARS-unique domain (SUD have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389-652 ("SUD(core" of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 A resolution, respectively revealed that SUD(core forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUD(core as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5-6 nucleotides, but more extended G-stretches are found in the 3'-nontranslated regions of mRNAs coding for certain host-cell proteins

  1. SARS-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the MAVS/TRAF3/TRAF6 signalosome.

    Science.gov (United States)

    Shi, Chong-Shan; Qi, Hai-Yan; Boularan, Cedric; Huang, Ning-Na; Abu-Asab, Mones; Shelhamer, James H; Kehrl, John H

    2014-09-15

    Coronaviruses (CoV) have recently emerged as potentially serious pathogens that can cause significant human morbidity and death. The severe acute respiratory syndrome (SARS)-CoV was identified as the etiologic agent of the 2002-2003 international SARS outbreak. Yet, how SARS evades innate immune responses to cause human disease remains poorly understood. In this study, we show that a protein encoded by SARS-CoV designated as open reading frame-9b (ORF-9b) localizes to mitochondria and causes mitochondrial elongation by triggering ubiquitination and proteasomal degradation of dynamin-like protein 1, a host protein involved in mitochondrial fission. Also, acting on mitochondria, ORF-9b targets the mitochondrial-associated adaptor molecule MAVS signalosome by usurping PCBP2 and the HECT domain E3 ligase AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF 6. This severely limits host cell IFN responses. Reducing either PCBP2 or AIP4 expression substantially reversed the ORF-9b-mediated reduction of MAVS and the suppression of antiviral transcriptional responses. Finally, transient ORF-9b expression led to a strong induction of autophagy in cells. The induction of autophagy depended upon ATG5, a critical autophagy regulator, but the inhibition of MAVS signaling did not. These results indicate that SARS-CoV ORF-9b manipulates host cell mitochondria and mitochondrial function to help evade host innate immunity. This study has uncovered an important clue to the pathogenesis of SARS-CoV infection and illustrates the havoc that a small ORF can cause in cells.

  2. Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus

    Science.gov (United States)

    2011-01-06

    West Nile viruses . In contrast, they do not inhibit replication of murine leukemia virus (MLV), or the entry processes of amphotropic MLV, Machupo virus ...MACV), Lassa virus (LASV), or lympho- cytic choriomeningitis virus (LCMV). Although IFITM proteins are induced by type I and II interferons, most...processes of several highly pathogenic viruses – Marburg virus , Ebola virus , and SARS coronavirus – are similarly disrupted by IFITM proteins. We

  3. The effect of inhibition of PP1 and TNFα signaling on pathogenesis of SARS coronavirus

    Energy Technology Data Exchange (ETDEWEB)

    McDermott, Jason E.; Mitchell, Hugh D.; Gralinski, Lisa E.; Eisfeld, Amie J.; Josset, Laurence; Bankhead, Armand; Neumann, Gabriele; Tilton, Susan C.; Schäfer, Alexandra; Li, Chengjun; Fan, Shufang; McWeeney, Shannon; Baric, Ralph S.; Katze, Michael G.; Waters, Katrina M.

    2016-09-23

    The complex interplay between viral replication and host immune response during infection remains poorly understood. While many viruses are known to employ antiimmune strategies to facilitate their replication, highly pathogenic virus infections can also cause an excessive immune response that exacerbates, rather than reduces pathogenicity. To investigate this dichotomy in severe acute respiratory syndrome coronavirus (SARS-CoV), we developed a transcriptional network model of SARS-CoV infection in mice and used the model to prioritize candidate regulatory targets for further investigation. We validated our predictions in 18 different knockout (KO) mouse strains, showing that network topology provides significant predictive power to identify genes that are important for viral infection. We identified a novel player in the immune response to virus infection, Kepi, an inhibitory subunit of the protein phosphatase 1 (PP1) complex, which protects against SARS-CoV pathogenesis. We also found that receptors for the proinflammatory cytokine, tumor necrosis factor alpha (TNFα), promote pathogenesis through a parallel feed-forward circuit that promotes inflammation. These results are consistent with previous studies showing the role of over-stimulation of the inflammatory response to SARS-CoV in pathogenesis. We conclude that the critical balance between immune response and inflammation can be manipulated to improve the outcome of the infection. Further, our study provides two potential therapeutic strategies for mitigating the effects of SARS-CoV infection, and may provide insight into treatment strategies for Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

  4. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide

    Directory of Open Access Journals (Sweden)

    Roh C

    2012-05-01

    Full Text Available Changhyun RohDivision of Biotechnology, Advanced Radiation Technology Institute (ARTI, Korea Atomic Energy Research Institute (KAERI, Jeongeup, Republic of KoreaAbstract: Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS, and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV nucleocapsid (N protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (--catechin gallate and (--gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (--catechin gallate and (--gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 µg mL–1, (--catechin gallate and (--gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.Keywords: SARS, RNA oligonucleotide, quantum dots, inhibitor, screening

  5. Excretion and detection of SARS coronavirus and its nucleic acid from digestive system

    Institute of Scientific and Technical Information of China (English)

    Xin-Wei Wang; Xiao-Ming Wu; Wen-Jun Xiao; Xiu-Mei Zhu; Chang-Qing Gu; Jing Yin; Wei Wei; Wei Yao; Chao Liu; Jian-Feng Li; Guo-Rong Ou; Jin-Song Li; Min-Nian Wang; Tong-Yu Fang; Gui-Jie Wang; Yao-Hui Qiu; Huai-Huan Wu; Fu-Huan Chao; Jun-Wen Li; Ting-Kai Guo; Bei Zhen; Qing-Xin Kong; Bin Yi; Zhong Li; Nong Song; Min Jin

    2005-01-01

    AIM: To study whether severe acute respiratory syndrome coronavirus (SARS-CoV) could be excreted from digestive system.METHODS: Cell culture and semi-nested RT-PCR were used to detect SARS-CoV and its RNA from 21 stool and urine samples, and a kind of electropositive filter media particles was used to concentrate the virus in 10 sewage samples from two hospitals receiving SAPS patients in Beijing in China.RESULTS: It was demonstrated that there was no live SARS-CoV in all samples collected, but the RNA of SARS-CoV could be detected in seven stool samples from SARS patients with any one of the symptoms of fever, malaise,cough, or dyspnea, in 10 sewage samples before disinfection and 3 samples after disinfection from the two hospitals.The RNA could not be detected in urine and stool samples from patients recovered from SARS.CONCLUSION: Nucleic acid of SARS-CoV can be excreted through the stool of patients into sewage system, and the possibility of SARS-CoV transmitting through digestive system cannot be excluded.

  6. Phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected SARS coronavirus.

    Science.gov (United States)

    Yasui, Fumihiko; Kohara, Michinori; Kitabatake, Masahiro; Nishiwaki, Tetsu; Fujii, Hideki; Tateno, Chise; Yoneda, Misako; Morita, Kouichi; Matsushima, Kouji; Koyasu, Shigeo; Kai, Chieko

    2014-04-01

    While the 2002-2003 outbreak of severe acute respiratory syndrome (SARS) resulted in 774 deaths, patients who were affected with mild pulmonary symptoms successfully recovered. The objective of the present work was to identify, using SARS coronavirus (SARS-CoV) mouse infection models, immune factors responsible for clearing of the virus. The elimination of pulmonary SARS-CoV infection required the activation of B cells by CD4(+) T cells. Furthermore, passive immunization (post-infection) with homologous (murine) anti-SARS-CoV antiserum showed greater elimination efficacy against SARS-CoV than that with heterologous (rabbit) antiserum, despite the use of equivalent titers of neutralizing antibodies. This distinction was mediated by mouse phagocytic cells (monocyte-derived infiltrating macrophages and partially alveolar macrophages, but not neutrophils), as demonstrated both by adoptive transfer from donors and by immunological depletion of selected cell types. These results indicate that the cooperation of anti-SARS-CoV antibodies and phagocytic cells plays an important role in the elimination of SARS-CoV. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Automated extraction protocol for quantification of SARS-Coronavirus RNA in serum: an evaluation study

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    Lui Wing-bong

    2006-02-01

    Full Text Available Abstract Background We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR. In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay. Methods An automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen for quantitative performance, analytical sensitivity and precision. Results The newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method. Conclusion As the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

  8. SARS coronavirus papain-like protease up-regulates the collagen expression through non-Samd TGF-β1 signaling.

    Science.gov (United States)

    Wang, Ching-Ying; Lu, Chien-Yi; Li, Shih-Wen; Lai, Chien-Chen; Hua, Chun-Hung; Huang, Su-Hua; Lin, Ying-Ju; Hour, Mann-Jen; Lin, Cheng-Wen

    2017-05-02

    SARS coronavirus (CoV) papain-like protease (PLpro) reportedly induced the production of TGF-β1 through p38 MAPK/STAT3-meidated Egr-1-dependent activation (Sci. Rep. 6, 25754). This study investigated the correlation of PLpro-induced TGF-β1 with the expression of Type I collagen in human lung epithelial cells and mouse pulmonary tissues. Specific inhibitors for TGF-βRI, p38 MAPK, MEK, and STAT3 proved that SARS-CoV PLpro induced TGF-β1-dependent up-regulation of Type I collagen in vitro and in vivo. Subcellular localization analysis of SMAD3 and SMAD7 indicated that non-SMAD pathways in TGF-β1 signaling involved in the production of Type I collagen in transfected cells with pSARS-PLpro. Comprehensive analysis of ubiquitin-conjugated proteins using immunoprecipitation and nanoLC-MS/MS indicated that SARS-CoV PLpro caused the change in the ubiquitination profile of Rho GTPase family proteins, in which linked with the increase of Rho-like GTPase family proteins. Moreover, selective inhibitors TGF-βRI and STAT6 (AS1517499) ascertained that STAT6 activation was required for PLpro-induced TGF-β1-dependent up-regulation of Type I collagen in human lung epithelial cells. The results showed that SARS-CoV PLpro stimulated TGF-β1-dependent expression of Type I collagen via activating STAT6 pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway

    Institute of Scientific and Technical Information of China (English)

    Hongliang Wang; Peng Yang; Kangtai Liu; Feng Guo; Yanli Zhang; Gongyi Zhang; Chengyu Jiang

    2008-01-01

    While severe acute respiratory syndrome coronavirus (SARS-CoV)fwas initially thought to enter cells through direct fusion with the plasma membrane, more recent evidence suggests that virus entry may also involve endocytosis. We have found that SARS-CoV enters cells via pH- and receptor-dependent endocytosis. Treatment of cells with either SARS-CoV spike protein or spike-bearing pseudoviruses resulted in the translocation of angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV, from the cell surface to endosomes. In addition, the spike-bearing pseudoviruses and early endosome antigen 1 were found to colocalize in endosomes. Further analyses using specific endocytic pathway inhibitors and dominant-negative Eps15 as well as caveolin-1 colocalization study suggested that virus entry was mediated by a clathrin- and caveolae-independent mechanism. Moreover, cholesterol- and sphingolipid-rich lipid raft microdomains in the plasma membrane, which have been shown to act as platforms for many physiological signaling pathways, were shown to be involved in virus entry. Endocytic entry of SARS-CoV may expand the cellular range of SARS-CoV infection, and our findings here contribute to the understanding of SARS-CoV pathogenesis, providing new information for anti-viral drug research.

  10. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide.

    Science.gov (United States)

    Roh, Changhyun

    2012-01-01

    Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS), and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV) nucleocapsid (N) protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (-)-catechin gallate and (-)-gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (-)-catechin gallate and (-)-gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 μg mL(-1), (-)-catechin gallate and (-)-gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.

  11. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Kèvin Knoops

    2008-09-01

    Full Text Available 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.

  12. Substitution at Aspartic Acid 1128 in the SARS Coronavirus Spike Glycoprotein Mediates Escape from a S2 Domain-Targeting Neutralizing Monoclonal Antibody

    Science.gov (United States)

    Ng, Oi-Wing; Keng, Choong-Tat; Leung, Cynthia Sau-Wai; Peiris, J. S. Malik; Poon, Leo Lit Man; Tan, Yee-Joo

    2014-01-01

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941–50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111–1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus. PMID:25019613

  13. Substitution at aspartic acid 1128 in the SARS coronavirus spike glycoprotein mediates escape from a S2 domain-targeting neutralizing monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Oi-Wing Ng

    Full Text Available The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs, are believed to be the natural reservoir. The SARS-CoV surface Spike (S protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2: 941-50. In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111-1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.

  14. Substitution at aspartic acid 1128 in the SARS coronavirus spike glycoprotein mediates escape from a S2 domain-targeting neutralizing monoclonal antibody.

    Science.gov (United States)

    Ng, Oi-Wing; Keng, Choong-Tat; Leung, Cynthia Sau-Wai; Peiris, J S Malik; Poon, Leo Lit Man; Tan, Yee-Joo

    2014-01-01

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941-50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111-1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.

  15. Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs

    Institute of Scientific and Technical Information of China (English)

    Yi SHI; De Hua YANG; Jie XIONG; Jie JIA; Bing HUANG; You Xin JIN

    2005-01-01

    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. dsRNAmediated 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 0~60 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.

  16. Reconstruction of the most recent common ancestor sequences of SARS-Cov S gene and detection of adaptive evolution in the spike protein

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yuan; ZHENG Nan; HAO Pei; ZHONG Yang

    2004-01-01

    @@ The genome organization and expression strategy of severe acute respiratory syndrome coronavirus (SARSCoV) have been described extensivelyl1- 10]. As a structural glycoprotein on the virion surface, the spike protein is responsible for binding to host cellular receptors and for the fusion between the viral envelope and the cellular membrane. It also induces neutralizing antibodies in the host and mediates cellular immunity[11]. Previous studies suggested that amino acid replacements in the spike protein could dramatically alter the pathogenesis and virulence of some coronaviruses[11]. It is therefore reasonable to test the hypothesis that radical amino acid replacements in the spike protein, favored by environmental selective pressure during the process of SARS-CoV interspecific transmission[10], might make this pathogen adapt to a new host. In this study, we investigated a total of 108complete sequences of the SARS-CoV S gene from GenBank (until March 23, 2004). After omission of those records containing frame-shift mutations or low quality sequences, e.g. ZJ01, and selection of one sequence for identical records, an alignment of 42 sequences was obtained using the program Clustal-X[13]. Then, we reconstructed the most recent common ancestor (MRCA) sequences of the SARS-CoV S gene and detected the adaptive evolution in the spike protein.

  17. Establishment of a fluorescent polymerase chain reaction method for the detection of the SARS-associated coronavirus and its clinical application

    Institute of Scientific and Technical Information of China (English)

    吴新伟; 程钢; 狄飚; 尹爱华; 何蕴韶; 王鸣; 周新宇; 何丽娟; 罗凯; 杜琳

    2003-01-01

    Objective To establish a fluorescent polymerase chain reaction (F-PCR) method for detecting the coronavirus related to severe acute respiratory syndrome (SARS) and to evaluate its value for clinical application. Methods The primers and the fluorescence-labeled probe were designed and synthesized according to the published sequence of the SARS-associated coronavirus genes. A F-PCR diagnosis kit for detecting the coronavirus was developed, and 115 clinical nasopharyngeal gargling liquid samples were tested. Results The sequence of PCR amplified products completely matched the related sequence of the SARS-associated coronavirus genome. Forty-nine out of 67 samples from identified SARS patients and 8 of 18 samples from persons having close contact with SARS patients showed positive results. All 30 samples from healthy controls were negative. Conclusion The F-PCR method established may be a rapid, accurate and efficient way for screening and for the early diagnosis of SARS patients.

  18. A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice.

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    Anjeanette Roberts

    2007-01-01

    Full Text Available No single animal model for severe acute respiratory syndrome (SARS reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15 that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15, duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as

  19. Protein Subcellular Localization Prediction and Genomic Polymorphism Analysis of the SARS Coronavirus

    Institute of Scientific and Technical Information of China (English)

    季星来; 柳树群; 李岭; 孙之荣

    2004-01-01

    The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus (CoV).Several sequences of the complete genome of SARS-CoV have been determined.The subcellular localization (SubLocation) of annotated open-reading frames of the SARS-CoV genome was predicted using a support vector machine.Several gene products were predicted to locate in the Golgi body and cell nucleus.The SubLocation information was combined with predicted transmembrane information to develop a model of the viral life cycle.The results show that this information can be used to predict the functions of genes and even the virus pathogenesis.In addition,the entire SARS viral genome sequences currently available in GenBank were compared to identify the sequence variations among different isolates.Some variations in the Hong Kong strains may be related to the special clinical manifestations and provide clues for understanding the relationship between gene functions and evolution.These variations reflect the evolution of the SARS virus in human populations and may help development of a vaccine.

  20. Immune Responses and Histopathological Changes in Rabbits Immunized with Inactivated SARS Coronavirus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To evaluate the immunogenicity of inactivated SARS coronavirus (SARS-CoV), three groups of rabbits were immunized three times at 2-week intervals with inactivated vaccine + adjuvant, adjuvant,and normal saline respectively. Eight batchs of serum were sampled from the auricular vein at day 7 to day 51, and specific IgG antibody titers and neutralizing antibody titers were detected by indirect ELISA and micro-cytopathic effect neutralizing test. Antibody specificity was identified by proteinchip assay.Histopathological changes were detected by H&E staining. The results showed that, rabbits in the experimental group immunized with inactivated SARS-CoV all generated specific IgG antibodies with neutralizing activity, which suggested the inactivated SARS-CoV could preserve its antigenicity well and elicit an effective humoral immune responses. The peak titer value of specific IgG antibody and neutralizing antibody reached 1:40960 and 1:2560 respectively. In the experimental group, no obvious histopathological changes was detected in the H&E stained slides of heart, spleen, kidney and testis samples, but the livers had slight histopathological changes, and the lungs presented remarkable histopathological changes. These findings are of importance for SARS-CoV inactivated vaccine development.

  1. Stability of SARS Coronavirus in Human Specimens and Environment and Its Sensitivity to Heating and UV Irradiation

    Institute of Scientific and Technical Information of China (English)

    SHU-MING DUAN; XIAO-PING DONG; SARS RESEARCH TEAM; XIN-SHENG ZHAO; RUI-FU WEN; JING-JING HUANG; GUO-HUA PI; SU-XIANG ZHANG; JUN HAN; SHENG-LI BI; LI RUAN

    2003-01-01

    The causal agent for SARS is considered as a novel coronavirus that has never been described both in human and animals previously. The stability of SARS coronavirus in human specimens and in environments was studied. Methods Using a SARS coronavirus strain CoV-P9,which was isolated from pharyngeal swab of a probable SARS case in Beijing, its stability in mimic human specimens and in mimic environment including surfaces of commonly used materials or in household conditions, as well as its resistances to temperature and UV irradiation were analyzed. A total of 106 TCID50 viruses were placed in each tested condition, and changes of the viral infectivity in samples after treatments were measured by evaluating cytopathic effect (CPE) in cell line Vero-E6 at 48 h after infectionn. Results The results showed that SARS coronavirus in the testing condition could survive in serum, 1:20 diluted sputum and feces for at least 96 h, whereas it could remain alive in urine for at least 72 h with a low level of infectivity. The survival abilities on the surfaces of eight different materials and in water were quite comparable, revealing reduction of infectivity after 72 to 96 h exposure. Viruses stayed stable at 4℃, at room temperature (20℃) and at 37℃ for at least 2 h without remarkable change in the infectious ability in cells, but were convened to be non-infectious after 90-, 60- and 30-min exposure at 56℃, at 67℃ and at 75℃, respectively. Irradiation of UV for 60 min on the virus in culture medium resulted in the destruction of viral infectivity at an undetectable level. Conclusion The survival ability of SARS coronavirus in human specimens and in environments seems to be relatively strong. Heating and UV irradiation can efficiently eliminate the viral infectivity.

  2. Identification of Immunogenic Determinants of the Spike Protein of SARS-like Coronavirus

    Institute of Scientific and Technical Information of China (English)

    Peng Zhou; Zhenggang Han; Lin-Fa Wang; Zhengli Shi

    2013-01-01

    Bat SARS-like coronavirus (SL-CoV) has a genome organization almost identical to that of SARS-CoV,but the N-terminus of the Spike (S) proteins,which interacts with host receptor and is a major target of neutralizing antibodies against CoVs,of the two viruses has only 63-64% sequence identity.Although there have been reports studying the overall immunogenicity of SSL,knowledge on the precise location of immunodominant determinants for SSL is still lacking.In this study,using a series of truncated expressed SSL fragments and SsL specific mouse sera,we identified two immunogenic determinants for SSL.Importantly,one of the two regions seems to be located in a region not shared by known immunogenic determinants of the SSARS.This finding will be of potential use in future monitoring of SL-CoV infection in bats and spillover animals and in development of more effective vaccine to cover broad protection against this new group of coronaviruses.

  3. Structure and inhibition of the SARS coronavirus envelope protein ion channel.

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    Konstantin Pervushin

    2009-07-01

    Full Text Available The envelope (E protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA, but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV that the transmembrane domain of E protein (ETM forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293 cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA, but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

  4. VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability.

    Science.gov (United States)

    Yu, Xiao; Chen, Shuliang; Hou, Panpan; Wang, Min; Chen, Yu; Guo, Deyin

    2015-04-03

    Eukaryotic cellular and most viral RNAs carry a 5'-terminal cap structure, a 5'-5' triphosphate linkage between the 5' end of the RNA and a guanosine nucleotide (cap-0). SARS coronavirus (SARS-CoV) nonstructural protein nsp16 functions as a methyltransferase, to methylate mRNA cap-0 structure at the ribose 2'-O position of the first nucleotide to form cap-1 structures. However, whether there is interplay between nsp16 and host proteins was not yet clear. In this report, we identified several potential cellular nsp16-interacting proteins from a human thymus cDNA library by yeast two-hybrid screening. VHL, one of these proteins, was proven to interact with nsp16 both in vitro and in vivo. Further studies showed that VHL can inhibit SARS-CoV replication by regulating nsp16 ubiquitination and promoting its degradation. Our results have revealed the role of cellular VHL in the regulation of SARS-CoV replication. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.

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    I-Chueh Huang

    Full Text Available Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3 are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV hemagglutinin (HA protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP(1,2 of Marburg and Ebola filoviruses (MARV, EBOV. Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV and entry mediated by the SARS-CoV spike (S protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression.

  6. The effect of inhibition of PP1 and TNFα signaling on pathogenesis of SARS coronavirus.

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    McDermott, Jason E; Mitchell, Hugh D; Gralinski, Lisa E; Eisfeld, Amie J; Josset, Laurence; Bankhead, Armand; Neumann, Gabriele; Tilton, Susan C; Schäfer, Alexandra; Li, Chengjun; Fan, Shufang; McWeeney, Shannon; Baric, Ralph S; Katze, Michael G; Waters, Katrina M

    2016-09-23

    The complex interplay between viral replication and host immune response during infection remains poorly understood. While many viruses are known to employ anti-immune strategies to facilitate their replication, highly pathogenic virus infections can also cause an excessive immune response that exacerbates, rather than reduces pathogenicity. To investigate this dichotomy in severe acute respiratory syndrome coronavirus (SARS-CoV), we developed a transcriptional network model of SARS-CoV infection in mice and used the model to prioritize candidate regulatory targets for further investigation. We validated our predictions in 18 different knockout (KO) mouse strains, showing that network topology provides significant predictive power to identify genes that are important for viral infection. We identified a novel player in the immune response to virus infection, Kepi, an inhibitory subunit of the protein phosphatase 1 (PP1) complex, which protects against SARS-CoV pathogenesis. We also found that receptors for the proinflammatory cytokine tumor necrosis factor alpha (TNFα) promote pathogenesis, presumably through excessive inflammation. The current study provides validation of network modeling approaches for identifying important players in virus infection pathogenesis, and a step forward in understanding the host response to an important infectious disease. The results presented here suggest the role of Kepi in the host response to SARS-CoV, as well as inflammatory activity driving pathogenesis through TNFα signaling in SARS-CoV infections. Though we have reported the utility of this approach in bacterial and cell culture studies previously, this is the first comprehensive study to confirm that network topology can be used to predict phenotypes in mice with experimental validation.

  7. High-yield expression of recombinant SARS coronavirus nucleocapsid protein in methylotrophic yeast Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Ru-Shi Liu; Kun-Yu Yang; Jian Lin; Yi-Wei Lin; Zhi-Hong Zhang; Jun Zhang; Ning-Shao Xia

    2004-01-01

    AIM: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), theantigenicity of the protein is better than spike (S) protein.This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS.METHODS: Full length cDNA of SCoV nucleocapsid (SCoVN)protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into Pichia pastoris (P. pastoris) GS115 (HisMut+) by electroporation. His+Mut+recombinant strains were identified by PCR and cultivated on MM/MD plates. The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied. The expression level and activation were detected by SDS-PAGE and Western-blot respectively.RESULTS: All of the recombinants were His+Mut+ aftertransformation of P. pastoriswith linearized plasmids. The BMMY medium was optimal for recombinant ScoVN (rSCoVN)protein expression and growth of the recombinant strains.The final optimal concentration of methanol was 20 mL/L,the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved,and a maximum cell ,A at 600 nm of 62 was achieved in shake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum.The biological activity of rSCoVN expressed in P. pastoris was about 4-fold higher than that expressed in E.coliwhen the same rSCoVN protein

  8. Identification of phosphorylation sites in the nucleocapsid protein (N protein) of SARS-coronavirus

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    Lin, Liang; Shao, Jianmin; Sun, Maomao; Liu, Jinxiu; Xu, Gongjin; Zhang, Xumin; Xu, Ningzhi; Wang, Rong; Liu, Siqi

    2007-12-01

    After decoding the genome of SARS-coronavirus (SARS-CoV), next challenge is to understand how this virus causes the illness at molecular bases. Of the viral structural proteins, the N protein plays a pivot role in assembly process of viral particles as well as viral replication and transcription. The SARS-CoV N proteins expressed in the eukaryotes, such as yeast and HEK293 cells, appeared in the multiple spots on two-dimensional electrophoresis (2DE), whereas the proteins expressed in E. coli showed a single 2DE spotE These 2DE spots were further examined by Western blot and MALDI-TOF/TOF MS, and identified as the N proteins with differently apparent pI values and similar molecular mass of 50 kDa. In the light of the observations and other evidences, a hypothesis was postulated that the SARS-CoV N protein could be phosphorylated in eukaryotes. To locate the plausible regions of phosphorylation in the N protein, two truncated N proteins were generated in E. coli and treated with PKC[alpha]. The two truncated N proteins after incubation of PKC[alpha] exhibited the differently electrophoretic behaviors on 2DE, suggesting that the region of 1-256 aa in the N protein was the possible target for PKC[alpha] phosphorylation. Moreover, the SARS-CoV N protein expressed in yeast were partially digested with trypsin and carefully analyzed by MALDI-TOF/TOF MS. In contrast to the completely tryptic digestion, these partially digested fragments generated two new peptide mass signals with neutral loss, and MS/MS analysis revealed two phosphorylated peptides located at the "dense serine" island in the N protein with amino acid sequences, GFYAEGSRGGSQASSRSSSR and GNSGNSTPGSSRGNSPARMASGGGK. With the PKC[alpha] phosphorylation treatment and the partially tryptic digestion, the N protein expressed in E. coli released the same peptides as observed in yeast cells. Thus, this investigation provided the preliminary data to determine the phosphorylation sites in the SARS-CoV N protein, and

  9. The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase.

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    Wei, Ping; Fan, Keqiang; Chen, Hao; Ma, Liang; Huang, Changkang; Tan, Lei; Xi, Dong; Li, Chunmei; Liu, Ying; Cao, Aoneng; Lai, Luhua

    2006-01-20

    The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. Accurate determination of the dimer dissociation constant and the role of the N-finger (residues 1-7) will provide more insights into the enzyme catalytic mechanism of SARS 3CL proteinase. The dimer dissociation constant of the wild-type protein was determined to be 14.0microM by analytical ultracentrifugation method. The N-finger fragment of the enzyme plays an important role in enzyme dimerization as shown in the crystal structure. Key residues in the N-finger have been studied by site-directed mutagenesis, enzyme assay, and analytical ultracentrifugation. A single mutation of M6A was found to be critical to maintain the dimer structure of the enzyme. The N-terminal octapeptide N8 and its mutants were also synthesized and tested for their potency as dimerization inhibitors. Peptide cleavage assay confirms that peptide N8 is a dimerization inhibitor with a K(i) of 2.20mM. The comparison of the inhibitory activities of N8 and its mutants indicates that the hydrophobic interaction of Met-6 and the electrostatic interaction of Arg-4 contribute most for inhibitor binding. This study describes the first example of inhibitors targeting the dimeric interface of SARS 3CL proteinase, providing a novel strategy for drug design against SARS and other coronaviruses.

  10. Ezrin interacts with the SARS coronavirus Spike protein and restrains infection at the entry stage.

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    Jean Kaoru Millet

    Full Text Available BACKGROUND: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S. There are still many unknowns on the implication of cellular factors that regulate the entry process. METHODOLOGY/PRINCIPAL FINDINGS: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion. CONCLUSIONS/SIGNIFICANCE: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.

  11. Ezrin Interacts with the SARS Coronavirus Spike Protein and Restrains Infection at the Entry Stage

    Science.gov (United States)

    Millet, Jean Kaoru; Kien, François; Cheung, Chung-Yan; Siu, Yu-Lam; Chan, Wing-Lim; Li, Huiying; Leung, Hiu-Lan; Jaume, Martial; Bruzzone, Roberto; Malik Peiris, Joseph S.; Altmeyer, Ralf Marius; Nal, Béatrice

    2012-01-01

    Background Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. Methodology/Principal Findings We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion. Conclusions/Significance Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection. PMID:23185364

  12. Molecular signature of clinical severity in recovering patients with severe acute respiratory syndrome coronavirus (SARS-CoV

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    Wu Ting-Shu

    2005-09-01

    Full Text Available Abstract Background Severe acute respiratory syndrome (SARS, a recent epidemic human disease, is caused by a novel coronavirus (SARS-CoV. First reported in Asia, SARS quickly spread worldwide through international travelling. As of July 2003, the World Health Organization reported a total of 8,437 people afflicted with SARS with a 9.6% mortality rate. Although immunopathological damages may account for the severity of respiratory distress, little is known about how the genome-wide gene expression of the host changes under the attack of SARS-CoV. Results Based on changes in gene expression of peripheral blood, we identified 52 signature genes that accurately discriminated acute SARS patients from non-SARS controls. While a general suppression of gene expression predominated in SARS-infected blood, several genes including those involved in innate immunity, such as defensins and eosinophil-derived neurotoxin, were upregulated. Instead of employing clustering methods, we ranked the severity of recovering SARS patients by generalized associate plots (GAP according to the expression profiles of 52 signature genes. Through this method, we discovered a smooth transition pattern of severity from normal controls to acute SARS patients. The rank of SARS severity was significantly correlated with the recovery period (in days and with the clinical pulmonary infection score. Conclusion The use of the GAP approach has proved useful in analyzing the complexity and continuity of biological systems. The severity rank derived from the global expression profile of significantly regulated genes in patients may be useful for further elucidating the pathophysiology of their disease.

  13. Immunodominant SARS Coronavirus Epitopes in Humans Elicited both Enhancing and Neutralizing Effects on Infection in Non-human Primates.

    Science.gov (United States)

    Wang, Qidi; Zhang, Lianfeng; Kuwahara, Kazuhiko; Li, Li; Liu, Zijie; Li, Taisheng; Zhu, Hua; Liu, Jiangning; Xu, Yanfeng; Xie, Jing; Morioka, Hiroshi; Sakaguchi, Nobuo; Qin, Chuan; Liu, Gang

    2016-05-13

    Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) and has the potential to threaten global public health and socioeconomic stability. Evidence of antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro and in non-human primates clouds the prospects for a safe vaccine. Using antibodies from SARS patients, we identified and characterized SARS-CoV B-cell peptide epitopes with disparate functions. In rhesus macaques, the spike glycoprotein peptides S471-503, S604-625, and S1164-1191 elicited antibodies that efficiently prevented infection in non-human primates. In contrast, peptide S597-603 induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (ESD) mechanism. This peptide exhibited a high level of serological reactivity (64%), which resulted from the additive responses of two tandem epitopes (S597-603 and S604-625) and a long-term human B-cell memory response with antisera from convalescent SARS patients. Thus, peptide-based vaccines against SARS-CoV could be engineered to avoid ADE via elimination of the S597-603 epitope. We provide herein an alternative strategy to prepare a safe and effective vaccine for ADE of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection.

  14. Development of a single nucleotide polymorphism DNA microarray for the detection and genotyping of the SARS coronavirus.

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    Guo, Xi; Geng, Peng; Wang, Quan; Cao, Boyang; Liu, Bin

    2014-10-01

    Severe acute respiratory syndrome (SARS), a disease that spread widely in the world during late 2002 to 2004, severely threatened public health. Although there have been no reported infections since 2004, the extremely pathogenic SARS coronavirus (SARS-CoV), as the causative agent of SARS, has recently been identified in animals, showing the potential for the re-emergence of this disease. Previous studies showed that 27 single nucleotide polymorphism (SNP) mutations among the spike (S) gene of this virus are correlated closely with the SARS pathogenicity and epidemicity. We have developed a SNP DNA microarray in order to detect and genotype these SNPs, and to obtain related information on the pathogenicity and epidemicity of a given strain. The microarray was hybridized with PCR products amplified from cDNAs obtained from different SARS-CoV strains. We were able to detect 24 SNPs and determine the type of a given strain. The hybridization profile showed that 19 samples were detected and genotyped correctly by using our microarray, with 100% accuracy. Our microarray provides a novel method for the detection and epidemiological surveillance of SARS-CoV.

  15. Mechanism for controlling the monomer-dimer conversion of SARS coronavirus main protease.

    Science.gov (United States)

    Wu, Cheng Guo; Cheng, Shu Chun; Chen, Shiang Chuan; Li, Juo Yan; Fang, Yi Hsuan; Chen, Yau Hung; Chou, Chi Yuan

    2013-05-01

    The Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)) cleaves two virion polyproteins (pp1a and pp1ab); this essential process represents an attractive target for the development of anti-SARS drugs. The functional unit of M(pro) is a homodimer and each subunit contains a His41/Cys145 catalytic dyad. Large amounts of biochemical and structural information are available on M(pro); nevertheless, the mechanism by which monomeric M(pro) is converted into a dimer during maturation still remains poorly understood. Previous studies have suggested that a C-terminal residue, Arg298, interacts with Ser123 of the other monomer in the dimer, and mutation of Arg298 results in a monomeric structure with a collapsed substrate-binding pocket. Interestingly, the R298A mutant of M(pro) shows a reversible substrate-induced dimerization that is essential for catalysis. Here, the conformational change that occurs during substrate-induced dimerization is delineated by X-ray crystallography. A dimer with a mutual orientation of the monomers that differs from that of the wild-type protease is present in the asymmetric unit. The presence of a complete substrate-binding pocket and oxyanion hole in both protomers suggests that they are both catalytically active, while the two domain IIIs show minor reorganization. This structural information offers valuable insights into the molecular mechanism associated with substrate-induced dimerization and has important implications with respect to the maturation of the enzyme.

  16. The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain.

    Science.gov (United States)

    Minakshi, Rinki; Padhan, Kartika; Rehman, Safikur; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2014-10-13

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a positive stranded RNA virus with ∼30kb genome. Among all open reading frames (orfs) of this virus, the orf3a is the largest, and encodes a protein of 274 amino acids, named as 3a protein. Sequence analysis suggests that the orf3a aligned to one calcium pump present in Plasmodium falciparum and the enzyme glutamine synthetase found in Leptospira interrogans. This sequence similarity was found to be limited only to amino acid residues 209-264 which form the cytoplasmic domain of the orf3a. Furthermore, this region was predicted to be involved in the calcium binding. Owing to this hypothesis, we were driven to establish its calcium binding property in vitro. Here, we expressed and purified the cytoplasmic domain of the 3a protein, called Cyto3a, as a recombinant His-tagged protein in the E. coli. The calcium binding nature was established by performing various staining methods such as ruthenium red and stains-all. (45)Ca overlay method was also done to further support the data. Since the 3a protein forms ion channels, we were interested to see any conformational changes occurring in the Cyot3a upon calcium binding, using fluorescence spectroscopy and circular dichroism. These studies clearly indicate a significant change in the conformation of the Cyto3a protein after binding with calcium. Our results strongly suggest that the cytoplasmic domain of the 3a protein of SARS-CoV binds calcium in vitro, causing a change in protein conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome.

    Directory of Open Access Journals (Sweden)

    Albrecht von Brunn

    Full Text Available The severe acute respiratory syndrome coronavirus (SARS-CoV genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.

  18. Bat-to-human: spike features determining 'host jump' of coronaviruses SARS-CoV, MERS-CoV, and beyond.

    Science.gov (United States)

    Lu, Guangwen; Wang, Qihui; Gao, George F

    2015-08-01

    Both severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic pathogens that crossed the species barriers to infect humans. The mechanism of viral interspecies transmission is an important scientific question to be addressed. These coronaviruses contain a surface-located spike (S) protein that initiates infection by mediating receptor-recognition and membrane fusion and is therefore a key factor in host specificity. In addition, the S protein needs to be cleaved by host proteases before executing fusion, making these proteases a second determinant of coronavirus interspecies infection. Here, we summarize the progress made in the past decade in understanding the cross-species transmission of SARS-CoV and MERS-CoV by focusing on the features of the S protein, its receptor-binding characteristics, and the cleavage process involved in priming. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with SARS coronavirus

    Directory of Open Access Journals (Sweden)

    Weber Friedemann

    2006-03-01

    Full Text Available Abstract Background SARS coronavirus (SARS-CoV is the etiologic agent of the severe acute respiratory syndrome. SARS-CoV mainly infects tissues of non-lymphatic origin, and the cytokine profile of those cells can determine the course of disease. Here, we investigated the cytokine response of two human non-lymphatic cell lines, Caco-2 and HEK 293, which are fully permissive for SARS-CoV. Results A comparison with established cytokine-inducing viruses revealed that SARS-CoV only weakly triggered a cytokine response. In particular, SARS-CoV did not activate significant transcription of the interferons IFN-α, IFN-β, IFN-λ1, IFN-λ2/3, as well as of the interferon-induced antiviral genes ISG56 and MxA, the chemokine RANTES and the interleukine IL-6. Interestingly, however, SARS-CoV strongly induced the chemokines IP-10 and IL-8 in the colon carcinoma cell line Caco-2, but not in the embryonic kidney cell line 293. Conclusion Our data indicate that SARS-CoV suppresses the antiviral cytokine system of non-immune cells to a large extent, thus buying time for dissemination in the host. However, synthesis of IP-10 and IL-8, which are established markers for acute-stage SARS, escapes the virus-induced silencing at least in some cell types. Therefore, the progressive infiltration of immune cells into the infected lungs observed in SARS patients could be due to the production of these chemokines by the infected tissue cells.

  20. Understanding Viral Transmission Behavior via Protein Intrinsic Disorder Prediction: Coronaviruses

    Directory of Open Access Journals (Sweden)

    Gerard Kian-Meng Goh

    2012-01-01

    Full Text Available Besides being a common threat to farm animals and poultry, coronavirus (CoV was responsible for the human severe acute respiratory syndrome (SARS epidemic in 2002–4. However, many aspects of CoV behavior, including modes of its transmission, are yet to be fully understood. We show that the amount and the peculiarities of distribution of the protein intrinsic disorder in the viral shell can be used for the efficient analysis of the behavior and transmission modes of CoV. The proposed model allows categorization of the various CoVs by the peculiarities of disorder distribution in their membrane (M and nucleocapsid (N. This categorization enables quick identification of viruses with similar behaviors in transmission, regardless of genetic proximity. Based on this analysis, an empirical model for predicting the viral transmission behavior is developed. This model is able to explain some behavioral aspects of important coronaviruses that previously were not fully understood. The new predictor can be a useful tool for better epidemiological, clinical, and structural understanding of behavior of both newly emerging viruses and viruses that have been known for a long time. A potentially new vaccine strategy could involve searches for viral strains that are characterized by the evolutionary misfit between the peculiarities of the disorder distribution in their shells and their behavior.

  1. A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2.

    Science.gov (United States)

    Wong, Swee Kee; Li, Wenhui; Moore, Michael J; Choe, Hyeryun; Farzan, Michael

    2004-01-30

    The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a critical target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318-510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). Smaller S protein fragments, expressing residues 327-510 or 318-490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished association of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC(50) of less than 10 nm, whereas the IC(50) of the S1 domain was approximately 50 nm. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein.

  2. Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses.

    Directory of Open Access Journals (Sweden)

    Markus Hoffmann

    Full Text Available Bats (Chiroptera host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat or Yangochiroptera (genera Carollia and Tadarida for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV, a porcine coronavirus, or to infection mediated by the Spike (S protein of SARS-coronavirus (SARS-CoV incorporated into pseudotypes based on vesicular stomatitis virus (VSV. The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3 were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed.

  3. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1.

    Science.gov (United States)

    Ma-Lauer, Yue; Carbajo-Lozoya, Javier; Hein, Marco Y; Müller, Marcel A; Deng, Wen; Lei, Jian; Meyer, Benjamin; Kusov, Yuri; von Brunn, Brigitte; Bairad, Dev Raj; Hünten, Sabine; Drosten, Christian; Hermeking, Heiko; Leonhardt, Heinrich; Mann, Matthias; Hilgenfeld, Rolf; von Brunn, Albrecht

    2016-08-30

    Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PL(pro)), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95-144 of RCHY1 and 389-652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PL(pro)s from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD-PL(pro) fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PL(pro) alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

  4. Allelic Variation in the Toll-Like Receptor Adaptor Protein Ticam2 Contributes to SARS-Coronavirus Pathogenesis in Mice.

    Science.gov (United States)

    Gralinski, Lisa E; Menachery, Vineet D; Morgan, Andrew P; Totura, Allison L; Beall, Anne; Kocher, Jacob; Plante, Jessica; Harrison-Shostak, D Corinne; Schäfer, Alexandra; Pardo-Manuel de Villena, Fernando; Ferris, Martin T; Baric, Ralph S

    2017-06-07

    Host genetic variation is known to contribute to differential pathogenesis following infection. Mouse models allow direct assessment of host genetic factors responsible for susceptibility to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). Based on an assessment of early stage lines from the Collaborative Cross mouse multi-parent population, we identified two lines showing highly divergent susceptibilities to SARS-CoV: the resistant CC003/Unc and the susceptible CC053/Unc. We generated 264 F2 mice between these strains, and infected them with SARS-CoV. Weight loss, pulmonary hemorrhage, and viral load were all highly correlated disease phenotypes. We identified a quantitative trait locus of major effect on chromosome 18 (27.1-58.6 Mb) which affected weight loss, viral titer and hemorrhage. Additionally, each of these three phenotypes had distinct quantitative trait loci [Chr 9 (weight loss), Chrs 7 and 12 (virus titer), and Chr 15 (hemorrhage)]. We identified Ticam2, an adaptor protein in the TLR signaling pathways, as a candidate driving differential disease at the Chr 18 locus. Ticam2(-/-) mice were highly susceptible to SARS-CoV infection, exhibiting increased weight loss and more pulmonary hemorrhage than control mice. These results indicate a critical role for Ticam2 in SARS-CoV disease, and highlight the importance of host genetic variation in disease responses. Copyright © 2017 Gralinski et al.

  5. Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2'-O-methyltransferase activity of nsp10/nsp16 complex.

    Science.gov (United States)

    Ke, Min; Chen, Yu; Wu, Andong; Sun, Ying; Su, Ceyang; Wu, Hao; Jin, Xu; Tao, Jiali; Wang, Yi; Ma, Xiao; Pan, Ji-An; Guo, Deyin

    2012-08-01

    Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV). However, effective means for combating coronaviruses are still lacking. The interaction between nonstructural protein (nsp) 10 and nsp16 has been demonstrated and the crystal structure of SARS-CoV nsp16/10 complex has been revealed. As nsp10 acts as an essential trigger to activate the 2'-O-methyltransferase activity of nsp16, short peptides derived from nsp10 may have inhibitory effect on viral 2'-O-methyltransferase activity. In this study, we revealed that the domain of aa 65-107 of nsp10 was sufficient for its interaction with nsp16 and the region of aa 42-120 in nsp10, which is larger than the interaction domain, was needed for stimulating the nsp16 2'-O-methyltransferase activity. We further showed that two short peptides derived from the interaction domain of nsp10 could inhibit the 2'-O-methyltransferase activity of SARS-CoV nsp16/10 complex, thus providing a novel strategy and proof-of-principle study for developing peptide inhibitors against SARS-CoV. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex.

    Science.gov (United States)

    Kusov, Yuri; Tan, Jinzhi; Alvarez, Enrique; Enjuanes, Luis; Hilgenfeld, Rolf

    2015-10-01

    The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. The SARS Coronavirus S Glycoprotein Receptor Binding Domain: Fine Mapping and Functional Characterization

    Directory of Open Access Journals (Sweden)

    Xiao Xiaodong

    2005-08-01

    Full Text Available Abstract The entry of the SARS coronavirus (SCV into cells is initiated by binding of its spike envelope glycoprotein (S to a receptor, ACE2. We and others identified the receptor-binding domain (RBD by using S fragments of various lengths but all including the amino acid residue 318 and two other potential glycosylation sites. To further characterize the role of glycosylation and identify residues important for its function as an interacting partner of ACE2, we have cloned, expressed and characterized various soluble fragments of S containing RBD, and mutated all potential glycosylation sites and 32 other residues. The shortest of these fragments still able to bind the receptor ACE2 did not include residue 318 (which is a potential glycosylation site, but started at residue 319, and has only two potential glycosylation sites (residues 330 and 357. Mutation of each of these sites to either alanine or glutamine, as well as mutation of residue 318 to alanine in longer fragments resulted in the same decrease of molecular weight (by approximately 3 kDa suggesting that all glycosylation sites are functional. Simultaneous mutation of all glycosylation sites resulted in lack of expression suggesting that at least one glycosylation site (any of the three is required for expression. Glycosylation did not affect binding to ACE2. Alanine scanning mutagenesis of the fragment S319–518 resulted in the identification of ten residues (K390, R426, D429, T431, I455, N473, F483, Q492, Y494, R495 that significantly reduced binding to ACE2, and one residue (D393 that appears to increase binding. Mutation of residue T431 reduced binding by about 2-fold, and mutation of the other eight residues – by more than 10-fold. Analysis of these data and the mapping of these mutations on the recently determined crystal structure of a fragment containing the RBD complexed to ACE2 (Li, F, Li, W, Farzan, M, and Harrison, S. C., submitted suggested the existence of two hot

  8. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    Science.gov (United States)

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu, Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Pöhlmann, Stefan

    2011-01-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S-activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. PMID:21435673

  9. SARS Patients-derived Human Recombinant Antibodies to S and M Proteins Efficiently Neutralize SARS-Coronavirus Infectivity

    Institute of Scientific and Technical Information of China (English)

    MI-FANG LIANG; KONG-XING WU; ZHAO-HUI XIONG; QI JIN; DE-XIN LI; RUN-LEI DU; JING-ZHI LIU; CHUAN LI; QUAN-FU ZHANG; LU-LU HAN; JIAN-SHI YU; SHU-MIN DUAN; XIAO-FANG WANG

    2005-01-01

    Objective To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection. Methods By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. Coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined. Results After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike (S) proteins, two Fab antibodies were specific for the virus membrane (M) protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect (CPE) inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions

  10. Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan.

    Science.gov (United States)

    Shieh, Wun-Ju; Hsiao, Cheng-Hsiang; Paddock, Christopher D; Guarner, Jeannette; Goldsmith, Cynthia S; Tatti, Kathleen; Packard, Michelle; Mueller, Laurie; Wu, Mu-Zong; Rollin, Pierre; Su, Ih-Jen; Zaki, Sherif R

    2005-03-01

    This article describes the pathological studies of fatal severe acute respiratory syndrome (SARS) in a 73-year-old man during an outbreak of SARS in Taiwan, 2003. Eight days before onset of symptoms, he visited a municipal hospital that was later identified as the epicenter of a large outbreak of SARS. On admission to National Taiwan University Hospital in Taipei, the patient experienced chest tightness, progressive dyspnea, and low-grade fever. His condition rapidly deteriorated with increasing respiratory difficulty, and he died 7 days after admission. The most prominent histopathologic finding was diffuse alveolar damage of the lung. Immunohistochemical and in situ hybridization assays demonstrated evidence of SARS-associated coronavirus (SARS-CoV) infection in various respiratory epithelial cells, predominantly type II pneumocytes, and in alveolar macrophages in the lung. Electron microscopic examination also revealed coronavirus particles in the pneumocytes, and their identity was confirmed as SARS-CoV by immunogold labeling electron microscopy. This report is the first to describe the cellular localization of SARS-CoV in human lung tissue by using a combination of immunohistochemistry, double-stain immunohistochemistry, in situ hybridization, electron microscopy, and immunogold labeling electron microscopy. These techniques represent valuable laboratory diagnostic modalities and provide insights into the pathogenesis of this emerging infection.

  11. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion.

    Science.gov (United States)

    Madu, Ikenna G; Belouzard, Sandrine; Whittaker, Gary R

    2009-10-25

    The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

  12. Coronavirus 3CL(pro) proteinase cleavage sites: Possible relevance to SARS virus pathology

    DEFF Research Database (Denmark)

    Kiemer, Lars; Lund, Ole; Brunak, Søren

    2004-01-01

    such as the cystic fibrosis transmembrane conductance regulator ( CFTR), transcription factors CREB-RP and OCT-I, and components of the ubiquitin pathway. Conclusions: Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified...

  13. Cross-reactive antibodies in convalescent SARS patients' sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests.

    Science.gov (United States)

    Chan, Kwok-Hung; Chan, Jasper Fuk-Woo; Tse, Herman; Chen, Honglin; Lau, Candy Choi-Yi; Cai, Jian-Piao; Tsang, Alan Ka-Lun; Xiao, Xincai; To, Kelvin Kai-Wang; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Zheng, Bo-Jiang; Wang, Ming; Yuen, Kwok-Yung

    2013-08-01

    A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans. Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests. Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  14. The SARS coronavirus papain like protease can inhibit IRF3 at a post activation step that requires deubiquitination activity.

    Science.gov (United States)

    Matthews, Krystal; Schäfer, Alexandra; Pham, Alissa; Frieman, Matthew

    2014-12-07

    The outcome of a viral infection is regulated by complex interactions of viral and host factors. SARS coronavirus (SARS-CoV) engages and regulates several innate immune response pathways during infection. We have previously shown that the SARS-CoV Papain-like Protease (PLpro) inhibits type I interferon (IFN) by inhibiting IRF3 phosphorylation thereby blocking downstream Interferon induction. This finding prompted us to identify other potential mechanisms of inhibition of PLpro on IFN induction. We have used plasmids expressing PLpro and IRF3 including an IRF3 mutant that is constitutively active, called IRF3(5D). In these experiments we utilize transfections, chromatin immunoprecipitation, Electro-mobility Shift Assays (EMSA) and protein localization to identify where IRF3 and IRF3(5D) are inhibited by PLpro. Here we show that PLpro also inhibits IRF3 activation at a step after phosphorylation and that this inhibition is dependent on the de-ubiquitination (DUB) activity of PLpro. We found that PLpro is able to block the type I IFN induction of a constitutively active IRF3, but does not inhibit IRF3 dimerization, nuclear localization or DNA binding. However, inhibition of PLpro's DUB activity by mutagenesis blocked the IRF3 inhibition activity of PLpro, suggesting a role for IRF3 ubiquitination in induction of a type I IFN innate immune response. These results demonstrate an additional mechanism that PLpro is able to inhibit IRF3 signaling. These data suggest novel innate immune antagonism activities of PLpro that may contribute to SARS-CoV pathogenesis.

  15. MERS: Emergence of a novel human coronavirus

    NARCIS (Netherlands)

    V.S. Raj (Stalin); A.D.M.E. Osterhaus (Albert); R.A.M. Fouchier (Ron); B.L. Haagmans (Bart)

    2014-01-01

    textabstractA novel coronavirus (CoV) that causes a severe lower respiratory tract infection in humans, emerged in the Middle East region in 2012. This virus, named Middle East respiratory syndrome (MERS)-CoV, is phylogenetically related to bat CoVs, but other animal species like dromedary camels ma

  16. Canine coronaviruses: Epidemiology, evolution and pathobiology

    NARCIS (Netherlands)

    Decaro, N.

    2009-01-01

    Coronaviruses (CoVs; order Nidovirales, family Coronaviridae) are viruses exceptionally prone to genetic evolution through the continual accumulation of mutations and by homologous recombination between related members. CoVs are organised into three antigenic groups of which group 1 is subdivided in

  17. Dynamics of the coronavirus replicative structures

    NARCIS (Netherlands)

    Hagemeijer, M.C.

    2011-01-01

    Coronaviruses (CoV) are positive-strand RNA (+RNA) viruses that are important infectious agents in both animals and man. Upon infection, CoVs generate large multicomponent protein complexes, consisting of 16 nonstructural proteins (nsp’s) and yet to be identified cellular proteins, dedicated to the

  18. Neotropical Bats from Costa Rica harbour Diverse Coronaviruses.

    Science.gov (United States)

    Moreira-Soto, A; Taylor-Castillo, L; Vargas-Vargas, N; Rodríguez-Herrera, B; Jiménez, C; Corrales-Aguilar, E

    2015-11-01

    Bats are hosts of diverse coronaviruses (CoVs) known to potentially cross the host-species barrier. For analysing coronavirus diversity in a bat species-rich country, a total of 421 anal swabs/faecal samples from Costa Rican bats were screened for CoV RNA-dependent RNA polymerase (RdRp) gene sequences by a pancoronavirus PCR. Six families, 24 genera and 41 species of bats were analysed. The detection rate for CoV was 1%. Individuals (n = 4) from four different species of frugivorous (Artibeus jamaicensis, Carollia perspicillata and Carollia castanea) and nectivorous (Glossophaga soricina) bats were positive for coronavirus-derived nucleic acids. Analysis of 440 nt. RdRp sequences allocated all Costa Rican bat CoVs to the α-CoV group. Several CoVs sequences clustered near previously described CoVs from the same species of bat, but were phylogenetically distant from the human CoV sequences identified to date, suggesting no recent spillover events. The Glossophaga soricina CoV sequence is sufficiently dissimilar (26% homology to the closest known bat CoVs) to represent a unique coronavirus not clustering near other CoVs found in the same bat species so far, implying an even higher CoV diversity than previously suspected.

  19. The Severe Acute Respiratory Syndrome (SARS-coronavirus 3a protein may function as a modulator of the trafficking properties of the spike protein

    Directory of Open Access Journals (Sweden)

    Tan Yee-Joo

    2005-02-01

    Full Text Available Abstract Background A recent publication reported that a tyrosine-dependent sorting signal, present in cytoplasmic tail of the spike protein of most coronaviruses, mediates the intracellular retention of the spike protein. This motif is missing from the spike protein of the severe acute respiratory syndrome-coronavirus (SARS-CoV, resulting in high level of surface expression of the spike protein when it is expressed on its own in vitro. Presentation of the hypothesis It has been shown that the severe acute respiratory syndrome-coronavirus genome contains open reading frames that encode for proteins with no homologue in other coronaviruses. One of them is the 3a protein, which is expressed during infection in vitro and in vivo. The 3a protein, which contains a tyrosine-dependent sorting signal in its cytoplasmic domain, is expressed on the cell surface and can undergo internalization. In addition, 3a can bind to the spike protein and through this interaction, it may be able to cause the spike protein to become internalized, resulting in a decrease in its surface expression. Testing the hypothesis The effects of 3a on the internalization of cell surface spike protein can be examined biochemically and the significance of the interplay between these two viral proteins during viral infection can be studied using reverse genetics methodology. Implication of the hypothesis If this hypothesis is proven, it will indicate that the severe acute respiratory syndrome-coronavirus modulates the surface expression of the spike protein via a different mechanism from other coronaviruses. The interaction between 3a and S, which are expressed from separate subgenomic RNA, would be important for controlling the trafficking properties of S. The cell surface expression of S in infected cells significantly impacts viral assembly, viral spread and viral pathogenesis. Modulation by this unique pathway could confer certain advantages during the replication of the severe

  20. Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts.

    Directory of Open Access Journals (Sweden)

    Stephanie Bertram

    Full Text Available The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2 in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host.

  1. Peptides derived from HIV-1, HIV-2, Ebola virus, SARS coronavirus and coronavirus 229E exhibit high affinity binding to the formyl peptide receptor

    Science.gov (United States)

    Mills, John S.

    2007-01-01

    Peptides derived from the membrane proximal region of fusion proteins of human immunodeficiency viruses 1 and 2, Coronavirus 229 E, severe acute respiratory syndrome coronavirus and Ebola virus were all potent antagonists of the formyl peptide receptor expressed in Chinese hamster ovary cells. Binding of viral peptides was affected by the naturally occurring polymorphisms at residues 190 and 192, which are located at second extracellular loop-transmembrane helix 5 interface. Substitution of R190 with W190 enhanced the affinity for a severe acute respiratory syndrome coronavirus peptide 6 fold but reduced the affinity for N-formyl-Nle–Leu-Phe by 2.5 fold. A 12 mer peptide derived from coronavirus 229E (ETYIKPWWVWL) was the most potent antagonist of the formyl peptide receptor W190 with a Ki of 230 nM. Fluorescently labeled ETYIKPWWVWL was effectively internalized by all three variants with EC50 of ~25 nM. An HKU-1 coronavirus peptide, MYVKWPWYVWL, was a potent antagonist but N-formyl-MYVKWPWYVWL was a potent agonist. ETYIKPWWVWL did not stimulate GTPγS binding but inhibited the stimulation by formyl-NleLeuPhe. It also blocked β arrestin translocation and receptor downregulation induced by formyl-Nle–Leu–Phe. This indicates that formyl peptide receptor may be important in viral infections and that variations in its sequence among individuals may affect their likelihood of viral and bacterial infections. PMID:16842982

  2. SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex.

    Science.gov (United States)

    Chen, Xiaojuan; Yang, Xingxing; Zheng, Yang; Yang, Yudong; Xing, Yaling; Chen, Zhongbin

    2014-05-01

    SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.

  3. Structure of a SARS coronavirus-derived peptide bound to the human major histocompatibility complex class I molecule HLA-B*1501

    DEFF Research Database (Denmark)

    Røder, Gustav; Kristensen, Ole; Kastrup, Jette S;

    2008-01-01

    , the crystal structure of HLA-B*1501 in complex with a SARS coronavirus-derived nonapeptide (VQQESSFVM) has been determined at high resolution (1.87 A). The peptide is deeply anchored in the B and F pockets, but with the Glu4 residue pointing away from the floor in the peptide-binding groove, making......The human leukocyte antigen (HLA) class I system comprises a highly polymorphic set of molecules that specifically bind and present peptides to cytotoxic T cells. HLA-B*1501 is a prototypical member of the HLA-B62 supertype and only two peptide-HLA-B*1501 structures have been determined. Here...

  4. SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6.

    Science.gov (United States)

    Li, Shih-Wen; Wang, Ching-Ying; Jou, Yu-Jen; Huang, Su-Hua; Hsiao, Li-Hsin; Wan, Lei; Lin, Ying-Ju; Kung, Szu-Hao; Lin, Cheng-Wen

    2016-05-05

    Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLPro) reportedly inhibits the production of type I interferons (IFNs) and pro-inflammatory cytokines in Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I) pathways. The study investigated the inhibitory effect and its antagonistic mechanism of SARS-CoV PLPro on TLR7-mediated cytokine production. TLR7 agonist (imiquimod (IMQ)) concentration-dependently induced activation of ISRE-, NF-κB- and AP-1-luciferase reporters, as well as the production of IFN-α, IFN-β, TNF-α, IL-6 and IL-8 in human promonocyte cells. However, SARS-CoV PLPro significantly inhibited IMQ-induced cytokine production through suppressing the activation of transcription factors IRF-3, NF-κB and AP-1. Western blot analysis with anti-Lys48 and anti-Lys63 ubiquitin antibodies indicated the SARS-CoV PLPro removed Lys63-linked ubiquitin chains of TRAF3 and TRAF6, but not Lys48-linked ubiquitin chains in un-treated and treated cells. The decrease in the activated state of TRAF3 and TRAF6 correlated with the inactivation of TBK1 in response to IMQ by PLPro. The results revealed that the antagonism of SARS-CoV PLPro on TLR7-mediated innate immunity was associated with the negative regulation of TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals.

  5. Detection of Coronaviruses in Bats of Various Species in Italy

    Directory of Open Access Journals (Sweden)

    Maria B. Boniotti

    2013-10-01

    Full Text Available Bats are natural reservoirs for many mammalian coronaviruses, which have received renewed interest after the discovery of the severe acute respiratory syndrome (SARS and the Middle East respiratory syndrome (MERS CoV in humans. This study describes the identification and molecular characterization of alphacoronaviruses and betacoronaviruses in bats in Italy, from 2010 to 2012. Sixty-nine faecal samples and 126 carcasses were tested using pan-coronavirus RT-PCR. Coronavirus RNAs were detected in seven faecal samples and nine carcasses. A phylogenetic analysis of RNA-dependent RNA polymerase sequence fragments aided in identifying two alphacoronaviruses from Kuhl’s pipistrelle (Pipistrellus kuhlii, three clade 2b betacoronaviruses from lesser horseshoe bats (Rhinolophus hipposideros, and 10 clade 2c betacoronaviruses from Kuhl’s pipistrelle, common noctule (Nyctalus noctula, and Savi’s pipistrelle (Hypsugo savii. This study fills a substantive gap in the knowledge on bat-CoV ecology in Italy, and extends the current knowledge on clade 2c betacoronaviruses with new sequences obtained from bats that have not been previously described as hosts of these viruses.

  6. Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptide.

    Science.gov (United States)

    Madu, Ikenna G; Roth, Shoshannah L; Belouzard, Sandrine; Whittaker, Gary R

    2009-08-01

    Many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides. While the coronavirus (CoV) spike (S) protein is known to be cleaved at the S1/S2 boundary, this cleavage site is not closely linked to a fusion peptide. However, a second cleavage site has been identified in the severe acute respiratory syndrome CoV (SARS-CoV) S2 domain (R797). Here, we investigated whether this internal cleavage of S2 exposes a viral fusion peptide. We show that the residues immediately C-terminal to the SARS-CoV S2 cleavage site SFIEDLLFNKVTLADAGF are very highly conserved across all CoVs. Mutagenesis studies of these residues in SARS-CoV S, followed by cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for residues L803, L804, and F805 in membrane fusion. Mutation of the most N-terminal residue (S798) had little or no effect on membrane fusion. Biochemical analyses of synthetic peptides corresponding to the proposed S2 fusion peptide also showed an important role for this region in membrane fusion and indicated the presence of alpha-helical structure. We propose that proteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S, which may be conserved across the Coronaviridae.

  7. Characterization of a Highly Conserved Domain within the Severe Acute Respiratory Syndrome Coronavirus Spike Protein S2 Domain with Characteristics of a Viral Fusion Peptide▿

    Science.gov (United States)

    Madu, Ikenna G.; Roth, Shoshannah L.; Belouzard, Sandrine; Whittaker, Gary R.

    2009-01-01

    Many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides. While the coronavirus (CoV) spike (S) protein is known to be cleaved at the S1/S2 boundary, this cleavage site is not closely linked to a fusion peptide. However, a second cleavage site has been identified in the severe acute respiratory syndrome CoV (SARS-CoV) S2 domain (R797). Here, we investigated whether this internal cleavage of S2 exposes a viral fusion peptide. We show that the residues immediately C-terminal to the SARS-CoV S2 cleavage site SFIEDLLFNKVTLADAGF are very highly conserved across all CoVs. Mutagenesis studies of these residues in SARS-CoV S, followed by cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for residues L803, L804, and F805 in membrane fusion. Mutation of the most N-terminal residue (S798) had little or no effect on membrane fusion. Biochemical analyses of synthetic peptides corresponding to the proposed S2 fusion peptide also showed an important role for this region in membrane fusion and indicated the presence of α-helical structure. We propose that proteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S, which may be conserved across the Coronaviridae. PMID:19439480

  8. SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway.

    Science.gov (United States)

    Li, Shih-Wein; Wang, Ching-Ying; Jou, Yu-Jen; Yang, Tsuey-Ching; Huang, Su-Hua; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

    2016-05-13

    SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between -175 to -60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo.

  9. Coronavirus Genomics and Bioinformatics Analysis

    Directory of Open Access Journals (Sweden)

    Kwok-Yung Yuen

    2010-08-01

    Full Text Available The drastic increase in the number of coronaviruses discovered and coronavirus genomes being sequenced have given us an unprecedented opportunity to perform genomics and bioinformatics analysis on this family of viruses. Coronaviruses possess the largest genomes (26.4 to 31.7 kb among all known RNA viruses, with G + C contents varying from 32% to 43%. Variable numbers of small ORFs are present between the various conserved genes (ORF1ab, spike, envelope, membrane and nucleocapsid and downstream to nucleocapsid gene in different coronavirus lineages. Phylogenetically, three genera, Alphacoronavirus, Betacoronavirus and Gammacoronavirus, with Betacoronavirus consisting of subgroups A, B, C and D, exist. A fourth genus, Deltacoronavirus, which includes bulbul coronavirus HKU11, thrush coronavirus HKU12 and munia coronavirus HKU13, is emerging. Molecular clock analysis using various gene loci revealed that the time of most recent common ancestor of human/civet SARS related coronavirus to be 1999-2002, with estimated substitution rate of 4´10-4 to 2´10-2 substitutions per site per year. Recombination in coronaviruses was most notable between different strains of murine hepatitis virus (MHV, between different strains of infectious bronchitis virus, between MHV and bovine coronavirus, between feline coronavirus (FCoV type I and canine coronavirus generating FCoV type II, and between the three genotypes of human coronavirus HKU1 (HCoV-HKU1. Codon usage bias in coronaviruses were observed, with HCoV-HKU1 showing the most extreme bias, and cytosine deamination and selection of CpG suppressed clones are the two major independent biological forces that shape such codon usage bias in coronaviruses.

  10. SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6

    Directory of Open Access Journals (Sweden)

    Shih-Wen Li

    2016-05-01

    Full Text Available Severe acute respiratory syndrome coronavirus (SARS-CoV papain-like protease (PLPro reportedly inhibits the production of type I interferons (IFNs and pro-inflammatory cytokines in Toll-like receptor 3 (TLR3 and retinoic acid-inducible gene 1 (RIG-I pathways. The study investigated the inhibitory effect and its antagonistic mechanism of SARS-CoV PLPro on TLR7-mediated cytokine production. TLR7 agonist (imiquimod (IMQ concentration-dependently induced activation of ISRE-, NF-κB- and AP-1-luciferase reporters, as well as the production of IFN-α, IFN-β, TNF-α, IL-6 and IL-8 in human promonocyte cells. However, SARS-CoV PLPro significantly inhibited IMQ-induced cytokine production through suppressing the activation of transcription factors IRF-3, NF-κB and AP-1. Western blot analysis with anti-Lys48 and anti-Lys63 ubiquitin antibodies indicated the SARS-CoV PLPro removed Lys63-linked ubiquitin chains of TRAF3 and TRAF6, but not Lys48-linked ubiquitin chains in un-treated and treated cells. The decrease in the activated state of TRAF3 and TRAF6 correlated with the inactivation of TBK1 in response to IMQ by PLPro. The results revealed that the antagonism of SARS-CoV PLPro on TLR7-mediated innate immunity was associated with the negative regulation of TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals.

  11. SARS-CoV regulates immune function-related gene expression in human monocytic cells.

    Science.gov (United States)

    Hu, Wanchung; Yen, Yu-Ting; Singh, Sher; Kao, Chuan-Liang; Wu-Hsieh, Betty A

    2012-08-01

    Severe acute respiratory syndrome (SARS) is characterized by acute respiratory distress syndrome (ARDS) and pulmonary fibrosis, and monocytes/macrophages are the key players in the pathogenesis of SARS. In this study, we compared the transcriptional profiles of SARS coronavirus (SARS-CoV)-infected monocytic cells against that infected by coronavirus 229E (CoV-229E). Total RNA was extracted from infected DC-SIGN-transfected monocytes (THP-1-DC-SIGN) at 6 and 24 h after infection, and the gene expression was profiled in oligonucleotide-based microarrays. Analysis of immune-related gene expression profiles showed that at 24 h after SARS-CoV infection: (1) IFN-α/β-inducible and cathepsin/proteasome genes were downregulated; (2) hypoxia/hyperoxia-related genes were upregulated; and (3) TLR/TLR-signaling, cytokine/cytokine receptor-related, chemokine/chemokine receptor-related, lysosome-related, MHC/chaperon-related, and fibrosis-related genes were differentially regulated. These results elucidate that SARS-CoV infection regulates immune-related genes in monocytes/macrophages, which may be important to the pathogenesis of SARS.

  12. Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of STING-mediated signaling.

    Directory of Open Access Journals (Sweden)

    Li Sun

    Full Text Available Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV NL63 and severe acute respiratory syndrome (SARS CoV papain-like proteases (PLP antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS. STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN. We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM or SARS-CoV (PLpro-TM inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction.

  13. Humoral immune responses in rabbits induced by an experimental inactivated severe acute respiratory syndrome coronavirus vaccine prepared from F69 strain

    Institute of Scientific and Technical Information of China (English)

    张传海; 郭中敏; 郑焕英; 陆家海; 王一飞; 鄢心革; 赵勇; 杜雄威; 张欣; 方苓; 凌文华; 戚树源; 余新炳; 钟南山

    2004-01-01

    Background The etiologic agent of severe acute respiratory syndrome (SARS) has been confirmed to be a novel coronavirus (CoV), namely SARS-CoV. Developing safe and effective SARS-CoV vaccines is essential for us to prevent the possible reemergence of its epidemic. Previous experiences indicate that inactivated vaccine is conventional and more hopeful to be successfully developed. Immunogenicity evaluation of an experimental inactivated SARS-CoV vaccine in rabbits was conducted and reported in this paper.Methods The large-scale cultured SARS-CoV F69 strain was inactivated with 0.4% formaldehyde and purified, then used as the immunogen combined with Freund's adjuvant. Eight adult New Zealand rabbits were immunized four times with this experimental inactivated vaccine. Twelve sets of rabbit serum were sampled from the third day to the seventy-fourth day after the first vaccination. The titers of specific anti-SARS-CoV IgG antibody were determined by indirect enzyme-linked immunosorbent assay, and the neutralizing antibody titers were detected with micro-cytopathic effect neutralization test.Conclusions The inactivated SARS-CoV vaccine made from F69 strain owns strong immunogenicity, and the cross neutralization response between the two different SARS-CoV strains gives a hint of the similar neutralizing epitopes, which provide stable bases for the development of inactivated SARS-CoV vaccines.

  14. The Conserved Coronavirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome Coronavirus Infection

    Directory of Open Access Journals (Sweden)

    Anthony R. Fehr

    2016-12-01

    Full Text Available ADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3. However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3’s de-ADP-ribosylation activity in vitro. Next, we created recombinant severe acute respiratory syndrome coronavirus (SARS-CoV strains with these mutations. These mutations led to virus attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant virus elicited an early, enhanced interferon (IFN, interferon-stimulated gene (ISG, and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs.

  15. Extraordinary GU-rich single-strand RNA identified from SARS coronavirus contributes an excessive innate immune response.

    Science.gov (United States)

    Li, Yan; Chen, Ming; Cao, Hongwei; Zhu, Yuanfeng; Zheng, Jiang; Zhou, Hong

    2013-02-01

    A dangerous cytokine storm occurs in the SARS involving in immune disorder, but many aspects of the pathogenetic mechanism remain obscure since its outbreak. To deeply reveal the interaction of host and SARS-CoV, based on the basic structural feature of pathogen-associated molecular pattern, we created a new bioinformatics method for searching potential pathogenic molecules and identified a set of SARS-CoV specific GU-rich ssRNA fragments with a high-density distribution in the genome. In vitro experiments, the result showed the representative SARS-CoV ssRNAs had powerful immunostimulatory activities to induce considerable level of pro-inflammatory cytokine TNF-a, IL-6 and IL-12 release via the TLR7 and TLR8, almost 2-fold higher than the strong stimulatory ssRNA40 that was found previously from other virus. Moreover, SARS-CoV ssRNA was able to cause acute lung injury in mice with a high mortality rate in vivo experiment. It suggests that SARS-CoV specific GU-rich ssRNA plays a very important role in the cytokine storm associated with a dysregulation of the innate immunity. This study not only presents new evidence about the immunopathologic damage caused by overactive inflammation during the SARS-CoV infection, but also provides a useful clue for a new therapeutic strategy. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  16. Circulation of Group 2 Coronaviruses in a Bat Species Common to Urban Areas in Western Europe

    NARCIS (Netherlands)

    Reusken, C.B.E.M.; Lina, P.H.C.; Pielaat, A.; Vries, de A.; Dam-Deisz, C.; Adema, J.; Drexler, J.F.; Drosten, C.; Kooi, E.A.

    2010-01-01

    Fecal samples of 211 bats representing 13 different bat species from 31 locations in the Netherlands were analyzed for the presence of coronaviruses (CoV) using a genus-wide reverse transcription (RT)-polymerase chain reaction. CoVs are known for their high potential for interspecies transmission, i

  17. The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor.

    Directory of Open Access Journals (Sweden)

    Rinki Minakshi

    Full Text Available The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV is reported to cause apoptosis of infected cells and several of its proteins including the 3a accessory protein, are pro-apoptotic. Since the 3a protein localizes to the endoplasmic reticulum (ER-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the 3a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR, which includes the inositol-requiring enzyme 1 (IRE-1, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK pathways, these were individually tested in 3a-expressing cells. Only the PERK pathway was found to be activated in 3a-expressing cells based on (1 increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha and inhibitory effects of a dominant-negative form of eIF2alpha on GRP78 promoter activity, (2 increased translation of activating transcription factor 4 (ATF4 mRNA, and (3 ATF4-dependent activation of the C/EBP homologous protein (CHOP gene promoter. Activation of PERK affects innate immunity by suppression of type 1 interferon (IFN signaling. The 3a protein was found to induce serine phosphorylation within the IFN alpha-receptor subunit 1 (IFNAR1 degradation motif and to increase IFNAR1 ubiquitination. Confocal microscopic analysis showed increased translocation of IFNAR1 into the lysosomal compartment and flow cytometry showed reduced levels of IFNAR1 in 3a-expressing cells. These results provide further mechanistic details of the pro-apoptotic effects of the SARS-CoV 3a protein, and suggest a potential role for it in attenuating interferon responses and innate immunity.

  18. Feline and Canine Coronaviruses: Common Genetic and Pathobiological Features

    OpenAIRE

    Sophie Le Poder

    2011-01-01

    A new human coronavirus responsible for severe acute respiratory syndrome (SARS) was identified in 2003, which raised concern about coronaviruses as agents of serious infectious disease. Nevertheless, coronaviruses have been known for about 50 years to be major agents of respiratory, enteric, or systemic infections of domestic and companion animals. Feline and canine coronaviruses are widespread among dog and cat populations, sometimes leading to the fatal diseases known as feline infectious ...

  19. Identification of a novel conserved HLA-A*0201-restricted epitope from the spike protein of SARS-CoV

    Directory of Open Access Journals (Sweden)

    Ni Bing

    2009-12-01

    Full Text Available Abstract Background The spike (S protein is a major structural glycoprotein of coronavirus (CoV, the causal agent of severe acute respiratory syndrome (SARS. The S protein is a potent target for SARS-specific cell-mediated immune responses. However, the mechanism CoV pathogenesis in SARS and the role of special CTLs in virus clearance are still largely uncharacterized. Here, we describe a study that leads to the identification of a novel HLA-A*0201-restricted epitope from conserved regions of S protein. Results First, different SARS-CoV sequences were analyzed to predict eight candidate peptides from conserved regions of the S protein based upon HLA-A*0201 binding and proteosomal cleavage. Four of eight candidate peptides were tested by HLA-A*0201 binding assays. Among the four candidate peptides, Sp8 (S958-966, VLNDILSRL induced specific CTLs both ex vivo in PBLs of healthy HLA-A2+ donors and in HLA-A2.1/Kb transgenic mice immunized with a plasmid encoding full-length S protein. The immunized mice released IFN-γ and lysed target cells upon stimulation with Sp8 peptide-pulsed autologous dendritic cells in comparison to other candidates. Conclusion These results suggest that Sp8 is a naturally processed epitope. We propose that Sp8 epitope should help in the characterization of mechanisms of virus control and immunopathology in SARS-CoV infection.

  20. The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines.

    Science.gov (United States)

    Regla-Nava, Jose A; Jimenez-Guardeño, Jose M; Nieto-Torres, Jose L; Gallagher, Thomas M; Enjuanes, Luis; DeDiego, Marta L

    2013-11-01

    Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease.

    Science.gov (United States)

    Yang, Xingxing; Chen, Xiaojuan; Bian, Guangxing; Tu, Jian; Xing, Yaling; Wang, Yayun; Chen, Zhongbin

    2014-03-01

    The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe pulmonary disease in humans and represents the second example of a highly pathogenic coronavirus (CoV) following severe acute respiratory syndrome coronavirus (SARS-CoV). Genomic studies revealed that two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), process the polyproteins encoded by the MERS-CoV genomic RNA. We previously reported that SARS-CoV PLpro acts as both deubiquitinase (DUB) and IFN antagonist, but the function of the MERS-CoV PLpro was poorly understood. In this study, we characterized MERS-CoV PLpro, which is a protease and can recognize and process the cleavage sites (CS) of nsp1-2, nsp2-3 and nsp3-4. The LXGG consensus cleavage sites in the N terminus of pp1a/1ab, which is generally essential for CoV PLpro-mediated processing, were also characterized in MERS-CoV. MERS-CoV PLpro, like human SARS-CoV PLpro and NL63-CoV PLP2, is a viral deubiquitinating enzyme. It acts on both K48- and K63-linked ubiquitination and ISG15-linked ISGylation. We confirmed that MERS-CoV PLpro acts as an IFN antagonist through blocking the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3). These findings indicate that MERS-CoV PLpro acts as a viral DUB and suppresses production of IFN-β by an interfering IRF3-mediated signalling pathway, in addition to recognizing and processing the CS at the N terminus of replicase polyprotein to release the non-structural proteins. The characterization of proteolytic processing, DUB and IFN antagonist activities of MERS-CoV PLpro would reveal the interactions between MERS-CoV and its host, and be applicable to develop strategies targeting PLpro for the effective control of MERS-CoV infection.

  2. The aetiology of SARS: Koch's postulates fulfilled

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); R.A.M. Fouchier (Ron); T. Kuiken (Thijs)

    2004-01-01

    textabstractProof that a newly identified coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV) is the primary cause of severe acute respiratory syndrome (SARS) came from a series of studies on experimentally infected cynomolgus macaques (Macaca, fascicularis). SARS-CoV-infected

  3. Efficient and Quick Inactivation of SARS Coronavirus and Other Microbes Exposed to the Surfaces of Some Metal Catalysts

    Institute of Scientific and Technical Information of China (English)

    JUN HAN; LAN CHEN; SHU-MIN DUAN; QING-XIANG YANG; MIN YANG; CHEN GAO; BAO-YUN ZHANG; HONG HE; XIAO-PING DONG

    2005-01-01

    Objective To study the two metal catalysts Ag/Al2O3 and Cu/Al2O3 that interdict the transmission pathway for SARS and other respiratory infectious diseases. Methods Two metal catalysts Ag/Al2O3 and Cu/Al2O3 were pressed into wafers. One hundred μL 106 TCID50/mL SARS-CoV, 100 μL 106 PFU/mL recombinant baculovirus expressing hamster's prion protein (haPrP) protein and roughly 106 E. coli were slowly dropped onto the surfaces of the catalyst wafers and exposed for 5 and 20 min, respectively. After eluted from the surfaces of wafers, the infectivity of viruses and propagation of bacteria were measured. The expression of PrP protein was determined by Western blot. The morphological changes of bacteria were observed by electronic microscopy. Results After exposure to the catalysts surfaces for 5 and 20 min, the infectivity of SARS-CoV in Vero cells and baculovirus in Sf9 cells dropped down to a very low and undetectable level, and no colony was detected using bacteria culture method. The expression of haPrP protein reduced to 21.8% in the preparation of Sf9 cells infected with recombinant baculovirus exposed for 5 min and was undetectable exposed for 20 min. Bacterial membranes seemed to be cracked and the cytoplasm seemed to be effluent from cell bodies. Conclusion Exposures to the surfaces of Ag/Al2O3 and Cu/Al2O3 destroy the replication and propagation abilities of SARS-CoV, baculovirus and E. coli. Inactivation ability of metal catalysts needs to interact with air, utilizing oxygen molecules in air. Efficiently killing viruses and bacteria on the surfaces of the two metal catalysts has a promising potential for air-disinfection in hospitals, communities, and households.

  4. Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: Engineering a recombination-resistant genome

    Science.gov (United States)

    Yount, Boyd; Roberts, Rhonda S.; Lindesmith, Lisa; Baric, Ralph S.

    2006-08-01

    Live virus vaccines provide significant protection against many detrimental human and animal diseases, but reversion to virulence by mutation and recombination has reduced appeal. Using severe acute respiratory syndrome coronavirus as a model, we engineered a different transcription regulatory circuit and isolated recombinant viruses. The transcription network allowed for efficient expression of the viral transcripts and proteins, and the recombinant viruses replicated to WT levels. Recombinant genomes were then constructed that contained mixtures of the WT and mutant regulatory circuits, reflecting recombinant viruses that might occur in nature. Although viable viruses could readily be isolated from WT and recombinant genomes containing homogeneous transcription circuits, chimeras that contained mixed regulatory networks were invariantly lethal, because viable chimeric viruses were not isolated. Mechanistically, mixed regulatory circuits promoted inefficient subgenomic transcription from inappropriate start sites, resulting in truncated ORFs and effectively minimize viral structural protein expression. Engineering regulatory transcription circuits of intercommunicating alleles successfully introduces genetic traps into a viral genome that are lethal in RNA recombinant progeny viruses. regulation | systems biology | vaccine design

  5. Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo

    Directory of Open Access Journals (Sweden)

    Grywna Klaus

    2009-08-01

    Full Text Available Abstract During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1. As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively. This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells. In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection in-vivo, ORF 7

  6. False-Positive Results in a Recombinant Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV) Nucleocapsid-Based Western Blot Assay Were Rectified by the Use of Two Subunits (S1 and S2) of Spike for Detection of Antibody to SARS-CoV

    Science.gov (United States)

    Maache, Mimoun; Komurian-Pradel, Florence; Rajoharison, Alain; Perret, Magali; Berland, Jean-Luc; Pouzol, Stéphane; Bagnaud, Audrey; Duverger, Blandine; Xu, Jianguo; Osuna, Antonio; Paranhos-Baccalà, Glaucia

    2006-01-01

    To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV. PMID:16522785

  7. Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication.

    Science.gov (United States)

    Reggiori, Fulvio; Monastyrska, Iryna; Verheije, Monique H; Calì, Tito; Ulasli, Mustafa; Bianchi, Siro; Bernasconi, Riccardo; de Haan, Cornelis A M; Molinari, Maurizio

    2010-06-25

    Coronaviruses (CoV), including SARS and mouse hepatitis virus (MHV), are enveloped RNA viruses that induce formation of double-membrane vesicles (DMVs) and target their replication and transcription complexes (RTCs) on the DMV-limiting membranes. The DMV biogenesis has been connected with the early secretory pathway. CoV-induced DMVs, however, lack conventional endoplasmic reticulum (ER) or Golgi protein markers, leaving their membrane origins in question. We show that MHV co-opts the host cell machinery for COPII-independent vesicular ER export of a short-living regulator of ER-associated degradation (ERAD), EDEM1, to derive cellular membranes for replication. MHV infection causes accumulation of EDEM1 and OS-9, another short-living ER chaperone, in the DMVs. DMVs are coated with the nonlipidated LC3/Atg8 autophagy marker. Downregulation of LC3, but not inactivation of host cell autophagy, protects cells from CoV infection. Our study identifies the host cellular pathway hijacked for supplying CoV replication membranes and describes an autophagy-independent role for nonlipidated LC3-I.

  8. Development of a molecular-beacon-based multi-allelic real-time RT-PCR assay for the detection of human coronavirus causing severe acute respiratory syndrome (SARS-CoV): a general methodology for detecting rapidly mutating viruses.

    Science.gov (United States)

    Hadjinicolaou, Andreas V; Farcas, Gabriella A; Demetriou, Victoria L; Mazzulli, Tony; Poutanen, Susan M; Willey, Barbara M; Low, Donald E; Butany, Jagdish; Asa, Sylvia L; Kain, Kevin C; Kostrikis, Leondios G

    2011-04-01

    Emerging infectious diseases have caused a global effort for development of fast and accurate detection techniques. The rapidly mutating nature of viruses presents a major difficulty, highlighting the need for specific detection of genetically diverse strains. One such infectious agent is SARS-associated coronavirus (SARS-CoV), which emerged in 2003. This study aimed to develop a real-time RT-PCR detection assay specific for SARS-CoV, taking into account its intrinsic polymorphic nature due to genetic drift and recombination and the possibility of continuous and multiple introductions of genetically non-identical strains into the human population, by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV S, E, M and N genes. These were applied in simple, reproducible duplex and multiplex real-time PCR assays on 25 post-mortem samples and constructed RNA controls, and they demonstrated high target detection ability and specificity. This assay can readily be adapted for detection of other emerging and rapidly mutating pathogens.

  9. The spike protein of severe acute respiratory syndrome (SARS) is cleaved in virus infected Vero-E6 cells

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Spike protein is one of the major structural proteins of severe acute respiratory syndrome-coronavirus. It is essential for the interaction of the virons with host cell receptors and subsequent fusion of the viral envelop with host cell membrane to allow infection. Some spike proteins of coronavirus, such as MHV, HCoV-OC43, AIBV and BcoV, are proteolytically cleaved into two subunits, S1 and S2. In contrast, TGV, FIPV and HCoV-229E are not. Many studies have shown that the cleavage of spike protein seriously affects its function. In order to investigate the maturation and proteolytic processing of the S protein of SARS CoV, we generated S1 and S2 subunit specific antibodies (Abs) as well as N, E and 3CL protein-specific Abs. Our results showed that the antibodies could efficiently and specifically bind to their corresponding proteins from E. coli expressed or lysate of SARS-CoV infected Vero-E6 cells by Western blot analysis. Furthermore, the anti-S 1 and S2 Abs were proved to be capable of binding to SARS CoV under electron microscope observation. When S2 Ab was used to perform immune precipitation with lysate of SARS-CoV infected cells, a cleaved S2 fragment was detected with S2-specific mAb by Western blot analysis. The data demonstrated that the cleavage of S protein was observed in the lysate, indicating that proteolytic processing of S protein is present in host cells.

  10. Incorporation of Spike and Membrane Glycoproteins into Coronavirus Virions

    Directory of Open Access Journals (Sweden)

    Makoto Ujike

    2015-04-01

    Full Text Available The envelopes of coronaviruses (CoVs contain primarily three proteins; the two major glycoproteins spike (S and membrane (M, and envelope (E, a non-glycosylated protein. Unlike other enveloped viruses, CoVs bud and assemble at the endoplasmic reticulum (ER-Golgi intermediate compartment (ERGIC. For efficient virion assembly, these proteins must be targeted to the budding site and to interact with each other or the ribonucleoprotein. Thus, the efficient incorporation of viral envelope proteins into CoV virions depends on protein trafficking and protein–protein interactions near the ERGIC. The goal of this review is to summarize recent findings on the mechanism of incorporation of the M and S glycoproteins into the CoV virion, focusing on protein trafficking and protein–protein interactions.

  11. Feline and canine coronaviruses: common genetic and pathobiological features.

    Science.gov (United States)

    Le Poder, Sophie

    2011-01-01

    A new human coronavirus responsible for severe acute respiratory syndrome (SARS) was identified in 2003, which raised concern about coronaviruses as agents of serious infectious disease. Nevertheless, coronaviruses have been known for about 50 years to be major agents of respiratory, enteric, or systemic infections of domestic and companion animals. Feline and canine coronaviruses are widespread among dog and cat populations, sometimes leading to the fatal diseases known as feline infectious peritonitis (FIP) and pantropic canine coronavirus infection in cats and dogs, respectively. In this paper, different aspects of the genetics, host cell tropism, and pathogenesis of the feline and canine coronaviruses (FCoV and CCoV) will be discussed, with a view to illustrating how study of FCoVs and CCoVs can improve our general understanding of the pathobiology of coronaviruses.

  12. Feline and Canine Coronaviruses: Common Genetic and Pathobiological Features

    Directory of Open Access Journals (Sweden)

    Sophie Le Poder

    2011-01-01

    Full Text Available A new human coronavirus responsible for severe acute respiratory syndrome (SARS was identified in 2003, which raised concern about coronaviruses as agents of serious infectious disease. Nevertheless, coronaviruses have been known for about 50 years to be major agents of respiratory, enteric, or systemic infections of domestic and companion animals. Feline and canine coronaviruses are widespread among dog and cat populations, sometimes leading to the fatal diseases known as feline infectious peritonitis (FIP and pantropic canine coronavirus infection in cats and dogs, respectively. In this paper, different aspects of the genetics, host cell tropism, and pathogenesis of the feline and canine coronaviruses (FCoV and CCoV will be discussed, with a view to illustrating how study of FCoVs and CCoVs can improve our general understanding of the pathobiology of coronaviruses.

  13. Chimeric severe acute respiratory syndrome coronavirus (SARS-CoV) S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV

    Science.gov (United States)

    Liu, Ye V.; Massare, Michael J.; Barnard, Dale L.; Kort, Thomas; Nathan, Margret; Wang, Lei; Smith, Gale

    2011-01-01

    SARS-CoV was the cause of the global pandemic in 2003 that infected over 8000 people in 8 months. Vaccines against SARS are still not available. We developed a novel method to produce high levels of a recombinant SARS virus-like particles (VLPs) vaccine containing the SARS spike (S) protein and the influenza M1 protein using the baculovirus insect cell expression system. These chimeric SARS VLPs have a similar size and morphology to the wild type SARS-CoV. We tested the immunogenicity and protective efficacy of purified chimeric SARS VLPs and full length SARS S protein vaccines in a mouse lethal challenge model. The SARS VLP vaccine, containing 0.8 μg of SARS S protein, completely protected mice from death when administered intramuscular (IM) or intranasal (IN) routes in the absence of an adjuvant. Likewise, the SARS VLP vaccine, containing 4 μg of S protein without adjuvant, reduced lung virus titer to below detectable level, protected mice from weight loss, and elicited a high level of neutralizing antibodies against SARS-CoV. Sf9 cell-produced full length purified SARS S protein was also an effective vaccine against SARS-CoV but only when co-administered IM with aluminum hydroxide. SARS-CoV VLPs are highly immunogenic and induce neutralizing antibodies and provide protection against lethal challenge. Sf9 cell-based VLP vaccines are a potential tool to provide protection against novel pandemic agents. PMID:21762752

  14. The nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same IFN-β antagonizing mechanism: attenuation of PACT-mediated RIG-I/ MDA5 activation.

    Science.gov (United States)

    Ding, Zhen; Fang, Liurong; Yuan, Shuangling; Zhao, Ling; Wang, Xunlei; Long, Siwen; Wang, Mohan; Wang, Dang; Foda, Mohamed Frahat; Xiao, Shaobo

    2017-07-25

    Coronaviruses (CoVs) are a huge threat to both humans and animals and have evolved elaborate mechanisms to antagonize interferons (IFNs). Nucleocapsid (N) protein is the most abundant viral protein in CoV-infected cells, and has been identified as an innate immunity antagonist in several CoVs, including mouse hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV. However, the underlying molecular mechanism(s) remain unclear. In this study, we found that MHV N protein inhibited Sendai virus and poly(I:C)-induced IFN-β production by targeting a molecule upstream of retinoic acid-induced gene I (RIG-I) and melanoma differentiation gene 5 (MDA5). Further studies showed that both MHV and SARS-CoV N proteins directly interacted with protein activator of protein kinase R (PACT), a cellular dsRNA-binding protein that can bind to RIG-I and MDA5 to activate IFN production. The N-PACT interaction sequestered the association of PACT and RIG-I/MDA5, which in turn inhibited IFN-β production. However, the N proteins from porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV), which are also classified in the order Nidovirales, did not interact and counteract with PACT. Taken together, our present study confirms that both MHV and SARS-CoV N proteins can perturb the function of cellular PACT to circumvent the innate antiviral response. However, this strategy does not appear to be used by all CoVs N proteins.

  15. [High-efficiency expression of a receptor-binding domain of SARS-CoV spike protein in tobacco chloroplasts].

    Science.gov (United States)

    Zhong, Xue; Qi, Guangxun; Yang, Jing; Xing, Guojie; Liu, Jianfeng; Yang, Xiangdong

    2014-06-01

    Chloroplast-based expression system is promising for the hyper-expression of plant-derived recombinant therapeutic proteins and vaccines. To verify the feasibility of obtaining high-level expression of the SARS subunit vaccine and to provide a suitable plant-derived vaccine production platform against the severe acute respiratory syndrome coronavirus (SARS-CoV), a 193-amino acid fragment of SARS CoV spike protein receptor-binding domain (RBD), fused with the peptide vector cholera toxin B subunit (CTB), was expressed in tobacco chloroplasts. Codon-optimized CTB-RBD sequence was integrated into the chloroplast genome and homoplasmy was obtained, as confirmed by PCR and Southern blot analysis. Western blot showed expression of the recombinant fusion protein mostly in soluble monomeric form. Quantification of the recombinant fusion protein CTB-RBD was conducted by ELISA analysis from the transplastomic leaves at different developmental stages, attachment positions and time points in a day and the different expression levels of the CTB-RBD were observed with the highest expression of 10.2% total soluble protein obtained from mature transplastomic leaves. Taken together, our results demonstrate the feasibility of highly expressing SARS subunit vaccine RBD, indicating its potential in subsequent development of a plant-derived recombinant subunit vaccine and reagents production for antibody detection in SARS serological tests.

  16. SARS-CoV-Encoded Small RNAs Contribute to Infection-Associated Lung Pathology.

    Science.gov (United States)

    Morales, Lucía; Oliveros, Juan Carlos; Fernandez-Delgado, Raúl; tenOever, Benjamin Robert; Enjuanes, Luis; Sola, Isabel

    2017-03-08

    Severe acute respiratory syndrome coronavirus (SARS-CoV) causes lethal disease in humans, which is characterized by exacerbated inflammatory response and extensive lung pathology. To address the relevance of small non-coding RNAs in SARS-CoV pathology, we deep sequenced RNAs from the lungs of infected mice and discovered three 18-22 nt small viral RNAs (svRNAs). The three svRNAs were derived from the nsp3 (svRNA-nsp3.1 and -nsp3.2) and N (svRNA-N) genomic regions of SARS-CoV. Biogenesis of CoV svRNAs was RNase III, cell type, and host species independent, but it was dependent on the extent of viral replication. Antagomir-mediated inhibition of svRNA-N significantly reduced in vivo lung pathology and pro-inflammatory cytokine expression. Taken together, these data indicate that svRNAs contribute to SARS-CoV pathogenesis and highlight the potential of svRNA-N antagomirs as antivirals. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Middle East respiratory syndrome coronavirus in children

    OpenAIRE

    Thabet, Farah; Chehab, May; Bafaqih, Hind; AlMohaimeed, Sulaiman

    2015-01-01

    The Middle East respiratory syndrome (MERS) is a new human disease caused by a novel coronavirus (CoV). The disease is reported mainly in adults. Data in children are scarce. The disease caused by MERS-CoV in children presents with a wide range of clinical manifestations, and it is associated with a lower mortality rate compared with adults. Poor outcome is observed mainly in admitted patients with medical comorbidities. We report a new case of MERS-CoV infection in a 9-month-old child compli...

  18. Innate immune response of human alveolar type II cells infected with severe acute respiratory syndrome-coronavirus.

    Science.gov (United States)

    Qian, Zhaohui; Travanty, Emily A; Oko, Lauren; Edeen, Karen; Berglund, Andrew; Wang, Jieru; Ito, Yoko; Holmes, Kathryn V; Mason, Robert J

    2013-06-01

    Severe acute respiratory syndrome (SARS)-coronavirus (CoV) produces a devastating primary viral pneumonia with diffuse alveolar damage and a marked increase in circulating cytokines. One of the major cell types to be infected is the alveolar type II cell. However, the innate immune response of primary human alveolar epithelial cells infected with SARS-CoV has not been defined. Our objectives included developing a culture system permissive for SARS-CoV infection in primary human type II cells and defining their innate immune response. Culturing primary human alveolar type II cells at an air-liquid interface (A/L) improved their differentiation and greatly increased their susceptibility to infection, allowing us to define their primary interferon and chemokine responses. Viral antigens were detected in the cytoplasm of infected type II cells, electron micrographs demonstrated secretory vesicles filled with virions, virus RNA concentrations increased with time, and infectious virions were released by exocytosis from the apical surface of polarized type II cells. A marked increase was evident in the mRNA concentrations of interferon-β and interferon-λ (IL-29) and in a large number of proinflammatory cytokines and chemokines. A surprising finding involved the variability of expression of angiotensin-converting enzyme-2, the SARS-CoV receptor, in type II cells from different donors. In conclusion, the cultivation of alveolar type II cells at an air-liquid interface provides primary cultures in which to study the pulmonary innate immune responses to infection with SARS-CoV, and to explore possible therapeutic approaches to modulating these innate immune responses.

  19. SARS病毒和其他冠状病毒中性突变速率初步的比较研究%The preliminary comparative study of the neutral mutation rate of SARS-CoV and other groups of Coronavirus

    Institute of Scientific and Technical Information of China (English)

    孙慧敏; 唐晓凤; 王波; 谭雅慧; 于晓寒; 张景霞; 闫永平; 夏结来; 徐德忠

    2011-01-01

    目的 比较SARS冠状病毒(SARS-CoV)与第1至第3组(其他3组)冠状病毒的中性突变速率之异同,为更深入研究SARS-CoV起源提供新的思路.方法 在美国国立生物技术信息中心中获取其他3组26条病毒序列,同时选取7条SARS-CoV序列.计算各病毒株5个主要基因串联序列的同义替换核苷酸的数量(Ks值),以此值与时间创建散点图,并进行直线拟合,取得各自中性突变速率并比较.结果SARS-CoV中性突变速率为7.33×10-6/位点·天,而其他3组冠状病毒为2×10-6/位点·天,仅约前者的1/3.5,但决定系数R2极低,无意义.经多种拟合检验,发现标本量和离散性不是本文R2低值的主要原因.结论用同一方法对SARS-CoV和其他3组冠状病毒的中性突变速率进行比较研究,结果显示两者明显不同或具不同的模式;提示和其他3组冠状病毒相比较,SARS-CoV在分子进化或起源模式上似有特别之处.%Objective To oompare the neutral mutation rate between the SARS-CoV and the other three groups (Gl, G2 and G3) of Coronavirus, in order to provide new ideas for research deeply into the origin of SARS-CoV . Methods We obtained 26 sequences of Gl, G2, G3 of Coronavirus and seven sequences of SARS-CoV from NCBI. The Pami-lo-Bianchi-Li model was used to calculate the number of synonymous substitutions per synonymous site, Ks, for the concatenated five known major coding sequences of Coronavirus. A plot of Ks for the concatenated coding sequences vs. The sampling dates was established. The slope of the fitted line from the linear regression model gives the estimation of the neutral mutation rate. Results The neutral mutation rate of SARS-CoV was 7.33 × 10~6nt-1· day-1', the other three groups was 2.0 × 10-6nt-1·day-1, and the later was only 1/3.5 of the former, but the coefficient of determination (R2) is low extremely, and meaningless. Fitted by a variety of tests, we found the number of specimens and the dispersion were

  20. Understanding SARS with Wolfram Approach

    Institute of Scientific and Technical Information of China (English)

    Da-WeiLI; Yu-XiPAN; YunDUAN; Zhen-DeHUNG; Ming-QingXU; LinHE

    2004-01-01

    Stepping acquired immunodeficiency syndrome (AIDS), severe acute respiratory syndrome (SARS) as another type of disease has been threatening mankind since late last year. Many scientists worldwide are making great efforts to study the etiology of this disease with different approaches. 13 species of SARS virus have been sequenced. However, most people still largely rely on the traditional methods with some disadvantages. In this work, we used Wolfram approach to study the relationship among SARS viruses and between SARS viruses and other types of viruses, the effect of variations on the whole genome and the advantages in the analysis of SARS based on this novel approach. As a result, the similarities between SARS viruses and other coronaviruses are not really higher than those between SARS viruses and non-coronaviruses.

  1. Study on the roles of HLA-A gene polymorphism in the susceptibility and symptom of SARS-Coronavirus infection%HLA-A基因多态性与SARS易感性及症状关系的研究

    Institute of Scientific and Technical Information of China (English)

    何丽; 魏茂提; 王世鑫; 胡役兰

    2011-01-01

    Objective:To study the association between HLA-A gene polym orphsm and the susceptibility and symptom of SARS-Coroavirus infection in Northern Chinese Han ethnics.Methods:HLA-A were typed in 53 SARS cases,44 high risks and 77 controls using PCR-SBT m ethods in two case-control studies.Data were analyzed using SPSS version 11.5 and chisquares and OR were used to show the differences and association .Results:The frequency of HLA-A * 2453 allele in SARS patients was markedly higher than that of the high risk care workers(P=0.039,0R=4.479,95%CI 0.95-21.015).How ever,the frequency of HLA-A * 2444 allele in SARS patients was lower than that of the health control( P=0.029 ) .There was no significant difference of the distribution of HLA-A allele frequency in mild cases and severe cases.Conclusion :Genotype of HLA-A * 2453 may be one risk factor for infection of SARS-Cov ,how ever,HLA-A * 2444 may be protect factor for it infection .The severity of SARS may not relate with the genotype of HLA-A.%目的:研究中国北方汉族人群HLA-A基因多态性与SARS-Cov易感性及症状的关系.方法:采用病例-对照研究设计,应用PCR-SBT的研究方法对53例SARS患者、44例高危人群和77例健康人员的HLA-A位点等位基因型分布进行研究,运用SPSS11.5软件包进行统计分析,组间比较采用χ2检验,计算比值比(OR)及其95%可信区间(95%CI).结果:与高危人群比较,SARS病人 HLA-A*2453(P=0.039,OR=4.479,95%CI 0.955~21.015)基因型出现频率较高,两者有统计学意义;与健康人群比较,SARS病人HLA-A*2444(P=0.029)基因型频率显著减低;SARS患者轻症组和重症组的HLA-A位点的等位基因频率分布相比较未发现统计学差异(P>0.05).结论:我国华北地区汉族人群中HLA-A*2453可能与SARS的易感性相关,而等位基因HLA-A*2444可能具有保护作用.HLA-A基因多态性与SARS患者病情严重程度可能无关.

  2. Discovery, diversity and evolution of novel coronaviruses sampled from rodents in China.

    Science.gov (United States)

    Wang, Wen; Lin, Xian-Dan; Guo, Wen-Ping; Zhou, Run-Hong; Wang, Miao-Ruo; Wang, Cai-Qiao; Ge, Shuang; Mei, Sheng-Hua; Li, Ming-Hui; Shi, Mang; Holmes, Edward C; Zhang, Yong-Zhen

    2015-01-01

    Although rodents are important reservoirs for RNA viruses, to date only one species of rodent coronavirus (CoV) has been identified. Herein, we describe a new CoV, denoted Lucheng Rn rat coronavirus (LRNV), and novel variants of two Betacoronavirus species termed Longquan Aa mouse coronavirus (LAMV) and Longquan Rl rat coronavirus (LRLV), that were identified in a survey of 1465 rodents sampled in China during 2011-2013. Phylogenetic analysis revealed that LAMV and LRLV fell into lineage A of the genus Betacoronavirus, which included CoVs discovered in humans and domestic and wild animals. In contrast, LRNV harbored by Rattus norvegicus formed a distinct lineage within the genus Alphacoronavirus in the 3CL(pro), RdRp, and Hel gene trees, but formed a more divergent lineage in the N and S gene trees, indicative of a recombinant origin. Additional recombination events were identified in LRLV. Together, these data suggest that rodents may carry additional unrecognized CoVs.

  3. ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells

    NARCIS (Netherlands)

    C. Burkard (Christine); M.H. Verheije (Monique); B.L. Haagmans (Bart); F.J.M. van Kuppeveld (Frank ); P.J.M. Rottier (Peter); B.J. Bosch (Berend Jan); C.A.M. de Haan (Cornelis)

    2015-01-01

    textabstractIn addition to transporting ions, the multisubunit Na+,K+-ATPase also functions by relaying cardiotonic steroid (CTS)-binding- induced signals into cells. In this study, we analyzed the role of Na+,K+-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection.

  4. CovS Simultaneously Activates and Inhibits the CovR-Mediated Repression of Distinct Subsets of Group A Streptococcus Virulence Factor-Encoding Genes▿ †

    OpenAIRE

    Treviño, Jeanette; Perez, Nataly; Ramirez-Peña, Esmeralda; Liu, Zhuyun; Shelburne, Samuel A.; Musser, James M.; Sumby, Paul

    2009-01-01

    To colonize and cause disease at distinct anatomical sites, bacterial pathogens must tailor gene expression in a microenvironment-specific manner. The molecular mechanisms that control the ability of the human bacterial pathogen group A Streptococcus (GAS) to transition between infection sites have yet to be fully elucidated. A key regulator of GAS virulence gene expression is the CovR-CovS two-component regulatory system (also known as CsrR-CsrS). covR and covS mutant strains arise spontaneo...

  5. Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late Endosome/Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV).

    Science.gov (United States)

    Zhou, Nan; Pan, Ting; Zhang, Junsong; Li, Qianwen; Zhang, Xue; Bai, Chuan; Huang, Feng; Peng, Tao; Zhang, Jianhua; Liu, Chao; Tao, Liang; Zhang, Hui

    2016-04-22

    Ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. The outbreaks of Ebola viruses in 2014 represented the most serious Ebola epidemics in history and greatly threatened public health worldwide. The development of additional effective anti-Ebola therapeutic agents is therefore quite urgent. In this study, via high throughput screening of Food and Drug Administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of Ebola envelope pseudotyped viruses into the cytoplasm. Furthermore, teicoplanin also has an inhibitory effect on transcription- and replication-competent virus-like particles, with an IC50 as low as 330 nm Comparative analysis further demonstrated that teicoplanin is able to block the entry of Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) envelope pseudotyped viruses as well. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection.

  6. Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin

    Science.gov (United States)

    Kumaki, Yohichi; Wandersee, Miles K.; Smith, Aaron J.; Zhou, Yanchen; Simmons, Graham; Nelson, Nathan M.; Bailey, Kevin W.; Vest, Zachary G.; Li, Joseph K.-K.; Chan, Paul Kay-Sheung; Smee, Donald F.; Barnard, Dale L.

    2011-01-01

    Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 µg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 hours before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (pSARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells. PMID:21338626

  7. Coronaviruses: emerging and re-emerging pathogens in humans and animals.

    Science.gov (United States)

    Lau, Susanna K P; Chan, Jasper F W

    2015-12-22

    The severe acute respiratory syndrome coronavirus (SARS-CoV) and recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics have proven the ability of coronaviruses to cross species barrier and emerge rapidly in humans. Other coronaviruses such as porcine epidemic diarrhea virus (PEDV) are also known to cause major disease epidemics in animals with huge economic loss. This special issue in Virology Journal aims to highlight the advances and key discoveries in the animal origin, viral evolution, epidemiology, diagnostics and pathogenesis of the emerging and re-emerging coronaviruses in both humans and animals.

  8. Identification of avian coronavirus in wild aquatic birds of the central and eastern USA.

    Science.gov (United States)

    Jordan, Brian J; Hilt, Deborah A; Poulson, Rebecca; Stallknecht, David E; Jackwood, Mark W

    2015-01-01

    Coronaviruses (CoVs) are worldwide in distribution, highly infectious, and difficult to control because of their extensive genetic diversity, short generation time, and high mutation rates. Genetically diverse CoVs have been reported from wild aquatic birds that may represent a potential reservoir for avian CoVs as well as hosts for mutations and recombination events leading to new serotypes or genera. We tested 133 pooled samples representing 700 first-passage (in eggs) and 303 direct cloacal swab transport media samples from wild aquatic birds in the US that were avian influenza-negative. We isolated RNA from frozen samples and performed reverse transcriptase-PCR using a published universal CoV primer set. Of the samples tested, one from a Ruddy Turnstone (Arenaria interpres) was positive for CoV, showing nucleotide sequence similarity to a duck coronavirus (DK/CH/HN/ZZ2004). These data indicate a possible low prevalence of CoVs circulating in wild aquatic birds in the eastern half of the US.

  9. HTCC: Broad Range Inhibitor of Coronavirus Entry.

    Directory of Open Access Journals (Sweden)

    Aleksandra Milewska

    Full Text Available To date, six human coronaviruses have been known, all of which are associated with respiratory infections in humans. With the exception of the highly pathogenic SARS and MERS coronaviruses, human coronaviruses (HCoV-NL63, HCoV-OC43, HCoV-229E, and HCoV-HKU1 circulate worldwide and typically cause the common cold. In most cases, infection with these viruses does not lead to severe disease, although acute infections in infants, the elderly, and immunocompromised patients may progress to severe disease requiring hospitalization. Importantly, no drugs against human coronaviruses exist, and only supportive therapy is available. Previously, we proposed the cationically modified chitosan, N-(2-hydroxypropyl-3-trimethylammonium chitosan chloride (HTCC, and its hydrophobically-modified derivative (HM-HTCC as potent inhibitors of the coronavirus HCoV-NL63. Here, we show that HTCC inhibits interaction of a virus with its receptor and thus blocks the entry. Further, we demonstrate that HTCC polymers with different degrees of substitution act as effective inhibitors of all low-pathogenic human coronaviruses.

  10. Residue analysis of a CTL epitope of SARS-CoV spike protein by IFN-gamma production and bioinformatics prediction

    Directory of Open Access Journals (Sweden)

    Huang Jun

    2012-09-01

    Full Text Available Abstract Background Severe acute respiratory syndrome (SARS is an emerging infectious disease caused by the novel coronavirus SARS-CoV. The T cell epitopes of the SARS CoV spike protein are well known, but no systematic evaluation of the functional and structural roles of each residue has been reported for these antigenic epitopes. Analysis of the functional importance of side-chains by mutational study may exaggerate the effect by imposing a structural disturbance or an unusual steric, electrostatic or hydrophobic interaction. Results We demonstrated that N50 could induce significant IFN-gamma response from SARS-CoV S DNA immunized mice splenocytes by the means of ELISA, ELISPOT and FACS. Moreover, S366-374 was predicted to be an optimal epitope by bioinformatics tools: ANN, SMM, ARB and BIMAS, and confirmed by IFN-gamma response induced by a series of S358-374-derived peptides. Furthermore, each of S366-374 was replaced by alanine (A, lysine (K or aspartic acid (D, respectively. ANN was used to estimate the binding affinity of single S366-374 mutants to H-2 Kd. Y367 and L374 were predicated to possess the most important role in peptide binding. Additionally, these one residue mutated peptides were synthesized, and IFN-gamma production induced by G368, V369, A371, T372 and K373 mutated S366-374 were decreased obviously. Conclusions We demonstrated that S366-374 is an optimal H-2 Kd CTL epitope in the SARS CoV S protein. Moreover, Y367, S370, and L374 are anchors in the epitope, while C366, G368, V369, A371, T372, and K373 may directly interact with TCR on the surface of CD8-T cells.

  11. Bats and SARS

    Centers for Disease Control (CDC) Podcasts

    2006-11-08

    Bats are a natural reservoir for emerging viruses, among them henipaviruses and rabies virus variants. Dr. Nina Marano, Chief, Geographic Medicine and Health Promotion Branch, Division of Global Migration and Quarantine, CDC, explains connection between horseshoe bats and SARS coronavirus transmission.  Created: 11/8/2006 by Emerging Infectious Diseases.   Date Released: 11/17/2006.

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

    NARCIS (Netherlands)

    P.A. Rota (Paul); M.S. Oberste (Steven); S.S. Monroe (Stephan); W.A. Nix (Allan); R. Campagnoli (Ray); J.P. Icenogle (Joseph); S. Penaranda; B. Bankamp (Bettina); K. Maher (Kaija); M.H. Chen (Min-hsin); S. Tong (Suxiong); A. Tamin (Azaibi); L. Lowe (Luis); M. Frace (Michael); J.L. DeRisi (Joseph); Q. Chen (Qi); D. Wang (David); D.D. Erdman (Dean); T.C. Peret (Teresa); C. Burns (Cara); T.G. Ksiazek (Thomas); P.E. Rollin (Pierre); A. Sanchez (Berenguer); S. Liffick (Stephanie); B. Holloway (Brian); J. Limor (Josef); K. McCaustland (Karen); M. Olsen-Rasmussen (Mellissa); S. Gunther; A.D.M.E. Osterhaus (Albert); C. Drosten (Christian); M.A. Pallansch (Mark); L.J. Anderson (Larry); W.J. Belline; R.A.M. Fouchier (Ron)

    2003-01-01

    textabstractIn 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 geno

  13. Serotype- and strain- dependent contribution of the sensor kinase CovS of the CovRS two-component system to Streptococcus pyogenes pathogenesis

    Directory of Open Access Journals (Sweden)

    Podbielski Andreas

    2010-02-01

    Full Text Available Abstract Background The Streptococcus pyogenes (group A streptococci, GAS two-component signal transduction system CovRS has been described to be important for pathogenesis of this exclusively human bacterial species. If this system acts uniquely in all serotypes is currently unclear. Presence of serotype- or strain-dependent regulatory circuits and polarity is an emerging scheme in Streptococcus pyogenes pathogenesis. Thus, the contribution of the sensor kinase (CovS of the global regulatory two-component signal transduction system CovRS on pathogenesis of several M serotypes was investigated. Results CovS mutation uniformly repressed capsule expression and hampered keratinocyte adherence in all tested serotypes. However, a serotype- and even strain-dependent contribution on survival in whole human blood and biofilm formation was noted, respectively. Conclusions These data provide new information on the action of the CovS sensor kinase and revealed that its activity on capsule expression and keratinocyte adherence is uniform across serotypes, whereas the influence on biofilm formation and blood survival is serotype or even strain dependent. This adds the CovRS system to a growing list of serotype-specific acting regulatory loci in S. pyogenes.

  14. Detection of feline coronavirus using microcantilever sensors

    Science.gov (United States)

    Velanki, Sreepriya; Ji, Hai-Feng

    2006-11-01

    This work demonstrated the feasibility of detecting severe acute respiratory syndrome associated coronavirus (SARS-CoV) using microcantilever technology by showing that the feline coronavirus (FIP) type I virus can be detected by a microcantilever modified by feline coronavirus (FIP) type I anti-viral antiserum. A microcantilever modified by FIP type I anti-viral antiserum was developed for the detection of FIP type I virus. When the FIP type I virus positive sample is injected into the fluid cell where the microcantilever is held, the microcantilever bends upon the recognition of the FIP type I virus by the antiserum on the surface of the microcantilever. A negative control sample that does not contain FIP type I virus did not cause any bending of the microcantilever. The detection limit of the sensor was 0.1 µg ml-1 when the assay time was <1 h.

  15. Biological Characteristics and Etiological Significance of Porcine Respiratory Coronavirus(PRCV)

    Institute of Scientific and Technical Information of China (English)

    FAN Xiuping; FENG Li; SHI Hongyan; CHEN Jianfei

    2009-01-01

    Porcine respiratory coronavirus (PRCV), a spike (S) gene natural deletion mutant of transmissible gastroenteritis virus (TGEV), causes porcine respiratory disease complex. Research advances on porcine respiratory coronavirus were reviewed from four aspects of biological character, the model function for SARS-CoV research, contribution of the immunity to PRCV to protection against TGEV challenge exposure and other etiological significance.

  16. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    Science.gov (United States)

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R.; Simmons, Graham

    2016-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess, whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  17. A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant.

    Science.gov (United States)

    Agnihothram, Sudhakar; Yount, Boyd L; Donaldson, Eric F; Huynh, Jeremy; Menachery, Vineet D; Gralinski, Lisa E; Graham, Rachel L; Becker, Michelle M; Tomar, Sakshi; Scobey, Trevor D; Osswald, Heather L; Whitmore, Alan; Gopal, Robin; Ghosh, Arun K; Mesecar, Andrew; Zambon, Maria; Heise, Mark; Denison, Mark R; Baric, Ralph S

    2014-03-25

    Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. IMPORTANCE The 2012 outbreak of MERS-CoV raises the specter

  18. Suppression of Coronavirus Replication by Cyclophilin Inhibitors

    Directory of Open Access Journals (Sweden)

    Takashi Sasaki

    2013-05-01

    Full Text Available Coronaviruses infect a variety of mammalian and avian species and cause serious diseases in humans, cats, mice, and birds in the form of severe acute respiratory syndrome (SARS, feline infectious peritonitis (FIP, mouse hepatitis, and avian infectious bronchitis, respectively. No effective vaccine or treatment has been developed for SARS-coronavirus or FIP virus, both of which cause lethal diseases. It has been reported that a cyclophilin inhibitor, cyclosporin A (CsA, could inhibit the replication of coronaviruses. CsA is a well-known immunosuppressive drug that binds to cellular cyclophilins to inhibit calcineurin, a calcium-calmodulin-activated serine/threonine-specific phosphatase. The inhibition of calcineurin blocks the translocation of nuclear factor of activated T cells from the cytosol into the nucleus, thus preventing the transcription of genes encoding cytokines such as interleukin-2. Cyclophilins are peptidyl-prolyl isomerases with physiological functions that have been described for many years to include chaperone and foldase activities. Also, many viruses require cyclophilins for replication; these include human immunodeficiency virus, vesicular stomatitis virus, and hepatitis C virus. However, the molecular mechanisms leading to the suppression of viral replication differ for different viruses. This review describes the suppressive effects of CsA on coronavirus replication.

  19. Coronavirus envelope (E) protein remains at the site of assembly

    Energy Technology Data Exchange (ETDEWEB)

    Venkatagopalan, Pavithra [The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287-5401 (United States); School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401 (United States); Microbiology Graduate Program, Arizona State University, Tempe, AZ 85287-5401 (United States); Daskalova, Sasha M. [The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287-5401 (United States); Department of Biochemistry and Chemistry, Arizona State University, Tempe, AZ 85287-5401 (United States); Lopez, Lisa A. [The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287-5401 (United States); School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401 (United States); Molecular and Cellular Biology Graduate Program, Arizona State University, Tempe, AZ 85287-5401 (United States); Dolezal, Kelly A. [The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287-5401 (United States); School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401 (United States); Microbiology Graduate Program, Arizona State University, Tempe, AZ 85287-5401 (United States); Hogue, Brenda G., E-mail: Brenda.Hogue@asu.edu [The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287-5401 (United States); School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401 (United States)

    2015-04-15

    Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteine tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. - Highlights: • Mouse hepatitis coronavirus (MHV-CoV) E protein localizes in the ERGIC and Golgi. • MHV-CoV E does not transport to the cell surface. • MHV-CoV can be genetically engineered with a tetracysteine tag appended to E. • First FRAP and correlative light electron microscopy of a CoV E protein. • Live-cell imaging shows that E is mobile in ERGIC/Golgi membranes.

  20. Antiviral activity of cepharanthine against severe acute respiratory syndrome coronavirus in vitro

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chuan-hai; XIONG Sheng; LI Jiu-xiang; QI Shu-yuan; WANG Yi-fei; LIU Xin-jian; LU Jia-hai; QIAN Chui-wen; WAN Zhuo-yue; YAN Xin-ge; ZHENG Huan-ying; ZHANG Mei-ying

    2005-01-01

    @@ Severe acute respiratory syndrome (SARS) is the first severe viral epidemic we encountered this century, which once spread in more than thirty countries in 2003.1 The etiological agent of SARS has been confirmed to be a novel coronavirus, namely SARS coronavirus (SARS-CoV),2,3 and the first outbreak of SARS has been successfully controlled worldwide, but the identification of SARS-CoV isolated from wild animals, the emergence of some sporadic SARS cases later after that outbreak, all suggest that the recurrence of such an epidemic is not unlikely in the future. In this case, development of SARS vaccines and specific drugs is undoubtedly essential to the control and prevention from the possible outbreak.4,5

  1. A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D Streptococcal pyogenes strain influences virulence-associated genes.

    Science.gov (United States)

    Liang, Zhong; Zhang, Yueling; Agrahari, Garima; Chandrahas, Vishwanatha; Glinton, Kristofor; Donahue, Deborah L; Balsara, Rashna D; Ploplis, Victoria A; Castellino, Francis J

    2013-03-01

    A skin-tropic invasive group A Streptococcus pyogenes (GAS) strain, AP53, contains a natural inactivating mutation in the covS gene (covS(M)) of the two-component responder (CovR)/sensor (CovS) gene regulatory system. The effects of this mutation on specific GAS virulence determinants have been assessed, with emphasis on expression of the extracellular protease, streptococcal pyrogenic exotoxin B (SpeB), capsular hyaluronic acid, and proteins that allow host plasmin assembly on the bacterial surface, viz. a high affinity plasminogen (Pg)/plasmin receptor, Pg-binding group A streptococcal M protein (PAM), and the human Pg activator streptokinase. To further illuminate mechanisms of the functioning of CovRS in the virulence of AP53, two AP53 isogenic strains were generated, one in which the natural covS(M) gene was mutated to WT-covS (AP53/covS(WT)) and a strain that contained an inactivated covR gene (AP53/ΔcovR). Two additional strains that do not contain PAM, viz. WT-NS931 and NS931/covS(M), were also employed. SpeB was not measurably expressed in strains containing covR(WT)/covS(M), whereas in strains with natural or engineered covR(WT)/covS(WT), SpeB expression was highly up-regulated. Alternatively, capsule synthesis via the hasABC operon was enhanced in strain AP53/covS(M), whereas streptokinase expression was only slightly affected by the covS inactivation. PAM expression was not substantially influenced by the covS mutation, suggesting that covRS had minimal effects on the mga regulon that controls PAM expression. These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase.

  2. Pains and Gains from China's Experiences with Emerging Epidemics: From SARS to H7N9

    Science.gov (United States)

    Hua, Jinwen; Yu, Weijia; Chen, Jiajie; Kang, Kang; Qiu, Congling

    2016-01-01

    Over the recent decades, China experienced several emerging virus outbreaks including those caused by the severe acute respiratory syndrome- (SARS-) coronavirus (Cov), H5N1 virus, and H7N9 virus. The SARS tragedy revealed faults in China's infectious disease prevention system, propelling the Chinese government to enact reforms that enabled better combating of the subsequent H1N1 and H7N9 avian flu epidemics. The system is buttressed by three fundamental, mutually reinforcing components: (1) enduring government administration reforms, including legislation establishing a unified public health emergency management system; (2) prioritized funding for biotechnology and biomedicine industrialization, especially in the areas of pathogen identification, drug production, and the development of vaccines and diagnostics; and (3) increasing investment for public health and establishment of a rapid-response infectious diseases prevention and control system. China is now using its hard-gained experience to support the fight against Ebola in Africa and the Middle East Respiratory Syndrome in its own country. PMID:27525272

  3. Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Juan Reguera

    Full Text Available The coronaviruses (CoVs are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10-20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN, a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs of two closely related CoV strains, transmissible gastroenteritis virus (TGEV and porcine respiratory CoV (PRCV, in complex with their receptor, porcine APN (pAPN, or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.

  4. Synthesis and evaluation of phenylisoserine derivatives for the SARS-CoV 3CL protease inhibitor.

    Science.gov (United States)

    Konno, Hiroyuki; Onuma, Takumi; Nitanai, Ikumi; Wakabayashi, Masaki; Yano, Shigekazu; Teruya, Kenta; Akaji, Kenichi

    2017-06-15

    Synthesis and evaluation of new scaffold phenylisoserine derivatives connected with the essential functional groups against SARS CoV 3CL protease are described. The phenylisoserine backbone was found by simulation on GOLD software and the structure activity relationship study of phenylisoserine derivatives gave SK80 with an IC50 value of 43μM against SARS CoV 3CL R188I mutant protease. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Discovery, Synthesis, And Structure-Based Optimization of a Series of N-(tert-Butyl)-2-(N-arylamido)-2-(pyridin-3-yl) Acetamides (ML188) as Potent Noncovalent Small Molecule Inhibitors of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CL Protease

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, Jon [Vanderbilt Univ., Nashville, TN (United States); Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN (United States); Grum-Tokars, Valerie [Northwestern Univ., Chicago, IL (United States); Zhou, Ya [Vanderbilt Univ., Nashville, TN (United States); Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN (United States); Turlington, Mark [Vanderbilt Univ., Nashville, TN (United States); Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN (United States); Saldanha, S. Adrian [Sripps Research Inst. Molecular Screening Center, Jupiter, FL (United States); Chase, Peter [Sripps Research Inst. Molecular Screening Center, Jupiter, FL (United States); Eggler, Aimee [Purdue Univ., West Lafayette, IN (United States); Dawson, Eric S. [Vanderbilt Univ., Nashville, TN (United States); Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN (United States); Baez-Santos, Yahira M. [Purdue Univ., West Lafayette, IN (United States); Tomar, Sakshi [Purdue Univ., West Lafayette, IN (United States); Mielech, Anna M. [Loyola Univ. Medical Center, Maywood, IL (United States); Baker, Susan C. [Loyola Univ. Medical Center, Maywood, IL (United States); Lindsley, Craig W. [Vanderbilt Univ., Nashville, TN (United States); Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN (United States); Hodder, Peter [Sripps Research Inst. Molecular Screening Center, Jupiter, FL (United States); Mesecar, Andrew [Purdue Univ., West Lafayette, IN (United States); Stauffer, Shaun R. [Vanderbilt Univ., Nashville, TN (United States); Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN (United States)

    2012-12-11

    A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). But, unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure–activity relationships within S1', S1, and S2enzyme binding pockets. Moreover, the X-ray structure of SARS-CoV 3CLpro bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action.

  6. Pains and Gains from China’s Experiences with Emerging Epidemics: From SARS to H7N9

    Directory of Open Access Journals (Sweden)

    Pengfei Wei

    2016-01-01

    Full Text Available Over the recent decades, China experienced several emerging virus outbreaks including those caused by the severe acute respiratory syndrome- (SARS- coronavirus (Cov, H5N1 virus, and H7N9 virus. The SARS tragedy revealed faults in China’s infectious disease prevention system, propelling the Chinese government to enact reforms that enabled better combating of the subsequent H1N1 and H7N9 avian flu epidemics. The system is buttressed by three fundamental, mutually reinforcing components: (1 enduring government administration reforms, including legislation establishing a unified public health emergency management system; (2 prioritized funding for biotechnology and biomedicine industrialization, especially in the areas of pathogen identification, drug production, and the development of vaccines and diagnostics; and (3 increasing investment for public health and establishment of a rapid-response infectious diseases prevention and control system. China is now using its hard-gained experience to support the fight against Ebola in Africa and the Middle East Respiratory Syndrome in its own country.

  7. The inhibitory effect of Chinese herb on SARS virus infection

    Institute of Scientific and Technical Information of China (English)

    Rika; Furuta; Jyunichi; Fujisawa; Toshio; Hattori

    2005-01-01

    [Subject]Severe acute respiratory syndrome(SARS)is a contagious atypical pneumonia with a high mortality rate.SARS coronavirus(SARS-CoV)is the pathogenof SARS.We established SARS-CoVS/HIVpseudotyped(SHP)virussystemandthe cell fusion assay systemto screeninhibitors for entry of SARS-CoV.[Materials and methods]SHPor VSV-Gpseudotype(VHP)virus was made bytransfecting pCMVΔR8·2,pHR’CMV-Luc and pCMV/R-SARS-S or pMDGplasmids into293Tcells.5ng p24of SHPor VHPvirus was addedfor eachinfec-tion.Twelve Chinese herbs,wh...

  8. Human cell tropism and innate immune system interactions of human respiratory coronavirus EMC compared to those of severe acute respiratory syndrome coronavirus.

    Science.gov (United States)

    Zielecki, Florian; Weber, Michaela; Eickmann, Markus; Spiegelberg, Larissa; Zaki, Ali Moh; Matrosovich, Mikhail; Becker, Stephan; Weber, Friedemann

    2013-05-01

    Infections with human coronavirus EMC (HCoV-EMC) are associated with severe pneumonia. We demonstrate that HCoV-EMC resembles severe acute respiratory syndrome coronavirus (SARS-CoV) in productively infecting primary and continuous cells of the human airways and in preventing the induction of interferon regulatory factor 3 (IRF-3)-mediated antiviral alpha/beta interferon (IFN-α/β) responses. However, HCoV-EMC was markedly more sensitive to the antiviral state established by ectopic IFN. Thus, HCoV-EMC can utilize a broad range of human cell substrates and suppress IFN induction, but it does not reach the IFN resistance of SARS-CoV.

  9. Virtual screening for SARS-CoV protease based on KZ7088 pharmacophore points.

    Science.gov (United States)

    Sirois, Suzanne; Wei, Dong-Qing; Du, Qishi; Chou, Kuo-Chen

    2004-01-01

    Pharmacophore modeling can provide valuable insight into ligand-receptor interactions. It can also be used in 3D (dimensional) database searching for potentially finding biologically active compounds and providing new research ideas and directions for drug-discovery projects. To stimulate the structure-based drug design against SARS (severe acute respiratory syndrome), a pharmacophore search was conducted over 3.6 millions of compounds based on the atomic coordinates of the complex obtained by docking KZ7088 (a derivative of AG7088) to SARS CoV M(pro) (coronavirus main proteinase), as reportedly recently (Chou, K. C.; Wei, D. Q.; Zhong, W. Z. Biochem. Biophys. Res. Commun. 2003, 308, 148-151). It has been found that, of the 3.6 millions of compounds screened, 0.07% are with the score satisfying five of the six pharmacophore points. Moreover, each of the hit compounds has been evaluated for druggability according to 13 metrics based on physical, chemical, and structural properties. Of the 0.07% compounds thus retrieved, 17% have a perfect score of 1.0; while 23% with one druggable rule violation, 13% two violations, and 47% more than two violations. If the criterion for druggability is set at a maximum allowance of two rule violations, we obtain that only about 0.03% of the compounds screened are worthy of further tests by experiments. These findings will significantly narrow down the search scope for potential compounds, saving substantial time and money. Finally, the featured templates derived from the current study will also be very useful for guiding the design and synthesis of effective drugs for SARS therapy.

  10. Reovirus, isolated from SARS patients

    Institute of Scientific and Technical Information of China (English)

    DUAN Qing; SONG Lihua; GAN Yonghua; TAN Hua; JIN Baofeng; LI Huiyan; ZUO Tingting; CHEN Dehui; ZHANG Xuemin; ZHU Hong; YANG Yi; LI Weihua; ZHOU Yusen; HE Jun; HE Kun; ZHANG Haojie; ZHOU Tao

    2003-01-01

    Beijing has been severely affected by SARS, and SARS-associated coronavirus has been confirmed as its cause. However, clinical and experimental evidence implicates the possibility of co-infection. In this report, reovirus was isolated from throat swabs of SARS patients, including the first case in Beijing andher mother. Identification with the electron microscopy revealed the characteristic features of reovirus. 24 of 38 samples from other SARS cases were found to have serologic responses to the reovirus. Primers designed for reovirus have amplified several fragments of DNA, one of which was sequenced (S2 gene fragment), which indicates it as a unique reovirus (orthoreovirus). Preliminary animal experiment showed that inoculation of the reovirus in mice caused death with atypical pneumonia. Nevertheless, the association of reovirus with SARS outbreak requires to be further investigated.

  11. Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture

    NARCIS (Netherlands)

    A.H. de Wilde (Adriaan); D. Jochmans (Dirk); C.C. Posthuma (Clara); J.C. Zevenhoven-Dobbe (Jessika); S. van Nieuwkoop (Stefan); T.M. Bestebroer (Theo); B.G. van den Hoogen (Bernadette); J. Neyts; E.J. Snijder (Eric)

    2014-01-01

    textabstractCoronaviruses can cause respiratory and enteric disease in a wide variety of human and animal hosts. The 2003 outbreak of severe acute respiratory syndrome (SARS) first demonstrated the potentially lethal consequences of zoonotic coronavirus infections in humans. In 2012, a similar previ

  12. Genome organization of the SARS-CoV

    DEFF Research Database (Denmark)

    Xu, Jing; Hu, Jianfei; Wang, Jing;

    2003-01-01

    Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or devel...

  13. SARS Basics

    Science.gov (United States)

    ... and Resources Related Links Clinician Registry Travelers' Health SARS Basics Fact Sheet Language: English Español (Spanish) Format: ... 3 pages] SARS [3 pages] SARS [3 pages] SARS? Severe acute respiratory syndrome (SARS) is a viral ...

  14. Elevated plasma surfactant protein D (SP-D) levels and a direct correlation with anti-severe acute respiratory syndrome coronavirus-specific IgG antibody in SARS patients

    DEFF Research Database (Denmark)

    Wu, Y P; Liu, Z H; Wei, R

    2009-01-01

    Pulmonary SP-D is a defence lectin promoting clearance of viral infections. SP-D is recognized to bind the S protein of SARS-CoV and enhance phagocytosis. Moreover, systemic SP-D is widely used as a biomarker of alveolar integrity. We investigated the relation between plasma SP-D, SARS-type pneum...

  15. Human coronavirus EMC is not the same as severe acute respiratory syndrome coronavirus.

    Science.gov (United States)

    Perlman, Stanley; Zhao, Jincun

    2013-01-15

    A newly identified betacoronavirus, human coronavirus EMC (HCoV-EMC), has been isolated from several patients with respiratory and renal disease in the Middle East. While only a few infected patients have been identified, the mortality of the infection is greater than 50%. Like its better-known cousin severe acute respiratory syndrome coronavirus (SARS-CoV), HCoV-EMC appears to have originated from bats. In a recent article in mBio, Müller et al. described several important differences between the two viruses [M. A. Müller et al., mBio 3(6):e00515-12, 2012, doi:10.1128/mBio.00515-12]. Unlike SARS-CoV, HCoV-EMC can directly infect bat cells. As important, HCoV-EMC does not enter cells using the SARS-CoV receptor, human angiotensin-converting receptor-2 (hACE2). These results provide a strong incentive for identifying the host cell receptor used by HCoV-EMC. Identification of the receptor will provide insight into the pathogenesis of pulmonary and renal disease and may also suggest novel therapeutic interventions.

  16. Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site.

    Science.gov (United States)

    Churchward, Gordon; Bates, Christopher; Gusa, Asiya A; Stringer, Virginia; Scott, June R

    2009-02-01

    The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) produces many virulence factors that are regulated by the two-component signal transduction system CovRS (CsrRS). Dissemination of GAS infection originating at the skin has been shown to require production of streptokinase, whose transcription is repressed by CovR. In this work we have studied the interaction of CovR and phosphorylated CovR (CovR-P) with the promoter for streptokinase, Pska. We found that, in contrast to the other CovR-repressed promoters, Pska regulation by CovR occurs through binding at a single ATTARA consensus binding sequence (CB) that overlaps the -10 region of the promoter. Binding of CovR to other nearby consensus sequences occurs upon phosphorylation of the protein, but these other CBs do not contribute to the regulation of Pska by CovR. Thus, binding at a specific site does not necessarily indicate that the site is involved in regulation by CovR. In addition, at Pska, CovR binding to the different sites does not appear to involve cooperative interactions, which simplifies the analysis of CovR binding and gives us insight into the modes of interaction that occur between CovR and its specific DNA-binding sites. Finally, the observation that regulation of transcription from Pska occurs at a very low concentration of phosphorylated CovR may have important implications for the regulation of virulence gene expression during GAS infection.

  17. Alphacoronaviruses Detected in French Bats Are Phylogeographically Linked to Coronaviruses of European Bats.

    Science.gov (United States)

    Goffard, Anne; Demanche, Christine; Arthur, Laurent; Pinçon, Claire; Michaux, Johan; Dubuisson, Jean

    2015-12-02

    Bats are a reservoir for a diverse range of viruses, including coronaviruses (CoVs). To determine the presence of CoVs in French bats, fecal samples were collected between July and August of 2014 from four bat species in seven different locations around the city of Bourges in France. We present for the first time the presence of alpha-CoVs in French Pipistrellus pipistrellus bat species with an estimated prevalence of 4.2%. Based on the analysis of a fragment of the RNA-dependent RNA polymerase (RdRp) gene, phylogenetic analyses show that alpha-CoVs sequences detected in French bats are closely related to other European bat alpha-CoVs. Phylogeographic analyses of RdRp sequences show that several CoVs strains circulate in European bats: (i) old strains detected that have probably diverged a long time ago and are detected in different bat subspecies; (ii) strains detected in Myotis and Pipistrellus bat species that have more recently diverged. Our findings support previous observations describing the complexity of the detected CoVs in bats worldwide.

  18. Alphacoronaviruses Detected in French Bats Are Phylogeographically Linked to Coronaviruses of European Bats

    Science.gov (United States)

    Goffard, Anne; Demanche, Christine; Arthur, Laurent; Pinçon, Claire; Michaux, Johan; Dubuisson, Jean

    2015-01-01

    Bats are a reservoir for a diverse range of viruses, including coronaviruses (CoVs). To determine the presence of CoVs in French bats, fecal samples were collected between July and August of 2014 from four bat species in seven different locations around the city of Bourges in France. We present for the first time the presence of alpha-CoVs in French Pipistrellus pipistrellus bat species with an estimated prevalence of 4.2%. Based on the analysis of a fragment of the RNA-dependent RNA polymerase (RdRp) gene, phylogenetic analyses show that alpha-CoVs sequences detected in French bats are closely related to other European bat alpha-CoVs. Phylogeographic analyses of RdRp sequences show that several CoVs strains circulate in European bats: (i) old strains detected that have probably diverged a long time ago and are detected in different bat subspecies; (ii) strains detected in Myotis and Pipistrellus bat species that have more recently diverged. Our findings support previous observations describing the complexity of the detected CoVs in bats worldwide. PMID:26633467

  19. Alphacoronaviruses Detected in French Bats Are Phylogeographically Linked to Coronaviruses of European Bats

    Directory of Open Access Journals (Sweden)

    Anne Goffard

    2015-12-01

    Full Text Available Bats are a reservoir for a diverse range of viruses, including coronaviruses (CoVs. To determine the presence of CoVs in French bats, fecal samples were collected between July and August of 2014 from four bat species in seven different locations around the city of Bourges in France. We present for the first time the presence of alpha-CoVs in French Pipistrellus pipistrellus bat species with an estimated prevalence of 4.2%. Based on the analysis of a fragment of the RNA-dependent RNA polymerase (RdRp gene, phylogenetic analyses show that alpha-CoVs sequences detected in French bats are closely related to other European bat alpha-CoVs. Phylogeographic analyses of RdRp sequences show that several CoVs strains circulate in European bats: (i old strains detected that have probably diverged a long time ago and are detected in different bat subspecies; (ii strains detected in Myotis and Pipistrellus bat species that have more recently diverged. Our findings support previous observations describing the complexity of the detected CoVs in bats worldwide.

  20. T-cell immunity of SARS-CoV: Implications for vaccine development against MERS-CoV.

    Science.gov (United States)

    Liu, William J; Zhao, Min; Liu, Kefang; Xu, Kun; Wong, Gary; Tan, Wenjie; Gao, George F

    2017-01-01

    Over 12 years have elapsed since severe acute respiratory syndrome (SARS) triggered the first global alert for coronavirus infections. Virus transmission in humans was quickly halted by public health measures and human infections of SARS coronavirus (SARS-CoV) have not been observed since. However, other coronaviruses still pose a continuous threat to human health, as exemplified by the recent emergence of Middle East respiratory syndrome (MERS) in humans. The work on SARS-CoV widens our knowledge on the epidemiology, pathophysiology and immunology of coronaviruses and may shed light on MERS coronavirus (MERS-CoV). It has been confirmed that T-cell immunity plays an important role in recovery from SARS-CoV infection. Herein, we summarize T-cell immunological studies of SARS-CoV and discuss the potential cross-reactivity of the SARS-CoV-specific immunity against MERS-CoV, which may provide useful recommendations for the development of broad-spectrum vaccines against coronavirus infections. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. The nucleocapsid protein of human coronavirus NL63.

    Directory of Open Access Journals (Sweden)

    Kaja Zuwała

    Full Text Available Human coronavirus (HCoV NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E. Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.

  2. Phylogeny of SARS-CoV as inferred from complete genome comparison

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    SARS-CoV, as the pathogeny of severe acute respiratory syndrome (SARS), is a mystery that the origin of the virus is still unknown even a few isolates of the virus were completely sequenced. To explore the genesis of SARS-CoV, the FDOD method previously developed by us was applied to comparing complete genomes from 12 SARS-CoV isolates to those from 12 previously identified coronaviruses and an unrooted phylogenetic tree was constructed. Our results show that all SARS-CoV isolates were clustered into a clique and previously identified coronaviruses formed the other clique. Meanwhile, the three groups of coronaviruses depart from each other clearly in our tree that is consistent with the results of prevenient papers. Differently, from the topology of the phylogenetic tree we found that SARS-CoV is more close to group 1 within genus coronavirus. The topology map also shows that the 12 SARS-CoV isolates may be divided into two groups determined by the association with the SARS-CoV from the Hotel M in Hong Kong that may give some information about the infectious relationship of the SARS.

  3. AIdentification of encoding proteins related to SARS-CoV

    Institute of Scientific and Technical Information of China (English)

    MEI Hu; SUN Lili; ZHOU Yuan; XIONG Qing; LI Zhiliang

    2004-01-01

    By sampling 100 encoding proteins from SARS-coronavirus (SARS-CoV, NC 004718) and other six coronaviruses and selecting 23 variables through stepwise multiple regression (SMR) from 172 variables, the multiple linear regression (MLR) model was established with good results of the quantitative modelling correlation coefficient R2 = 0.645 and the cross-validation correlation coefficient 0.375. After removing 4 outliers, the quantitative modelling and cross-validation correlation coefficients were R2 = 0.743 and R2CV=0.543, respectively.

  4. Cytoplasmic tail of coronavirus spike protein has intracellular targeting signals

    Indian Academy of Sciences (India)

    JIBIN SADASIVAN; MANMEET SINGH; JAYASRI DAS SARMA

    2017-06-01

    Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is localized in the ER or ERGIC compartment and OC43 spike protein is predominantly localized in thelysosome. Differential localization can be explained by signal sequence. The sequence alignment using Clustal Wshows that the signal sequence present at the cytoplasmic tail plays an important role in spike protein localization. Aunique GYQEL motif is identified at the cytoplasmic terminal of OC43 spike protein which helps in localization in thelysosome, and a novel KLHYT motif is identified in the cytoplasmic tail of SARS spike protein which helps in ER orERGIC localization. This study sheds some light on the role of cytoplasmic tail of spike protein in cell-to-cell fusion,coronavirus host cell fusion and subsequent pathogenicity.

  5. Coronavirus spike-receptor interactions

    NARCIS (Netherlands)

    Mou, H.

    2015-01-01

    Coronaviruses cause important diseases in humans and animals. Coronavirus infection starts with the virus binding with its spike proteins to molecules present on the surface of host cells that act as receptors. This spike-receptor interaction is highly specific and determines the virus’ cell, tissue

  6. Simultaneous isolation of emm89-type Streptococcus pyogenes strains with a wild-type or mutated covS gene from a single streptococcal toxic shock syndrome patient.

    Science.gov (United States)

    Masuno, Katsuaki; Okada, Ryo; Zhang, Yan; Isaka, Masanori; Tatsuno, Ichiro; Shibata, Shinichiro; Hasegawa, Tadao

    2014-04-01

    Streptococcal toxic shock syndrome (STSS) is a re-emerging infectious disease in many developed countries. Recent studies have suggested that mutations in CovRS, a two-component regulatory system in Streptococcus pyogenes, play important roles in the pathogenesis of STSS. However, in vivo evidence of the significance of CovRS in human infections has not been fully demonstrated. We investigated five S. pyogenes strains isolated simultaneously from the pharynx, sputum, knee joint, cerebrospinal fluid and blood of a single STSS patient. All were emm89-type strains, and multilocus sequence typing (MLST) analysis revealed that the strains of pharynx and blood were isogenic. The growth rates of the strains from pharynx and sputum were faster than those of the other strains. Protein profiles of the culture supernatants of strains from the pharynx and sputum were also different from those of the other strains. Sequence analyses revealed that strains from the knee joint, cerebrospinal fluid and blood contained a single nucleotide difference in the covS coding region, resulting in one amino acid change, compared with the other strains. Introduction of a plasmid containing the covS gene from the pharynx strain to the blood strain increased the production of SpeB protein. This suggests that the one amino acid alteration in CovS was relevant to pathogenesis. This report supports the idea that mutated CovS plays important roles in vivo in the dissemination of S. pyogenes from the upper respiratory tract of human to aseptic tissues such as blood and cerebrospinal fluid.

  7. Stability of bovine coronavirus on lettuce surfaces under household refrigeration conditions.

    Science.gov (United States)

    Mullis, Lisa; Saif, Linda J; Zhang, Yongbin; Zhang, Xuming; Azevedo, Marli S P

    2012-05-01

    Fecal suspensions with an aerosol route of transmission were responsible for a cluster of severe acute respiratory syndrome (SARS) cases in 2003 in Hong Kong. Based on that event, the World Health Organization recommended that research be implemented to define modes of transmission of SARS coronavirus through sewage, feces, food and water. Environmental studies have shown that animal coronaviruses remain infectious in water and sewage for up to a year depending on the temperature and humidity. In this study, we examined coronavirus stability on lettuce surfaces. A cell culture adapted bovine coronavirus, diluted in growth media or in bovine fecal suspensions to simulate fecal contamination was used to spike romaine lettuce. qRT-PCR detected viral RNA copy number ranging from 6.6 × 10⁴ to 1.7 × 10⁶ throughout the experimental period of 30 days. Whereas infectious viruses were detected for at least 14 days, the amount of infectious virus varied, depending upon the diluent used for spiking the lettuce. UV and confocal microscopic observation indicated attachment of residual labeled virions to the lettuce surface after the elution procedure, suggesting that rates of inactivation or detection of the virus may be underestimated. Thus, it is possible that contaminated vegetables may be potential vehicles for coronavirus zoonotic transmission to humans.

  8. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC.

    Science.gov (United States)

    Raj, V Stalin; Mou, Huihui; Smits, Saskia L; Dekkers, Dick H W; Müller, Marcel A; Dijkman, Ronald; Muth, Doreen; Demmers, Jeroen A A; Zaki, Ali; Fouchier, Ron A M; Thiel, Volker; Drosten, Christian; Rottier, Peter J M; Osterhaus, Albert D M E; Bosch, Berend Jan; Haagmans, Bart L

    2013-03-14

    Most human coronaviruses cause mild upper respiratory tract disease but may be associated with more severe pulmonary disease in immunocompromised individuals. However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread. Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection. Viral genome analysis revealed close relatedness to coronaviruses found in bats. Here we identify dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for hCoV-EMC. DPP4 specifically co-purified with the receptor-binding S1 domain of the hCoV-EMC spike protein from lysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibited hCoV-EMC infection of primary human bronchial epithelial cells and Huh-7 cells. Expression of human and bat (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells enabled infection by hCoV-EMC. The use of the evolutionarily conserved DPP4 protein from different species as a functional receptor provides clues about the host range potential of hCoV-EMC. In addition, it will contribute critically to our understanding of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention strategies.

  9. Unraveling the complexities of the interferon response during SARS-CoV infection

    NARCIS (Netherlands)

    A. de Lang (Anna); T. Baas (Tracey); S.L. Smits (Saskia); M.G. Katze (Michael); A.D.M.E. Osterhaus (Albert); B.L. Haagmans (Bart)

    2009-01-01

    textabstractViruses employ different strategies to circumvent the antiviral actions of the innate immune response. SARS coronavirus (SARS-CoV), a virus that causes severe lung damage encodes an array of proteins able to inhibit induction and signaling of type-I interferons. However, recent studies h

  10. Unraveling the complexities of the interferon response during SARS-CoV infection

    NARCIS (Netherlands)

    A. de Lang (Anna); T. Baas (Tracey); S.L. Smits (Saskia); M.G. Katze (Michael); A.D.M.E. Osterhaus (Albert); B.L. Haagmans (Bart)

    2009-01-01

    textabstractViruses employ different strategies to circumvent the antiviral actions of the innate immune response. SARS coronavirus (SARS-CoV), a virus that causes severe lung damage encodes an array of proteins able to inhibit induction and signaling of type-I interferons. However, recent studies

  11. Evolution and Variation of the SARS-CoV Genome

    Institute of Scientific and Technical Information of China (English)

    Jianfei Hu; Zizhang Zhang; Wei Wei; Songgang Li; Jun Wang; Jian Wang; Jun Yu; Huanming Yang; Jing Wang; Jing Xu; Wei Li; Yujun Han; Yan Li; Jia Ji; Jia Ye; Zhao Xu

    2003-01-01

    Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARSCoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us.

  12. Severe acute respiratory syndrome coronavirus persistence in Vero cells

    Institute of Scientific and Technical Information of China (English)

    Gustavo Palacios; Omar Jabado; Neil Renwick; Thomas Briese; W. Ian Lipkin

    2005-01-01

    Background Several coronaviruses establish persistent infections in vitro and in vivo, however it is unknown whether persistence is a feature of the severe acute respiratory syndorme coronavirus (SARS-CoV) life cycle. This study was conducted to investigate viral persistence.Methods We inoculated confluent monolayers of Vero cells with SARS-CoV at a multiplicity of infection of 0.1 TCID50 and passaged the remaining cells every 4 to 8 days for a total of 11 passages. Virus was titrated at each passage by limited dilution assay and nucleocapsid antigen was detected by Western blot and immunofluoresence assays. The presence of viral particles in passage 11 cells was assessed by electron microscopy. Changes in viral genomic sequences during persistent infection were examined by DNA sequencing. Results Cytopathic effect was extensive after initial inoculation but diminished with serial passages. Infectious virus was detected after each passage and viral growth curves were identical for parental virus stock and virus obtained from passage 11 cells. Nucleocapsid antigen was detected in the majority of cells after initial inoculation but in only 10%-40% of cells at passages 2-11. Electron microscopy confirmed the presence of viral particles in passage 11 cells. Sequence analysis at passage 11 revealed fixed mutations in the spike (S) gene and ORFs 7a-8b but not in the nucleocapsid (N) gene. Conclusions SARS-CoV can establish a persistent infection in vitro. The mechanism for viral persistence is consistent with the formation of a carrier culture whereby a limited number of cells are infected with each round of virus replication and release. Persistence is associated with selected mutations in the SARS-CoV genome. This model may provide insight into SARS-related lung pathology and mechanisms by which humans and animals can serve as reservoirs for infection.

  13. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

    Directory of Open Access Journals (Sweden)

    Christine Burkard

    2014-11-01

    Full Text Available Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs. Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV. Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

  14. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

    Science.gov (United States)

    Burkard, Christine; Verheije, Monique H; Wicht, Oliver; van Kasteren, Sander I; van Kuppeveld, Frank J; Haagmans, Bart L; Pelkmans, Lucas; Rottier, Peter J M; Bosch, Berend Jan; de Haan, Cornelis A M

    2014-11-01

    Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

  15. Severe acute respiratory syndrome (SARS) in Hong Kong.

    Science.gov (United States)

    Tsang, Kenneth W; Mok, Thomas Y; Wong, Poon C; Ooi, Gaik C

    2003-09-01

    Severe acute respiratory syndrome (SARS) is a recently recognized and highly contagious pneumonic illness, caused by a novel coronavirus. While developments in diagnostic, clinical and other aspects of SARS research are well underway, there is still great difficulty for frontline clinicians as validated rapid diagnostic tests or effective treatment regimens are lacking. This article attempts to summarize some of the recent developments in this newly recognized condition from the Asia Pacific perspective.

  16. Activation of the SMU.1882 transcription by CovR in Streptococcus mutans.

    Directory of Open Access Journals (Sweden)

    Patrick Chong

    Full Text Available In Streptococcus mutans, the global response regulator CovR plays an important role in biofilm formation, stress-tolerance response, and caries production. We have previously shown that CovR acts as a transcriptional repressor by binding to the upstream promoter regions of its target genes. Here, we report that in vivo, CovR activates the transcription of SMU.1882, which encodes a small peptide containing a double-glycine motif. We also show that SMU.1882 is transcriptionally linked to comA that encodes a putative ABC transporter protein. Several genes from man gene clusters that encode mannose phosphotranferase system flank SMU.1882 -comA genes. Genomic comparison with other streptococci indicates that SMU.1882 is uniquely present in S. mutans, while the man operon is conserved among all streptococci, suggesting that a genetic rearrangement might have taken place at this locus. With the use of a transcriptional reporter system and semi-quantitative RT-PCR, we demonstrated the transcriptional regulation of SMU.1882 by CovR. In vitro gel shift and DNase I foot-printing analyses with purified CovR suggest that CovR binds to a large region surrounding the -10 region of the P(1882. Using this information and comparing with other CovR regulated promoters, we have developed a putative consensus binding sequence for CovR. Although CovR binds to P(1882, in vitro experiments using purified S. mutans RpoD, E. coli RNA polymerase, and CovR did not activate transcription from this promoter. Thus, we speculate that in vivo, CovR may interfere with the binding of a repressor or requires a cofactor.

  17. Coronavirus avian infectious bronchitis virus

    National Research Council Canada - National Science Library

    Cavanagh, Dave

    2007-01-01

    Infectious bronchitis virus (IBV), the coronavirus of the chicken (Gallus gallus), is one of the foremost causes of economic loss within the poultry industry, affecting the performance of both meat-type and egg-laying birds...

  18. Severe acute respiratory syndrome--a new coronavirus from the Chinese dragon's lair.

    Science.gov (United States)

    Knudsen, T B; Kledal, T N; Andersen, O; Eugen-Olsen, J; Kristiansen, T B

    2003-09-01

    The recent identification of a novel clinical entity, the severe acute respiratory syndrome (SARS), the rapid subsequent spread and case fatality rates of 14-15% have prompted a massive international collaborative investigation facilitated by a network of laboratories established by the World Health Organization (WHO). As SARS has the potential of becoming the first pandemic of the new millennium, a global warning by the WHO was issued on 12 March 2003. The disease, which is believed to have its origin in the Chinese Guangdong province, spread from Hong Kong via international airports to its current worldwide distribution. The concerted efforts of a globally united scientific community have led to the independent isolation and identification of a novel coronavirus from SARS patients by several groups. The extraordinarily rapid isolation of a causative agent of this newly emerged infectious disease constitutes an unprecedented scientific achievement. The main scope of the article is to provide the clinician with an overview of the natural history, epidemiology and clinical characteristics of SARS. On the basis of the recently published viral genome and structural features common to the members of the coronavirus family, a model for host cell-virus interaction and possible targets for antiviral drugs are presented. The epidemiological consequences of introducing a novel pathogen in a previously unexposed population and the origin and evolution of a new and more pathogenic strain of coronavirus are discussed.

  19. Generalized interpretation scheme for arbitrary HR InSAR image pairs

    Science.gov (United States)

    Boldt, Markus; Thiele, Antje; Schulz, Karsten

    2013-10-01

    Land cover classification of remote sensing imagery is an important topic of research. For example, different applications require precise and fast information about the land cover of the imaged scenery (e.g., disaster management and change detection). Focusing on high resolution (HR) spaceborne remote sensing imagery, the user has the choice between passive and active sensor systems. Passive systems, such as multispectral sensors, have the disadvantage of being dependent from weather influences (fog, dust, clouds, etc.) and time of day, since they work in the visible part of the electromagnetic spectrum. Here, active systems like Synthetic Aperture Radar (SAR) provide improved capabilities. As an interactive method analyzing HR InSAR image pairs, the CovAmCohTM method was introduced in former studies. CovAmCoh represents the joint analysis of locality (coefficient of variation - Cov), backscatter (amplitude - Am) and temporal stability (coherence - Coh). It delivers information on physical backscatter characteristics of imaged scene objects or structures and provides the opportunity to detect different classes of land cover (e.g., urban, rural, infrastructure and activity areas). As example, railway tracks are easily distinguishable from other infrastructure due to their characteristic bluish coloring caused by the gravel between the sleepers. In consequence, imaged objects or structures have a characteristic appearance in CovAmCoh images which allows the development of classification rules. In this paper, a generalized interpretation scheme for arbitrary InSAR image pairs using the CovAmCoh method is proposed. This scheme bases on analyzing the information content of typical CovAmCoh imagery using the semisupervised k-means clustering. It is shown that eight classes model the main local information content of CovAmCoh images sufficiently and can be used as basis for a classification scheme.

  20. Coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines.

    Directory of Open Access Journals (Sweden)

    Roland Züst

    2007-08-01

    Full Text Available Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1. In cell culture, nsp1 of mouse hepatitis virus (MHV, like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines.

  1. SARS-CoV ORF1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets.

    Science.gov (United States)

    Subissi, Lorenzo; Imbert, Isabelle; Ferron, François; Collet, Axelle; Coutard, Bruno; Decroly, Etienne; Canard, Bruno

    2014-01-01

    The SARS (severe acute respiratory syndrome) pandemic caused ten years ago by the SARS-coronavirus (SARS-CoV) has stimulated a number of studies on the molecular biology of coronaviruses. This research has provided significant new insight into many mechanisms used by the coronavirus replication-transcription complex (RTC). The RTC directs and coordinates processes in order to replicate and transcribe the coronavirus genome, a single-stranded, positive-sense RNA of outstanding length (∼27-32kilobases). Here, we review the up-to-date knowledge on SARS-CoV replicative enzymes encoded in the ORF1b, i.e., the main RNA-dependent RNA polymerase (nsp12), the helicase/triphosphatase (nsp13), two unusual ribonucleases (nsp14, nsp15) and RNA-cap methyltransferases (nsp14, nsp16). We also review how these enzymes co-operate with other viral co-factors (nsp7, nsp8, and nsp10) to regulate their activity. These last ten years of research on SARS-CoV have considerably contributed to unravel structural and functional details of one of the most fascinating replication/transcription machineries of the RNA virus world. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses". Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Functional genomics highlights differential induction of antiviral pathways in the lungs of SARS-CoV-infected macaques.

    NARCIS (Netherlands)

    A. de Lang (Anna); T. Baas (Tracey); T.H. Teal (Thomas); L.M.E. Leijten (Lonneke); B. Rain (Brandon); A.D.M.E. Osterhaus (Albert); B.L. Haagmans (Bart); M.G. Katze (Michael)

    2007-01-01

    textabstractThe pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is likely mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. Using functional genomics, we analyzed early host responses to SARS-CoV infection in the lungs o

  3. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication

    Science.gov (United States)

    Kindler, Eveline; Gil-Cruz, Cristina; Spanier, Julia; Li, Yize; Wilhelm, Jochen; Rabouw, Huib H.; Züst, Roland; Marti, Sabrina; Habjan, Matthias; Cervantes-Barragan, Luisa; Elliot, Ruth; Karl, Nadja; Gaughan, Christina; Silverman, Robert H.; Keller, Markus; Ludewig, Burkhard; Bergmann, Cornelia C.; Ziebuhr, John; Kalinke, Ulrich

    2017-01-01

    Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis–within the replicase complex—suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses. PMID:28158275

  4. Involvement of Autophagy in Coronavirus Replication

    Directory of Open Access Journals (Sweden)

    Paul Britton

    2012-11-01

    Full Text Available Coronaviruses are single stranded, positive sense RNA viruses, which induce the rearrangement of cellular membranes upon infection of a host cell. This provides the virus with a platform for the assembly of viral replication complexes, improving efficiency of RNA synthesis. The membranes observed in coronavirus infected cells include double membrane vesicles. By nature of their double membrane, these vesicles resemble cellular autophagosomes, generated during the cellular autophagy pathway. In addition, coronavirus infection has been demonstrated to induce autophagy. Here we review current knowledge of coronavirus induced membrane rearrangements and the involvement of autophagy or autophagy protein microtubule associated protein 1B light chain 3 (LC3 in coronavirus replication.

  5. Multiple Sequence Alignment of the M Proteinin SARS—Associated and Other Known Coronaviruses

    Institute of Scientific and Technical Information of China (English)

    史定华; 周晖杰; 王斌宾; 顾燕红; 王翼飞

    2003-01-01

    In this paper, we report a multiple sequence alignment result on the basis of 10 amino acid sequences of the M protein,which come from different coronaviruses (4 SARS-associated and 6 others known). The alignment model was based on the profile HMM (Hidden Markov Model), and the model training was implemented through the SAHMM (Self-Adapting Hidden Markov Model)software developed by the authors.

  6. CovR-controlled global regulation of gene expression in Streptococcus mutans.

    Directory of Open Access Journals (Sweden)

    Alexander Dmitriev

    Full Text Available CovR/S is a two-component signal transduction system (TCS that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (∼6.5% of the genome were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI. Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus.

  7. Bilateral Entry and Release of Middle East Respiratory Syndrome Coronavirus Induces Profound Apoptosis of Human Bronchial Epithelial Cells

    Science.gov (United States)

    Tao, Xinrong; Hill, Terence E.; Morimoto, Chikao; Peters, Clarence J.; Ksiazek, Thomas G.

    2013-01-01

    The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) infects human bronchial epithelial Calu-3 cells. Unlike severe acute respiratory syndrome (SARS)-CoV, which exclusively infects and releases through the apical route, this virus can do so through either side of polarized Calu-3 cells. Infection results in profound apoptosis within 24 h irrespective of its production of titers that are lower than those of SARS-CoV. Together, our results provide new insights into the dissemination and pathogenesis of MERS-CoV and may indicate that the virus differs markedly from SARS-CoV. PMID:23824802

  8. Sequence Analysis and Structural Prediction of the Severe Acute Respiratory Syndrome Coronavirus nsp5

    Institute of Scientific and Technical Information of China (English)

    Jia-Hai LU; Nan-Shan ZHONG; Ding-Mei ZHANG; Guo-Ling WANG; Zhong-Min GUO; Juan LI; Bing-Yan TAN; Li-Ping OU-YANG; Wen-Hua LING; Xin-Bing YU

    2005-01-01

    The non-structural proteins (nsp or replicase proteins) of coronaviruses are relatively conserved and can be effective targets for drugs. Few studies have been conducted into the function of the severe acute respiratory syndrome coronavirus (SARS-CoV) nsp5. In this study, bioinformatics methods were employed to predict the secondary structure and construct 3-D models of the SARS-CoV GD strain nsp5. Sequencing and sequential comparison was performed to analyze the mutation trend of the polymerase nsp5 gene during the epidemic process using a nucleotide-nucleotide basic local alignment search tool (BLASTN) and a protein-protein basic local alignment search tool (BLASTP). The results indicated that the nsp5 gene was steady during the epidemic process and the protein was homologous with other coronavirus nsp5 proteins. The protein encoded by the nsp5 gene was expressed in COS-7 cells and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This study provided the foundation for further exploration of the protein's biological function, and contributed to the search for anti-SARS-CoV drugs.

  9. Human Coronavirus 229E Remains Infectious on Common Touch Surface Materials.

    Science.gov (United States)

    Warnes, Sarah L; Little, Zoë R; Keevil, C William

    2015-11-10

    The evolution of new and reemerging historic virulent strains of respiratory viruses from animal reservoirs is a significant threat to human health. Inefficient human-to-human transmission of zoonotic strains may initially limit the spread of transmission, but an infection may be contracted by touching contaminated surfaces. Enveloped viruses are often susceptible to environmental stresses, but the human coronaviruses responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) have recently caused increasing concern of contact transmission during outbreaks. We report here that pathogenic human coronavirus 229E remained infectious in a human lung cell culture model following at least 5 days of persistence on a range of common nonbiocidal surface materials, including polytetrafluoroethylene (Teflon; PTFE), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. We have shown previously that noroviruses are destroyed on copper alloy surfaces. In this new study, human coronavirus 229E was rapidly inactivated on a range of copper alloys (within a few minutes for simulated fingertip contamination) and Cu/Zn brasses were very effective at lower copper concentration. Exposure to copper destroyed the viral genomes and irreversibly affected virus morphology, including disintegration of envelope and dispersal of surface spikes. Cu(I) and Cu(II) moieties were responsible for the inactivation, which was enhanced by reactive oxygen species generation on alloy surfaces, resulting in even faster inactivation than was seen with nonenveloped viruses on copper. Consequently, copper alloy surfaces could be employed in communal areas and at any mass gatherings to help reduce transmission of respiratory viruses from contaminated surfaces and protect the public health. Respiratory viruses are responsible for more deaths globally than any other infectious agent. Animal coronaviruses that "host jump" to humans result in

  10. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    Science.gov (United States)

    Irigoyen, Nerea; Firth, Andrew E; Jones, Joshua D; Chung, Betty Y-W; Siddell, Stuart G; Brierley, Ian

    2016-02-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  11. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    Directory of Open Access Journals (Sweden)

    Nerea Irigoyen

    2016-02-01

    Full Text Available Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV, are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59, a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the

  12. Protective Role of Toll-like Receptor 3-Induced Type I Interferon in Murine Coronavirus Infection of Macrophages

    Directory of Open Access Journals (Sweden)

    Sonia Navas-Martin

    2012-05-01

    Full Text Available Toll-like Receptors (TLRs sense viral infections and induce production of type I interferons (IFNs, other cytokines, and chemokines. Viral recognition by TLRs and other pattern recognition receptors (PRRs has been proven to be cell-type specific. Triggering of TLRs with selected ligands can be beneficial against some viral infections. Macrophages are antigen-presenting cells that express TLRs and have a key role in the innate and adaptive immunity against viruses. Coronaviruses (CoVs are single-stranded, positive-sense RNA viruses that cause acute and chronic infections and can productively infect macrophages. Investigation of the interplay between CoVs and PRRs is in its infancy. We assessed the effect of triggering TLR2, TLR3, TLR4, and TLR7 with selected ligands on the susceptibility of the J774A.1 macrophage cell line to infection with murine coronavirus (mouse hepatitis virus, [MHV]. Stimulation of TLR2, TLR4, or TLR7 did not affect MHV production. In contrast, pre-stimulation of TLR3 with polyinosinic-polycytidylic acid (poly I:C hindered MHV infection through induction of IFN-β in macrophages. We demonstrate that activation of TLR3 with the synthetic ligand poly I:C mediates antiviral immunity that diminishes (MHV-A59 or suppresses (MHV-JHM, MHV-3 virus production in macrophages.

  13. Human coronaviruses: insights into environmental resistance and its influence on the development of new antiseptic strategies.

    Science.gov (United States)

    Geller, Chloé; Varbanov, Mihayl; Duval, Raphaël E

    2012-11-12

    The Coronaviridae family, an enveloped RNA virus family, and, more particularly, human coronaviruses (HCoV), were historically known to be responsible for a large portion of common colds and other upper respiratory tract infections. HCoV are now known to be involved in more serious respiratory diseases, i.e. bronchitis, bronchiolitis or pneumonia, especially in young children and neonates, elderly people and immunosuppressed patients. They have also been involved in nosocomial viral infections. In 2002-2003, the outbreak of severe acute respiratory syndrome (SARS), due to a newly discovered coronavirus, the SARS-associated coronavirus (SARS-CoV); led to a new awareness of the medical importance of the Coronaviridae family. This pathogen, responsible for an emerging disease in humans, with high risk of fatal outcome; underline the pressing need for new approaches to the management of the infection, and primarily to its prevention. Another interesting feature of coronaviruses is their potential environmental resistance, despite the accepted fragility of enveloped viruses. Indeed, several studies have described the ability of HCoVs (i.e. HCoV 229E, HCoV OC43 (also known as betacoronavirus 1), NL63, HKU1 or SARS-CoV) to survive in different environmental conditions (e.g. temperature and humidity), on different supports found in hospital settings such as aluminum, sterile sponges or latex surgical gloves or in biological fluids. Finally, taking into account the persisting lack of specific antiviral treatments (there is, in fact, no specific treatment available to fight coronaviruses infections), the Coronaviridae specificities (i.e. pathogenicity, potential environmental resistance) make them a challenging model for the development of efficient means of prevention, as an adapted antisepsis-disinfection, to prevent the environmental spread of such infective agents. This review will summarize current knowledge on the capacity of human coronaviruses to survive in the

  14. Human Coronaviruses: Insights into Environmental Resistance and Its Influence on the Development of New Antiseptic Strategies

    Directory of Open Access Journals (Sweden)

    Mihayl Varbanov

    2012-11-01

    Full Text Available The Coronaviridae family, an enveloped RNA virus family, and, more particularly, human coronaviruses (HCoV, were historically known to be responsible for a large portion of common colds and other upper respiratory tract infections. HCoV are now known to be involved in more serious respiratory diseases, i.e. bronchitis, bronchiolitis or pneumonia, especially in young children and neonates, elderly people and immunosuppressed patients. They have also been involved in nosocomial viral infections. In 2002–2003, the outbreak of severe acute respiratory syndrome (SARS, due to a newly discovered coronavirus, the SARS-associated coronavirus (SARS-CoV; led to a new awareness of the medical importance of the Coronaviridae family. This pathogen, responsible for an emerging disease in humans, with high risk of fatal outcome; underline the pressing need for new approaches to the management of the infection, and primarily to its prevention. Another interesting feature of coronaviruses is their potential environmental resistance, despite the accepted fragility of enveloped viruses. Indeed, several studies have described the ability of HCoVs (i.e. HCoV 229E, HCoV OC43 (also known as betacoronavirus 1, NL63, HKU1 or SARS-CoV to survive in different environmental conditions (e.g. temperature and humidity, on different supports found in hospital settings such as aluminum, sterile sponges or latex surgical gloves or in biological fluids. Finally, taking into account the persisting lack of specific antiviral treatments (there is, in fact, no specific treatment available to fight coronaviruses infections, the Coronaviridae specificities (i.e. pathogenicity, potential environmental resistance make them a challenging model for the development of efficient means of prevention, as an adapted antisepsis-disinfection, to prevent the environmental spread of such infective agents. This review will summarize current knowledge on the capacity of human coronaviruses to

  15. Coronaviruses in polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Bekker, C P; Voorhout, W F; Horzinek, M C; Van der Ende, A; Strous, G J; Rottier, P J

    1995-01-01

    Coronaviruses have a marked tropism for epithelial cells. In this paper the interactions of the porcine transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV-A59) with epithelial cells are compared. Porcine (LLC-PK1) and murine (mTAL) epithelial cells were grown on permeable supp

  16. Altered Lipid Metabolism in Recovered SARS Patients Twelve Years after Infection.

    Science.gov (United States)

    Wu, Qi; Zhou, Lina; Sun, Xin; Yan, Zhongfang; Hu, Chunxiu; Wu, Junping; Xu, Long; Li, Xue; Liu, Huiling; Yin, Peiyuan; Li, Kuan; Zhao, Jieyu; Li, Yanli; Wang, Xiaolin; Li, Yu; Zhang, Qiuyang; Xu, Guowang; Chen, Huaiyong

    2017-08-22

    Severe acute respiratory syndrome-coronavirus (SARS-CoV) and SARS-like coronavirus are a potential threat to global health. However, reviews of the long-term effects of clinical treatments in SARS patients are lacking. Here a total of 25 recovered SARS patients were recruited 12 years after infection. Clinical questionnaire responses and examination findings indicated that the patients had experienced various diseases, including lung susceptibility to infections, tumors, cardiovascular disorders, and abnormal glucose metabolism. As compared to healthy controls, metabolomic analyses identified significant differences in the serum metabolomes of SARS survivors. The most significant metabolic disruptions were the comprehensive increase of phosphatidylinositol and lysophospha tidylinositol levels in recovered SARS patients, which coincided with the effect of methylprednisolone administration investigated further in the steroid treated non-SARS patients with severe pneumonia. These results suggested that high-dose pulses of methylprednisolone might cause long-term systemic damage associated with serum metabolic alterations. The present study provided information for an improved understanding of coronavirus-associated pathologies, which might permit further optimization of clinical treatments.

  17. SARS疫苗研究进展%The progress in research of SARS vaccine

    Institute of Scientific and Technical Information of China (English)

    张增峰

    2011-01-01

    Severe acute respiratory syndrome (SARS) is a serious infectious disease caused by SARSassociated coronavirus (SARS-CoV). There are no approved antiviral drugs that effectively target SARS-CoV,and vaccination is the most effective mode for preventing SARS in people. At present, SARS vaccines,including inactivated vaccines, attenuated vaccines, subunit vaccines and DNA vaccines, etc., are being developed. Progress has been made in animal models, and some of the vaccines have entered clinical trials. In this article, the current state of SARS vaccine development is reviewed.%严重急性呼吸综合征(severe acute respiratory syndrome,SARS)是由SARS相关冠状病毒(SARS-associated coronavirus,SARS-CoV)引起的一类严重的急性呼吸系统传染病.目前尚未研制出治疗SARS的有效药物,防范SARS-CoV感染最有效的方法是使用疫苗.正在研制的SARS疫苗有灭活疫苗、减毒活疫苗、亚单位疫苗和DNA疫苗等,这些疫苗在动物模型中取得一些进展,有的已进入人体试验.此文就近几年有关SARS疫苗的研发现状做一综述.

  18. Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture.

    Science.gov (United States)

    de Wilde, Adriaan H; Jochmans, Dirk; Posthuma, Clara C; Zevenhoven-Dobbe, Jessika C; van Nieuwkoop, Stefan; Bestebroer, Theo M; van den Hoogen, Bernadette G; Neyts, Johan; Snijder, Eric J

    2014-08-01

    Coronaviruses can cause respiratory and enteric disease in a wide variety of human and animal hosts. The 2003 outbreak of severe acute respiratory syndrome (SARS) first demonstrated the potentially lethal consequences of zoonotic coronavirus infections in humans. In 2012, a similar previously unknown coronavirus emerged, Middle East respiratory syndrome coronavirus (MERS-CoV), thus far causing over 650 laboratory-confirmed infections, with an unexplained steep rise in the number of cases being recorded over recent months. The human MERS fatality rate of ∼ 30% is alarmingly high, even though many deaths were associated with underlying medical conditions. Registered therapeutics for the treatment of coronavirus infections are not available. Moreover, the pace of drug development and registration for human use is generally incompatible with strategies to combat emerging infectious diseases. Therefore, we have screened a library of 348 FDA-approved drugs for anti-MERS-CoV activity in cell culture. If such compounds proved sufficiently potent, their efficacy might be directly assessed in MERS patients. We identified four compounds (chloroquine, chlorpromazine, loperamide, and lopinavir) inhibiting MERS-CoV replication in the low-micromolar range (50% effective concentrations [EC(50)s], 3 to 8 μM). Moreover, these compounds also inhibit the replication of SARS coronavirus and human coronavirus 229E. Although their protective activity (alone or in combination) remains to be assessed in animal models, our findings may offer a starting point for treatment of patients infected with zoonotic coronaviruses like MERS-CoV. Although they may not necessarily reduce viral replication to very low levels, a moderate viral load reduction may create a window during which to mount a protective immune response.

  19. Association of SARS susceptibility with single nucleic acid polymorphisms of OASI and MxA genes: A case-control study

    NARCIS (Netherlands)

    J. He (Jing); D. Feng (Dan); S.J. de Vlas (Sake); H. Wang (Hongwei); A. Fontanet (Arnaud); F. Zhang (Fang); S. Plancoulaine (Sabine); F. Tang (Fang); L. Zhan (Lin); H. Yang (Honghui); T. Wang (Teng); J.H. Richardus (Jan Hendrik); J.D.F. Habbema (Dik); W.-C. Cao (Wuchun)

    2006-01-01

    textabstractBackground: Host genetic factors may play a role in susceptibility and resistance to SARS associated coronavirus (SARS-CoV) infection. The study was carried out to investigate the association between the genetic polymorphisms of 2′,5′-oligoadenylate synthetase I (OASI) gene as well as my

  20. Association of SARS susceptibility with single nucleic acid polymorphisms of OAS1 and MxA genes : a case-control study

    NARCIS (Netherlands)

    He, Jing; Feng, Dan; de Vlas, Sake J.; Wang, Hongwei; Fontanet, Arnaud; Zhang, Panhe; Plancoulaine, Sabine; Tang, Fang; Zhan, Lin; Yang, Hong; Wang, Tianbao; Richardus, Jan H.; Habbema, J. Dik F.; Cao, Wuchun

    2006-01-01

    Background: Host genetic factors may play a role in susceptibility and resistance to SARS associated coronavirus (SARS-CoV) infection. The study was carried out to investigate the association between the genetic polymorphisms of 2',5'-oligoadenylate synthetase 1 (OAS1) gene as well as myxovirus resi

  1. Biodiversity impact of host interferon-stimulated-gene-product 15 on the coronavirus Papain-like protease deISGylase functions

    Science.gov (United States)

    Coronaviruses are single-stranded, positive sense RNA viruses whose members have severe impact on human health and cause significant economic hardships. Some pertinent examples include severe acute and Middle East respiratory syndromes (SARS-CoV; MERS-CoV), porcine epidemic diarrhea virus (PEDV), an...

  2. Coronavirus Infections in the Central Nervous System and Respiratory Tract Show Distinct Features in Hospitalized Children.

    Science.gov (United States)

    Li, Yuanyuan; Li, Haipeng; Fan, Ruyan; Wen, Bo; Zhang, Jian; Cao, Xiaoying; Wang, Chengwu; Song, Zhanyi; Li, Shuochi; Li, Xiaojie; Lv, Xinjun; Qu, Xiaowang; Huang, Renbin; Liu, Wenpei

    2016-01-01

    Coronavirus (CoV) infections induce respiratory tract illnesses and central nervous system (CNS) diseases. We aimed to explore the cytokine expression profiles in hospitalized children with CoV-CNS and CoV-respiratory tract infections. A total of 183 and 236 hospitalized children with acute encephalitis-like syndrome and respiratory tract infection, respectively, were screened for anti-CoV IgM antibodies. The expression profiles of multiple cytokines were determined in CoV-positive patients. Anti-CoV IgM antibodies were detected in 22/183 (12.02%) and 26/236 (11.02%) patients with acute encephalitis-like syndrome and respiratory tract infection, respectively. Cytokine analysis revealed that the level of serum granulocyte colony-stimulating factor (G-CSF) was significantly higher in both CoV-CNS and CoV-respiratory tract infection compared with healthy controls. Additionally, the serum level of granulocyte macrophage colony-stimulating factor (GM-CSF) was significantly higher in CoV-CNS infection than in CoV-respiratory tract infection. In patients with CoV-CNS infection, the levels of IL-6, IL-8, MCP-1, and GM-CSF were significantly higher in their cerebrospinal fluid samples than in matched serum samples. To the best of our knowledge, this is the first report showing a high incidence of CoV infection in hospitalized children, especially with CNS illness. The characteristic cytokine expression profiles in CoV infection indicate the importance of host immune response in disease progression. © 2017 S. Karger AG, Basel.

  3. Control of Streptococcus pyogenes virulence: modeling of the CovR/S signal transduction system.

    Science.gov (United States)

    Mitrophanov, Alexander Y; Churchward, Gordon; Borodovsky, Mark

    2007-05-07

    The CovR/S system in Streptococcus pyogenes (Group A Streptococcus, or GAS), a two-component signal transduction/transcription regulation system, controls the expression of major virulence factors. The presence of a negative feedback loop distinguishes the CovR/S system from the majority of bacterial two-component systems. We developed a deterministic model of the CovR/S system consisting of eight delay differential equations. Computational experiments showed that the system possessed a unique stable steady state. The dynamical behavior of the system showed a tendency for oscillations becoming more pronounced for longer but still biochemically realistic delays resulting from reductions in the rates of translation elongation. We have devised an efficient procedure for computing the system's steady state. Further, we have shown that the signal-response curves are hyperbolic for the default parameter values. However, in experiments with randomized parameters we demonstrated that sigmoidality of signal-response curves, implying a response threshold, is not only possible, but seems to be rather typical for CovR/S-like systems even when binding of the CovR response regulator protein to a promoter is non-cooperative. We used sensitivity analysis to simplify the model in order to make it analytically tractable. The existence and uniqueness of the steady state and hyperbolicity of signal-response curves for the majority of the variables was proved for the simplified model. Also, we found that provided CovS was active, the system was insensitive to changes in the concentration of any other phosphoryl donor such as acetyl phosphate.

  4. Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence.

    Science.gov (United States)

    Feng, Wenchao; Minor, Dylan; Liu, Mengyao; Li, Jinquan; Ishaq, Suzanne L; Yeoman, Carl; Lei, Benfang

    2017-01-01

    Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [Sse(A+)] and normal SpeB activity [SpeB(A+)]). Sse(A+) SpeB(A+) variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an Sse(A+) SpeB(A+) isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other Sse(A+) SpeB(A+) isolates also had nonsense mutations or small indels in rocA RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence. Copyright © 2016 American Society for Microbiology.

  5. The emerging novel Middle East respiratory syndrome coronavirus: The “knowns” and “unknowns”

    Directory of Open Access Journals (Sweden)

    Jasper Fuk-Woo Chan

    2013-07-01

    Full Text Available A novel lineage C betacoronavirus, originally named human coronavirus EMC/2012 (HCoV-EMC and recently renamed Middle East respiratory syndrome coronavirus (MERS-CoV, that is phylogenetically closely related to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coronavirus HKU5, which we discovered in 2007 from bats in Hong Kong, has recently emerged in the Middle East to cause a severe acute respiratory syndrome (SARS-like infection in humans. The first laboratory-confirmed case, which involved a 60-year-old man from Bisha, the Kingdom of Saudi Arabia (KSA, who died of rapidly progressive community-acquired pneumonia and acute renal failure, was announced by the World Health Organization (WHO on September 23, 2012. Since then, a total of 70 cases, including 39 fatalities, have been reported in the Middle East and Europe. Recent clusters involving epidemiologically-linked household contacts and hospital contacts in the Middle East, Europe, and Africa strongly suggested possible human-to-human transmission. Clinical and laboratory research data generated in the past few months have provided new insights into the possible animal reservoirs, transmissibility, and virulence of MERS-CoV, and the optimal laboratory diagnostic options and potential antiviral targets for MERS-CoV-associated infection.

  6. Bioinformatic Analysis of Putative Gene Products Encoded in SARS-HCoV Genome

    Institute of Scientific and Technical Information of China (English)

    赵心刚; 韩敬东; 宁元亨; 孟安明; 陈晔光

    2003-01-01

    The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus named as SARS-HCoV.Using bioinformatic methods, we have performed a detailed domain search.In addition to the viral structure proteins, we have found that several putative polypeptides share sequence similarity to known domains or proteins.This study may provide a basis for future studies on the infection and replication process of this notorious virus.

  7. Effects of air temperature and relative humidity on coronavirus survival on surfaces.

    Science.gov (United States)

    Casanova, Lisa M; Jeon, Soyoung; Rutala, William A; Weber, David J; Sobsey, Mark D

    2010-05-01

    Assessment of the risks posed by severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) on surfaces requires data on survival of this virus on environmental surfaces and on how survival is affected by environmental variables, such as air temperature (AT) and relative humidity (RH). The use of surrogate viruses has the potential to overcome the challenges of working with SARS-CoV and to increase the available data on coronavirus survival on surfaces. Two potential surrogates were evaluated in this study; transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) were used to determine effects of AT and RH on the survival of coronaviruses on stainless steel. At 4 degrees C, infectious virus persisted for as long as 28 days, and the lowest level of inactivation occurred at 20% RH. Inactivation was more rapid at 20 degrees C than at 4 degrees C at all humidity levels; the viruses persisted for 5 to 28 days, and the slowest inactivation occurred at low RH. Both viruses were inactivated more rapidly at 40 degrees C than at 20 degrees C. The relationship between inactivation and RH was not monotonic, and there was greater survival or a greater protective effect at low RH (20%) and high RH (80%) than at moderate RH (50%). There was also evidence of an interaction between AT and RH. The results show that when high numbers of viruses are deposited, TGEV and MHV may survive for days on surfaces at ATs and RHs typical of indoor environments. TGEV and MHV could serve as conservative surrogates for modeling exposure, the risk of transmission, and control measures for pathogenic enveloped viruses, such as SARS-CoV and influenza virus, on health care surfaces.

  8. Effects of Air Temperature and Relative Humidity on Coronavirus Survival on Surfaces▿

    Science.gov (United States)

    Casanova, Lisa M.; Jeon, Soyoung; Rutala, William A.; Weber, David J.; Sobsey, Mark D.

    2010-01-01

    Assessment of the risks posed by severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) on surfaces requires data on survival of this virus on environmental surfaces and on how survival is affected by environmental variables, such as air temperature (AT) and relative humidity (RH). The use of surrogate viruses has the potential to overcome the challenges of working with SARS-CoV and to increase the available data on coronavirus survival on surfaces. Two potential surrogates were evaluated in this study; transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) were used to determine effects of AT and RH on the survival of coronaviruses on stainless steel. At 4°C, infectious virus persisted for as long as 28 days, and the lowest level of inactivation occurred at 20% RH. Inactivation was more rapid at 20°C than at 4°C at all humidity levels; the viruses persisted for 5 to 28 days, and the slowest inactivation occurred at low RH. Both viruses were inactivated more rapidly at 40°C than at 20°C. The relationship between inactivation and RH was not monotonic, and there was greater survival or a greater protective effect at low RH (20%) and high RH (80%) than at moderate RH (50%). There was also evidence of an interaction between AT and RH. The results show that when high numbers of viruses are deposited, TGEV and MHV may survive for days on surfaces at ATs and RHs typical of indoor environments. TGEV and MHV could serve as conservative surrogates for modeling exposure, the risk of transmission, and control measures for pathogenic enveloped viruses, such as SARS-CoV and influenza virus, on health care surfaces. PMID:20228108

  9. Receptor-binding domain as a target for developing SARS vaccines.

    Science.gov (United States)

    Zhu, Xiaojie; Liu, Qi; Du, Lanying; Lu, Lu; Jiang, Shibo

    2013-08-01

    A decade ago, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a global pandemic with a mortality rate of 10%. Reports of recent outbreaks of a SARS-like disease caused by Middle East respiratory syndrome coronavirus (MERS-CoV) have raised serious concerns of a possible reemergence of SARS-CoV, either by laboratory escape or the presence of a natural reservoir. Therefore, the development of effective and safe SARS vaccines is still needed. Based on our previous studies, we believe that the receptor-binding domain (RBD) in the S1 subunit of the SARS-CoV spike (S) protein is the most important target for developing a SARS vaccine. In particular, RBD of S protein contains the critical neutralizing domain (CND), which is able to induce highly potent neutralizing antibody response and cross-protection against divergent SARS-CoV strains. Furthermore, a RBD-based subunit vaccine is expected to be safer than other vaccines that may induce Th2-type immunopathology. This review will discuss key advances in the development of RBD-based SARS vaccines and the possibility of using a similar strategy to develop vaccines against MERS-CoV.

  10. Modeling the Early Events of Severe Acute Respiratory Syndrome Coronavirus Infection In Vitro

    Science.gov (United States)

    Yen, Yu-Ting; Liao, Fang; Hsiao, Cheng-Hsiang; Kao, Chuan-Liang; Chen, Yee-Chun; Wu-Hsieh, Betty A.

    2006-01-01

    The clinical picture of severe acute respiratory syndrome (SARS) is characterized by pulmonary inflammation and respiratory failure, resembling that of acute respiratory distress syndrome. However, the events that lead to the recruitment of leukocytes are poorly understood. To study the cellular response in the acute phase of SARS coronavirus (SARS-CoV)-host cell interaction, we investigated the induction of chemokines, adhesion molecules, and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) by SARS-CoV. Immunohistochemistry revealed neutrophil, macrophage, and CD8 T-cell infiltration in the lung autopsy of a SARS patient who died during the acute phase of illness. Additionally, pneumocytes and macrophages in the patient's lung expressed P-selectin and DC-SIGN. In in vitro study, we showed that the A549 and THP-1 cell lines were susceptible to SARS-CoV. A549 cells produced CCL2/monocyte chemoattractant protein 1 (MCP-1) and CXCL8/interleukin-8 (IL-8) after interaction with SARS-CoV and expressed P-selectin and VCAM-1. Moreover, SARS-CoV induced THP-1 cells to express CCL2/MCP-1, CXCL8/IL-8, CCL3/MIP-1α, CXCL10/IP-10, CCL4/MIP-1β, and CCL5/RANTES, which attracted neutrophils, monocytes, and activated T cells in a chemotaxis assay. We also demonstrated that DC-SIGN was inducible in THP-1 as well as A549 cells after SARS-CoV infection. Our in vitro experiments modeling infection in humans together with the study of a lung biopsy of a patient who died during the early phase of infection demonstrated that SARS-CoV, through a dynamic interaction with lung epithelial cells and monocytic cells, creates an environment conducive for immune cell migration and accumulation that eventually leads to lung injury. PMID:16501078

  11. Proteolytic Activation of the Coronavirus Fusion Protein

    NARCIS (Netherlands)

    Wicht, O.

    2014-01-01

    Coronaviruses are enveloped viruses with a positive-stranded RNA genome. They have been isolated from various mammals and birds and can cause severe diseases among farm and companion animals. Cross-species transmission of animal viruses and genuine human coronavirus infections pose a potential

  12. Comparative properties of feline coronaviruses in vitro.

    Science.gov (United States)

    McKeirnan, A J; Evermann, J F; Davis, E V; Ott, R L

    1987-04-01

    Two feline coronaviruses were characterized to determine their biological properties in vitro and their antigenic relatedness to a previously recognized feline infectious peritonitis virus and canine coronavirus. The viruses, designated WSU 79-1146 and WSU 79-1683, were shown to have comparable growth curves with the prototype feline infectious peritonitis virus. Treatment of the feline infectious peritonitis virus strains with 0.25% trypsin indicated that they were relatively resistant to proteolytic inactivation when compared with the feline enteric coronavirus strain. This observation may serve as a useful in vitro marker to distinguish closely related members of the feline coronavirus group. Plaque assay results indicated that the feline infectious peritonitis virus strains produced large homogeneous plaques in comparison to the feline enteric coronavirus strain and canine coronavirus, which showed a heterogenous plaque size distribution. No naturally temperature sensitive mutants were detected in either of the feline coronavirus populations. Both of the viruses were antigenically related to feline infectious peritonitis virus and to a lesser extent to canine coronavirus by virus neutralization.

  13. Preparation and development of equine hyperimmune globulin F(ab')2 against severe acute respiratory syndrome coronavirus

    Institute of Scientific and Technical Information of China (English)

    Jia-hai LU; Bing L WONG; Nan-shan ZHONG; Zhong-min GUO; Wen-yu HAN; Guo-ling WANG; Ding-mei ZHANG; Yi-fei WANG; Sheng-yun SUN; Qin-he YANG; Huan-ying ZHENG

    2005-01-01

    Aim: The resurgence of severe acute respiratory syndrome (SARS) is still a threat because the causative agent remaining in animal reservoirs is not fully understood,and sporadic cases continue to be reported. Developing high titers of anti-SARS hyperimmune globulin to provide an alternative pathway for emergent future prevention and treatment of SARS. Methods: SARS coronavirus (CoV)F69 (AY313906)and Z2-Y3 (AY394989) were isolated and identified from 2 different Cantonese onset SARS patients. Immunogen was prepared from SARS-CoV F69 strain. Six health horses were immunized 4 times and serum was collected periodically to measure the profile of specific IgG and neutralizing antibodies using indirect enzyme-linked immunosorbent assay and a microneutralization test. Sera were collected in large amounts at the peak, where IgG was precipitated using ammonium sulphate and subsequently digested with pepsin. The product was then purified using anion-exchange chromatography to obtain F(ab')2 fragments. Results: The specific IgG and neutralizing antibody titers peaked at approximately week 7 after the first immunization, with a maximum value of 1:14210. The sera collected at the peak were then purified. Fragment of approximately 15 g F(ab')2 was obtained from 1 litre antiserum and the purity was above 90% with the titer of 1:5120, which could neutralize the other strain (SARS-CoV Z2-Y3) as well. Conclusion: This research provides a viable strategy for the prevention and treatment of SARS coronavirus infection with equine hyperimmune globulin, with the purpose of combating any resurgence of SARS.

  14. A Strategy Toward Convergent Combination Immunotherapy for SARS

    Institute of Scientific and Technical Information of China (English)

    Wayne; A.; Marasco

    2005-01-01

    Passive Immunotherapyfor viral infections withimmune humanimmunoglobulin has been usedfor many yearsinthe pro-phylaxis andtreatment of infectious disease such as RSV,CMV,rabies,hepatitis Aand B and others.Recently,ad-vances in antibody engineering have allowedthe rapid isolation and pre-clinical development of human monoclonal anti-bodies(Mab)for the treatment of humaninfectious diseases and other conditions.We have explored the use of humanmonoclonal antibodies against the newly emerged SARS coronavirus(Co...

  15. Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    Science.gov (United States)

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-01-01

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein, and may point to important differences in assembly and infectivity of these two coronaviruses. PMID:20580052

  16. Severe acute respiratory syndrome coronavirus protein 6 mediates ubiquitin-dependent proteosomal degradation of N-Myc(and STAT) interactor

    Institute of Scientific and Technical Information of China (English)

    Weijia; Cheng; Shiyou; Chen; Ruiling; Li; Yu; Chen; Min; Wang; Deyin; Guo

    2015-01-01

    Severe acute respiratory syndrome coronavirus(SARS-Co V) encodes eight accessory proteins, the functions of which are not yet fully understood. SARS-Co V protein 6(P6) is one of the previously studied accessory proteins that have been documented to enhance viral replication and suppress host interferon(IFN) signaling pathways. Through yeast two-hybrid screening, we identified eight potential cellular P6-interacting proteins from a human spleen c DNA library. For further investigation, we targeted the IFN signaling pathway-mediating protein, N-Myc(and STAT) interactor(Nmi). Its interaction with P6 was confirmed within cells. The results showed that P6 can promote the ubiquitin-dependent proteosomal degradation of Nmi. This study revealed a new mechanism of SARS-Co V P6 in limiting the IFN signaling to promote SARS-Co V survival in host cells.

  17. Combinatorial synthetic peptide vaccine strategy protects against hypervirulent CovR/S mutant streptococci

    DEFF Research Database (Denmark)

    Pandey, Manisha; Mortensen, Rasmus; Calcutt, Ainslie;

    2016-01-01

    Cluster of virulence responder/sensor (CovR/S) mutant group A streptococci (GAS) are serious human pathogens of multiple M protein strains that upregulate expression of virulence factors, including the IL-8 protease Streptococcus pyogenes cell envelope proteinase (SpyCEP), thus blunting neutrophi...

  18. Isolation and Characterization of Dromedary Camel Coronavirus UAE-HKU23 from Dromedaries of the Middle East: Minimal Serological Cross-Reactivity between MERS Coronavirus and Dromedary Camel Coronavirus UAE-HKU23.

    Science.gov (United States)

    Woo, Patrick C Y; Lau, Susanna K P; Fan, Rachel Y Y; Lau, Candy C Y; Wong, Emily Y M; Joseph, Sunitha; Tsang, Alan K L; Wernery, Renate; Yip, Cyril C Y; Tsang, Chi-Ching; Wernery, Ulrich; Yuen, Kwok-Yung

    2016-05-07

    Recently, we reported the discovery of a dromedary camel coronavirus UAE-HKU23 (DcCoV UAE-HKU23) from dromedaries in the Middle East. In this study, DcCoV UAE-HKU23 was successfully isolated in two of the 14 dromedary fecal samples using HRT-18G cells, with cytopathic effects observed five days after inoculation. Northern blot analysis revealed at least seven distinct RNA species, corresponding to predicted subgenomic mRNAs and confirming the core sequence of transcription regulatory sequence motifs as 5'-UCUAAAC-3' as we predicted previously. Antibodies against DcCoV UAE-HKU23 were detected in 58 (98.3%) and 59 (100%) of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, p < 0.0001). Immunization of mice using recombinant N proteins of DcCoV UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, and heat-inactivated DcCoV UAE-HKU23 showed minimal cross-antigenicity between DcCoV UAE-HKU23 and MERS-CoV by Western blot and neutralization antibody assays. Codon usage and genetic distance analysis of RdRp, S and N genes showed that the 14 strains of DcCoV UAE-HKU23 formed a distinct cluster, separated from those of other closely related members of Betacoronavirus 1, including alpaca CoV, confirming that DcCoV UAE-HKU23 is a novel member of Betacoronavirus 1.

  19. Isolation and Characterization of Dromedary Camel Coronavirus UAE-HKU23 from Dromedaries of the Middle East: Minimal Serological Cross-Reactivity between MERS Coronavirus and Dromedary Camel Coronavirus UAE-HKU23

    Directory of Open Access Journals (Sweden)

    Patrick C. Y. Woo

    2016-05-01

    Full Text Available Recently, we reported the discovery of a dromedary camel coronavirus UAE-HKU23 (DcCoV UAE-HKU23 from dromedaries in the Middle East. In this study, DcCoV UAE-HKU23 was successfully isolated in two of the 14 dromedary fecal samples using HRT-18G cells, with cytopathic effects observed five days after inoculation. Northern blot analysis revealed at least seven distinct RNA species, corresponding to predicted subgenomic mRNAs and confirming the core sequence of transcription regulatory sequence motifs as 5′-UCUAAAC-3′ as we predicted previously. Antibodies against DcCoV UAE-HKU23 were detected in 58 (98.3% and 59 (100% of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, p < 0.0001. Immunization of mice using recombinant N proteins of DcCoV UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV, respectively, and heat-inactivated DcCoV UAE-HKU23 showed minimal cross-antigenicity between DcCoV UAE-HKU23 and MERS-CoV by Western blot and neutralization antibody assays. Codon usage and genetic distance analysis of RdRp, S and N genes showed that the 14 strains of DcCoV UAE-HKU23 formed a distinct cluster, separated from those of other closely related members of Betacoronavirus 1, including alpaca CoV, confirming that DcCoV UAE-HKU23 is a novel member of Betacoronavirus 1.

  20. Isolation and Characterization of Dromedary Camel Coronavirus UAE-HKU23 from Dromedaries of the Middle East: Minimal Serological Cross-Reactivity between MERS Coronavirus and Dromedary Camel Coronavirus UAE-HKU23

    Science.gov (United States)

    Woo, Patrick C. Y.; Lau, Susanna K. P.; Fan, Rachel Y. Y.; Lau, Candy C. Y.; Wong, Emily Y. M.; Joseph, Sunitha; Tsang, Alan K. L.; Wernery, Renate; Yip, Cyril C. Y.; Tsang, Chi-Ching; Wernery, Ulrich; Yuen, Kwok-Yung

    2016-01-01

    Recently, we reported the discovery of a dromedary camel coronavirus UAE-HKU23 (DcCoV UAE-HKU23) from dromedaries in the Middle East. In this study, DcCoV UAE-HKU23 was successfully isolated in two of the 14 dromedary fecal samples using HRT-18G cells, with cytopathic effects observed five days after inoculation. Northern blot analysis revealed at least seven distinct RNA species, corresponding to predicted subgenomic mRNAs and confirming the core sequence of transcription regulatory sequence motifs as 5′-UCUAAAC-3′ as we predicted previously. Antibodies against DcCoV UAE-HKU23 were detected in 58 (98.3%) and 59 (100%) of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, p < 0.0001). Immunization of mice using recombinant N proteins of DcCoV UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, and heat-inactivated DcCoV UAE-HKU23 showed minimal cross-antigenicity between DcCoV UAE-HKU23 and MERS-CoV by Western blot and neutralization antibody assays. Codon usage and genetic distance analysis of RdRp, S and N genes showed that the 14 strains of DcCoV UAE-HKU23 formed a distinct cluster, separated from those of other closely related members of Betacoronavirus 1, including alpaca CoV, confirming that DcCoV UAE-HKU23 is a novel member of Betacoronavirus 1. PMID:27164099

  1. Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) the beginning of another SARS-like pandemic?

    Science.gov (United States)

    Chan, Jasper F W; Li, Kenneth S M; To, Kelvin K W; Cheng, Vincent C C; Chen, Honglin; Yuen, Kwok-Yung

    2012-12-01

    Fouchier et al. reported the isolation and genome sequencing of a novel coronavirus tentatively named "human betacoronavirus 2c EMC/2012 (HCoV-EMC)" from a Saudi patient presenting with pneumonia and renal failure in June 2012. Genome sequencing showed that this virus belongs to the group C species of the genus betacoronavirus and phylogenetically related to the bat coronaviruses HKU4 and HKU5 previously found in lesser bamboo bat and Japanese Pipistrelle bat of Hong Kong respectively. Another patient from Qatar with similar clinical presentation and positive RT-PCR test was reported in September 2012. We compare and contrast the clinical presentation, laboratory diagnosis and management of infection due to this novel coronavirus and that of SARS coronavirus despite the paucity of published information on the former. Since 70% of all emerging infectious pathogens came from animals, the emergence of this novel virus may represent another instance of interspecies jumping of betacoronavirus from animals to human similar to the group A coronavirus OC43 possibly from a bovine source in the 1890s and the group B SARS coronavirus in 2003 from bat to civet and human. Despite the apparently low transmissibility of the virus at this stage, research preparedness against another SARS-like pandemic is an important precautionary strategy.

  2. Cloaked similarity between HIV-1 and SARS-CoV suggests an anti-SARS strategy

    Directory of Open Access Journals (Sweden)

    Kliger Yossef

    2003-09-01

    Full Text Available Abstract Background Severe acute respiratory syndrome (SARS is a febrile respiratory illness. The disease has been etiologically linked to a novel coronavirus that has been named the SARS-associated coronavirus (SARS-CoV, whose genome was recently sequenced. Since it is a member of the Coronaviridae, its spike protein (S2 is believed to play a central role in viral entry by facilitating fusion between the viral and host cell membranes. The protein responsible for viral-induced membrane fusion of HIV-1 (gp41 differs in length, and has no sequence homology with S2. Results Sequence analysis reveals that the two viral proteins share the sequence motifs that construct their active conformation. These include (1 an N-terminal leucine/isoleucine zipper-like sequence, and (2 a C-terminal heptad repeat located upstream of (3 an aromatic residue-rich region juxtaposed to the (4 transmembrane segment. Conclusions This study points to a similar mode of action for the two viral proteins, suggesting that anti-viral strategy that targets the viral-induced membrane fusion step can be adopted from HIV-1 to SARS-CoV. Recently the FDA approved Enfuvirtide, a synthetic peptide corresponding to the C-terminal heptad repeat of HIV-1 gp41, as an anti-AIDS agent. Enfuvirtide and C34, another anti HIV-1 peptide, exert their inhibitory activity by binding to a leucine/isoleucine zipper-like sequence in gp41, thus inhibiting a conformational change of gp41 required for its activation. We suggest that peptides corresponding to the C-terminal heptad repeat of the S2 protein may serve as inhibitors for SARS-CoV entry.

  3. Spike protein homology between the SARS-associated virus and murine hepatitis virus implies existence of a putative receptor-binding region

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Coronavirus has been determined to be the cause of the recent outbreak of severe acute respiratory syndrome (SARS). Human coronavirus 229E had been studied well and its receptor-binding domain was restricted to aa417-547 of S protein. However, this region has no homology with the newly separated SARS-associated virus (Hong Kong isolate CUHK-W1). Then we analyzed the phylogenesis of S1 subunit of the coronavirus spike protein (SARS-associated virus, Hong Kong isolate CUHK-W1). Interestingly, the highest homology between murine hepatitis virus (MHV) and SARS-associated coronavirus was found. And the important sites (aa62-65 and aa214-216) on the spike protein of MHV with receptor-binding capacity were highly conservative in comparison with the newly separated SARS-asso- ciated virus (the corresponding sites are aa51-54 and aa195-197). These results from bioinformatics analysis might help us to study the receptor-binding sites of SARS-associ- ated virus and the mechanism of the virus entry into the target cell, and design antiviral drugs and potent vaccines.

  4. Association of human leukocyte antigen class II alleles with severe Middle East respiratory syndrome-coronavirus infection.

    Science.gov (United States)

    Hajeer, Ali H; Balkhy, Hanan; Johani, Sameera; Yousef, Mohammed Z; Arabi, Yaseen

    2016-01-01

    Middle East Respiratory Syndrome (MERS) is a disease of the lower respiratory tract and is characterized by high mortality. It is caused by a beta coronavirus (CoV) referred to as MERS-CoV. Majority of MERS-CoV cases have been reported from Saudi Arabia. We investigated the human leukocyte antigen (HLA) Class II alleles in patients with severe MERS who were admitted in our Intensive Care Unit. A total of 23 Saudi patients with severe MERS-CoV infection were typed for HLA class II, results were compared with those of 161 healthy controls. Two HLA class II alleles were associated with the disease; HLA-DRB1*11:01 and DQB1*02:02, but not with the disease outcome. Our results suggest that the HLA-DRB1*11:01 and DQB1*02:02 may be associated with susceptibility to MERS.

  5. IFITM Proteins Inhibit Entry Driven by the MERS-Coronavirus Spike Protein: Evidence for Cholesterol-Independent Mechanisms

    Directory of Open Access Journals (Sweden)

    Florian Wrensch

    2014-09-01

    Full Text Available The interferon-inducible transmembrane (IFITM proteins 1, 2 and 3 inhibit the host cell entry of several enveloped viruses, potentially by promoting the accumulation of cholesterol in endosomal compartments. IFITM3 is essential for control of influenza virus infection in mice and humans. In contrast, the role of IFITM proteins in coronavirus infection is less well defined. Employing a retroviral vector system for analysis of coronavirus entry, we investigated the susceptibility of human-adapted and emerging coronaviruses to inhibition by IFITM proteins. We found that entry of the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV is sensitive to inhibition by IFITM proteins. In 293T cells, IFITM-mediated inhibition of cellular entry of the emerging MERS- and SARS-CoV was less efficient than blockade of entry of the globally circulating human coronaviruses 229E and NL63. Similar differences were not observed in A549 cells, suggesting that cellular context and/or IFITM expression levels can impact inhibition efficiency. The differential IFITM-sensitivity of coronaviruses observed in 293T cells afforded the opportunity to investigate whether efficiency of entry inhibition by IFITMs and endosomal cholesterol accumulation correlate. No such correlation was observed. Furthermore, entry mediated by the influenza virus hemagglutinin was robustly inhibited by IFITM3 but was insensitive to accumulation of endosomal cholesterol, indicating that modulation of cholesterol synthesis/transport did not account for the antiviral activity of IFITM3. Collectively, these results show that the emerging MERS-CoV is a target of the antiviral activity of IFITM proteins and demonstrate that mechanisms other than accumulation of endosomal cholesterol can contribute to viral entry inhibition by IFITMs.

  6. Comparative properties of feline coronaviruses in vitro.

    OpenAIRE

    McKeirnan, A J; Evermann, J F; Davis, E. V.; Ott, R L

    1987-01-01

    Two feline coronaviruses were characterized to determine their biological properties in vitro and their antigenic relatedness to a previously recognized feline infectious peritonitis virus and canine coronavirus. The viruses, designated WSU 79-1146 and WSU 79-1683, were shown to have comparable growth curves with the prototype feline infectious peritonitis virus. Treatment of the feline infectious peritonitis virus strains with 0.25% trypsin indicated that they were relatively resistant to pr...

  7. Unraveling the Mysteries of Middle East Respiratory Syndrome Coronavirus

    Centers for Disease Control (CDC) Podcasts

    2014-03-11

    Dr. Aron Hall, a CDC coronavirus epidemiologist, discusses Middle East Respiratory Syndrome Coronavirus.  Created: 3/11/2014 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 3/11/2014.

  8. Searching for animal models and potential target species for emerging pathogens: Experience gained from Middle East respiratory syndrome (MERS coronavirus

    Directory of Open Access Journals (Sweden)

    Júlia Vergara-Alert

    2017-06-01

    Full Text Available Emerging and re-emerging pathogens represent a substantial threat to public health, as demonstrated with numerous outbreaks over the past years, including the 2013–2016 outbreak of Ebola virus in western Africa. Coronaviruses are also a threat for humans, as evidenced in 2002/2003 with infection by the severe acute respiratory syndrome coronavirus (SARS-CoV, which caused more than 8000 human infections with 10% fatality rate in 37 countries. Ten years later, a novel human coronavirus (Middle East respiratory syndrome coronavirus, MERS-CoV, associated with severe pneumonia, arose in the Kingdom of Saudi Arabia. Until December 2016, MERS has accounted for more than 1800 cases and 35% fatality rate. Finding an animal model of disease is key to develop vaccines or antivirals against such emerging pathogens and to understand its pathogenesis. Knowledge of the potential role of domestic livestock and other animal species in the transmission of pathogens is of importance to understand the epidemiology of the disease. Little is known about MERS-CoV animal host range. In this paper, experimental data on potential hosts for MERS-CoV is reviewed. Advantages and limitations of different animal models are evaluated in relation to viral pathogenesis and transmission studies. Finally, the relevance of potential new target species is discussed.

  9. Receptor-Dependent Coronavirus Infection of Dendritic Cells

    Science.gov (United States)

    Turner, Brian C.; Hemmila, Erin M.; Beauchemin, Nicole; Holmes, Kathryn V.

    2004-01-01

    In several mammalian species, including humans, coronavirus infection can modulate the host immune response. We show a potential role of dendritic cells (DC) in murine coronavirus-induced immune modulation and pathogenesis by demonstrating that the JAW SII DC line and primary DC from BALB/c mice and p/p mice with reduced expression of the murine coronavirus receptor, murine CEACAM1a, are susceptible to murine coronavirus infection by a receptor-dependent pathway. PMID:15113927

  10. Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

    OpenAIRE

    2011-01-01

    Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this ...

  11. Single crystal growth and magnetic excitations of transistion metal oxide CoV2 O6

    Science.gov (United States)

    Stockdale, Christopher; Wallington, F.; Taylor, J. W.; Garcia-Sakai, V.; Arevalo-Lopez, A. M.; Attfield, P.; Stock, C.

    2015-03-01

    Low-dimensional magnetic materials are an area of interest due to their unusual properties such as metamagnetism and magnetization plateaus. Solid state synthesis has produced polycrystalline CoV2O6 which exists in two polymorphs: one with a monoclinic structure, and the other with a triclinic structure. Single crystals have been grown from polycrystalline CoV2O6 using the flux method under vacuum and are large enough to aid in single crystal neutron diffraction. Magnetic excitations have been measured using powder neutron diffraction in the low temperatures regime with variable energy. The magnetic excitations have been compared between the two phases. The energy of the system has been modelled in terms of the spin-orbit coupling, structural distortions, and the crystal field and compared to neutron data.

  12. Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus

    Science.gov (United States)

    Gilardi, K.; Menachery, V. D.; Goldstein, T.; Ssebide, B.; Mbabazi, R.; Navarrete-Macias, I.; Liang, E.; Wells, H.; Hicks, A.; Petrosov, A.; Byarugaba, D. K.; Debbink, K.; Dinnon, K. H.; Scobey, T.; Randell, S. H.; Yount, B. L.; Cranfield, M.; Johnson, C. K.; Baric, R. S.; Lipkin, W. I.; Mazet, J. A. K.

    2017-01-01

    ABSTRACT The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as several 2c CoVs have been described from various species in the family Vespertilionidae. Here, we describe a MERS-like CoV identified from a Pipistrellus cf. hesperidus bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans. PMID:28377531

  13. Coronavirus MHV-A59 infects the lung and causes severe pneumonia in C57BL/6 mice

    Institute of Scientific and Technical Information of China (English)

    Zhangsheng; Yang; Jun; Du; Gang; Chen; Jie; Zhao; Xuanming; Yang; Lishan; Su; Genhong; Cheng; Hong; Tang

    2014-01-01

    It remains challenging to develop animal models of lung infection and severe pneumonia by severe acute respiratory syndrome coronavirus(SARS-CoV) and Middle East respiratory syndrome cornavirus(MERS-Co V) without high level of containment. This inevitably hinders understanding of virushost interaction and development of appropriate countermeasures. Here we report that intranasal inoculation of sublethal doses of murine coronavirus mouse hepatitis virus A-59(MHV-A59), a hepatic and neuronal tropic coronavirus, can induce acute pneumonia and severe lung injuries in C57BL/6 mice. Inflammatory leukocyte infiltrations, hemorrhages and fibrosis of alveolar walls can be observed 2-11 days after MHV-A59 infection. This pathological manifestation is associated with dramatical elevation of tissue IP-10 and IFN-γ and moderate increase of TNF-α and IL-1β, but inability of anti-viral type I interferon response. These results suggest that intranasal infection of MHV-A59 would serve as a surrogate mouse model of acute respiratory distress syndrome by SARS-CoV and MERS-CoV infections.

  14. Orbital degeneracy near the itinerant electron limit in CoV2 O4

    Science.gov (United States)

    Reig-I-Plessis, D.; Casavant, D.; Garlea, V. O.; Aczel, A. A.; Feygenson, M.; Neuefeind, J.; Zhou, H. D.; Nagler, S. E.; MacDougall, G. J.

    Vanadium spinels, AV2O4 have both magnetic frustration and orbital degeneracy on the V3+ sublattice, which lead to strong coupling of the orbital, lattice and spin degrees of freedom. Additionally, upon decreasing the V-V distance, the material is predicted to go from a Mott insulator to a metallic phase. Of all the materials in the AV2O4 series, CoV2O4 is closest to the predicted transition, and it's debated whether it may be fully described by either localized or itinerant electrons pictures. In all other studied vanadium spinels, there is a cubic to tetragonal transition associated with ordering of the degenerate V3+ orbitals, consistent with a local orbital picture but, this transition is surprisingly absent from CoV2O4 despite being an insulator with local spins. In this talk we present recent high resolution neutron diffraction and inelastic scattering measurements by our group on powders of CoV2O4. Diffraction data show there is small but clear first order structural transition present which correlates with canting of the V3+ spins, while inelastic data are well described by a local spinwave picture. We discuss how these results contribute evidence of a local orbital ordering phase in the region near electron itinerancy. This work was sponsored by NSF Grant DMR-145526.

  15. A novel pancoronavirus RT-PCR assay: frequent detection of human coronavirus NL63 in children hospitalized with respiratory tract infections in Belgium

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2005-02-01

    Full Text Available Abstract Background Four human coronaviruses are currently known to infect the respiratory tract: human coronaviruses OC43 (HCoV-OC43 and 229E (HCoV-229E, SARS associated coronavirus (SARS-CoV and the recently identified human coronavirus NL63 (HCoV-NL63. In this study we explored the incidence of HCoV-NL63 infection in children diagnosed with respiratory tract infections in Belgium. Methods Samples from children hospitalized with respiratory diseases during the winter seasons of 2003 and 2004 were evaluated for the presence of HCoV-NL63 using a optimized pancoronavirus RT-PCR assay. Results Seven HCoV-NL63 positive samples were identified, six were collected during January/February 2003 and one at the end of February 2004. Conclusions Our results support the notation that HCoV-NL63 can cause serious respiratory symptoms in children. Sequence analysis of the S gene showed that our isolates could be classified into two subtypes corresponding to the two prototype HCoV-NL63 sequences isolated in The Netherlands in 1988 and 2003, indicating that these two subtypes may currently be cocirculating.

  16. Use of a Phosphorylation Site Mutant To Identify Distinct Modes of Gene Repression by the Control of Virulence Regulator (CovR) in Streptococcus pyogenes.

    Science.gov (United States)

    Horstmann, Nicola; Sahasrabhojane, Pranoti; Yao, Hui; Su, Xiaoping; Shelburne, Samuel A

    2017-09-15

    Control of the virulence regulator/sensor kinase (CovRS) two-component system (TCS) serves as a model for investigating the impact of signaling pathways on the pathogenesis of Gram-positive bacteria. However, the molecular mechanisms by which CovR, an OmpR/PhoB family response regulator, controls virulence gene expression are poorly defined, partly due to the labile nature of its aspartate phosphorylation site. To better understand the regulatory effect of phosphorylated CovR, we generated the phosphorylation site mutant strain 10870-CovR-D53E, which we predicted to have a constitutive CovR phosphorylation phenotype. Interestingly, this strain showed CovR activity only for a subset of the CovR regulon, which allowed for classification of CovR-influenced genes into D53E-regulated and D53E-nonregulated groups. Inspection of the promoter sequences of genes belonging to each group revealed distinct promoter architectures with respect to the location and number of putative CovR-binding sites. Electrophoretic mobility shift analysis demonstrated that recombinant CovR-D53E protein retains its ability to bind promoter DNA from both CovR-D53E-regulated and -nonregulated groups, implying that factors other than mere DNA binding are crucial for gene regulation. In fact, we found that CovR-D53E is incapable of dimerization, a process thought to be critical to OmpR/PhoB family regulator function. Thus, our global analysis of CovR-D53E indicates dimerization-dependent and dimerization-independent modes of CovR-mediated repression, thereby establishing distinct mechanisms by which this critical regulator coordinates virulence gene expression.IMPORTANCEStreptococcus pyogenes causes a wide variety of diseases, ranging from superficial skin and throat infections to life-threatening invasive infections. To establish these various disease manifestations, Streptococcus pyogenes requires tightly coordinated production of its virulence factor repertoire. Here, the response regulator Cov

  17. Core Structure of S2 from the Human Coronavirus NL63 Spike Glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Zheng,Q.; Deng, Y.; Liu, J.; van der Hoek, L.; Berkhout, B.; Lu, M.

    2006-01-01

    Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an {alpha}-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 {sup o}C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.

  18. The role of viral population diversity in adaptation of bovine coronavirus to new host environments.

    Directory of Open Access Journals (Sweden)

    Monica K Borucki

    Full Text Available The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing and 38,000×(Illumina. The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were "selected" from a pre-existing pool rather than through de novo mutation and subsequent population fixation.

  19. Cross-Reaction of SARS-CoV Antigen with Autoantibodies in Autoimmune Diseases

    Institute of Scientific and Technical Information of China (English)

    YunshanWang; ShanhuiSun; HongShen; LihuaJiang; MaoxiuZhang; DongjieXiao; YangLiu; XiaoliMa; YongZhang; NongjianGuo; TanghongJia

    2004-01-01

    To investigate the significance of the SARS-associated coronavirus (SARS-CoV) antibody, detected by ELISA and indirect immunofluorescence assays (IFA) for the SARS-CoV Vero E6 cell lysates, in non-SARS subjects, 114 serum samples from healthy controls and 104 serum specimens from autoimmune disease patients were collected. The results of ELISA showed that among 114 sera from healthy controls, 4 (3.5%) were positive of SARS-CoV-IgG antibody and 114 (100%) were all negative of SARS-CoV-IgM antibody; the specificity of SARS-CoV-IgG antibody for SARS patients was 96.5%, but the specificity of both SARS-CoV-IgG and -IgM antibodies for SARS patients was 100%. In 58 cases with SLE, positive rates of SARS-CoV-IgG and -IgM antibodies were 32.8% (19/58) and 8.6% (5/58), respectively, in which 11 cases (19%) were positive of both SARS-CoV-IgG and -IgM antibodies; in 10 cases with SS, positive rate of both SARS-CoV-IgG and -IgM antibodies was 10% (1/10); in 16 cases with MCTD, positive rate of SARS-CoV-IgG was 37.5% (6/16), positive rate of both SARS-CoV-IgG and -IgM antibodies was 6.3% (1/16); in 20 cases with RA, one case was positive (5%) of SARS-CoV-IgC However, of all samples with positive SARS-CoV-IgG and -IgM antibodies for autoimmune diseases and healthy controls, SARS-CoV RNA and antibodies were all negative by RT-PCR and IFA. All sera for negative or positive ELISA results were also negative or positive results using ELISA with Vero E6 cells lysates. These studies showed that SARS-CoV Vero E6 cell lysates for the ELISA to detect SARS-CoV antibodies could lead to the false-positive reactions or cross-reactions of SARS-CoV antibodies in non-SARS diseases and healthy controls, and the false-positive reactions or cross-reactions were related to Vero E6 cell lysates and autoantibodies in non-SARS population. Cellular & Molecular Immunology.

  20. Cross-Reaction of SARS-CoV Antigen with Autoantibodies in Autoimmune Diseases

    Institute of Scientific and Technical Information of China (English)

    Yunshan Wang; Nongjian Guo; Tanghong Jia; Shanhui Sun; Hong Shen; Lihua Jiang; Maoxiu Zhang; Dongjie Xiao; Yang Liu; Xiaoli Ma; Yong Zhang

    2004-01-01

    To investigate the significance of the SARS-associated coronavirus (SARS-CoV) antibody, detected by ELISA and indirect immunofluorescence assays (IFA) for the SARS-CoV Vero E6 cell lysates, in non-SARS subjects,114 serum samples from healthy controls and 104 serum specimens from autoimmune disease patients were collected. The results of ELISA showed that among 114 sera from healthy controls, 4 (3.5 %) were positive of SARS-CoV-IgG antibody and 114 (100%) were all negative of SARS-CoV-IgM antibody; the specificity of SARS-CoV-IgG antibody for SARS patients was 96.5%, but the specificity of both SARS-CoV-IgG and -IgM antibodies for SARS patients was 100%. In 58 cases with SLE, positive rates of SARS-CoV-IgG and -IgM antibodies were 32.8% (19/58) and 8.6% (5/58), respectively, in which 11 cases (19%) were positive of both SARS-CoV-IgG and -IgM antibodies; in 10 cases with SS, positive rate of both SARS-CoV-IgG and -IgM antibodies was 10% (1/10); in 16 cases with MCTD, positive rate of SARS-CoV-IgG was 37.5% (6/16), positive rate of both SARS-CoV-IgG and -IgM antibodies was 6.3% (1/16); in 20 cases with RA, one case was positive (5%) of SARS-CoV-IgG. However, of all samples with positive SARS-CoV-IgG and -IgM antibodies for autoimmune diseases and healthy controls, SARS-CoV RNA and antibodies were all negative by RT-PCR and IFA. All sera for negative or positive ELISA results were also negative or positive results using ELISA with Vero E6 cells lysates. These studies showed that SARS-CoV Vero E6 cell lysates for the ELISA to detect SARS-CoV antibodies could lead to the false-positive reactions or cross-reactions of SARS-CoV antibodies in non-SARS diseases and healthy controls, and the false-positive reactions or cross-reactions were related to Vero E6 cell lysates and autoantibodies in non-SARS population.

  1. Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease.

    Science.gov (United States)

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C; Chang, Kyeong-Ok

    2013-02-01

    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against a feline coronavirus in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC(50) in a nanomolar range) and, furthermore, combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in a cell culture system.

  2. COV2HTML: a visualization and analysis tool of bacterial next generation sequencing (NGS) data for postgenomics life scientists.

    Science.gov (United States)

    Monot, Marc; Orgeur, Mickael; Camiade, Emilie; Brehier, Clément; Dupuy, Bruno

    2014-03-01

    COV2HTML is an interactive web interface, which is addressed to biologists, and allows performing both coverage visualization and analysis of NGS alignments performed on prokaryotic organisms (bacteria and phages). It combines two processes: a tool that converts the huge NGS mapping or coverage files into light specific coverage files containing information on genetic elements; and a visualization interface allowing a real-time analysis of data with optional integration of statistical results. To demonstrate the scope of COV2HTML, the program was tested with data from two published studies. The first data were from RNA-seq analysis of Campylobacter jejuni, based on comparison of two conditions with two replicates. We were able to recover 26 out of 27 genes highlighted in the publication using COV2HTML. The second data comprised of stranded TSS and RNA-seq data sets on the Archaea Sulfolobus solfataricus. COV2HTML was able to highlight most of the TSSs from the article and allows biologists to visualize both TSS and RNA-seq on the same screen. The strength of the COV2HTML interface is making possible NGS data analysis without software installation, login, or a long training period. A web version is accessible at https://mmonot.eu/COV2HTML/ . This website is free and open to users without any login requirement.

  3. Coronavirus infection, ER stress and Apoptosis

    Directory of Open Access Journals (Sweden)

    TO SING eFUNG

    2014-06-01

    Full Text Available The replication of coronavirus, a family of important animal and human pathogens, is closely associated with the cellular membrane compartments, especially the endoplasmic reticulum (ER. Coronavirus infection of cultured cells was previously shown to cause ER stress and induce the unfolded protein response (UPR, a process that aims to restore the ER homeostasis by global translation shutdown and increasing the ER folding capacity. However under prolonged ER stress, UPR can also induce apoptotic cell death. Accumulating evidence from recent studies has shown that induction of ER stress and UPR may constitute a major aspect of coronavirus-host interaction. Activation of the three branches of UPR modulates a wide variety of signaling pathways, such as mitogen-activated protein (MAP kinases activation, autophagy, apoptosis and innate immune response. ER stress and UPR activation may therefore contribute significantly to the viral replication and pathogenesis during coronavirus infection. In this review, we summarize current knowledge on coronavirus-induced ER stress and UPR activation, with emphasis on their cross-talking to apoptotic signaling.

  4. Cinanserin is an inhibitor of the 3C-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro.

    Science.gov (United States)

    Chen, Lili; Gui, Chunshan; Luo, Xiaomin; Yang, Qingang; Günther, Stephan; Scandella, Elke; Drosten, Christian; Bai, Donglu; He, Xichang; Ludewig, Burkhard; Chen, Jing; Luo, Haibin; Yang, Yiming; Yang, Yifu; Zou, Jianping; Thiel, Volker; Chen, Kaixian; Shen, Jianhua; Shen, Xu; Jiang, Hualiang

    2005-06-01

    The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for anti-SARS-CoV drugs due to its crucial role in the viral life cycle. In this study, a database containing structural information of more than 8,000 existing drugs was virtually screened by a docking approach to identify potential binding molecules of SARS-CoV 3CLpro. As a target for screening, both a homology model and the crystallographic structure of the binding pocket of the enzyme were used. Cinanserin (SQ 10,643), a well-characterized serotonin antagonist that has undergone preliminary clinical testing in humans in the 1960s, showed a high score in the screening and was chosen for further experimental evaluation. Binding of both cinanserin and its hydrochloride to bacterially expressed 3CLpro of SARS-CoV and the related human coronavirus 229E (HCoV-229E) was demonstrated by surface plasmon resonance technology. The catalytic activity of both enzymes was inhibited with 50% inhibitory concentration (IC50) values of 5 microM, as tested with a fluorogenic substrate. The antiviral activity of cinanserin was further evaluated in tissue culture assays, namely, a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E. All assays revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50 values ranging from 19 to 34 microM. These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase.

  5. Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

    Science.gov (United States)

    Plant, Ewan P; Rakauskaite, Rasa; Taylor, Deborah R; Dinman, Jonathan D

    2010-05-01

    In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

  6. Genome Organization of the SARS-CoV

    Institute of Scientific and Technical Information of China (English)

    Jing Xu; Zizhang Zhang; Wei Wei; Songgang Li; Jun Wang; Jian Wang; Jun Yu; Huanming Yang; Jianfei Hu; Jing Wang; Yujun Han; Yongwu Hu; Jie Wen; Yan Li; Jia Ji; Jia Ye

    2003-01-01

    Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves.Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions.The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs.

  7. Terrain Measurement with SAR/InSAR

    Science.gov (United States)

    Li, Deren; Liao, Mingsheng; Balz, Timo; Zhang, Lu; Yang, Tianliang

    2016-08-01

    Terrain measurement and surface motion estimation are the most important applications for commercial and scientific SAR missions. In Dragon-3, we worked on these applications, especially regarding DEM generation, surface motion estimation with SAR time- series for urban subsidence monitoring and landslide motion estimation, as well as developing tomographic SAR processing methods in urban areas.

  8. Srv mediated dispersal of streptococcal biofilms through SpeB is observed in CovRS+ strains.

    Directory of Open Access Journals (Sweden)

    Kristie L Connolly

    Full Text Available Group A Streptococcus (GAS is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1 and MGAS315 (serotype M3, both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds.

  9. Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV replication.

    Directory of Open Access Journals (Sweden)

    Ji'an Pan

    Full Text Available Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12 provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

  10. Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Kailang; Peng, Guiqing; Wilken, Matthew; Geraghty, Robert J.; Li, Fang (UMMC)

    2012-12-10

    The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

  11. The E Protein Is a Multifunctional Membrane Protein of SARS-CoV

    Institute of Scientific and Technical Information of China (English)

    Qingfa Wu; Jia Ji; Jing Xu; Jia Ye; Yongwu Hu; Wenjun Chen; Songgang Li; Jun Wang; Jian Wang; Shengli Bi; Huanming Yang; Yilin Zhang; Hong Lü; Jing Wang; Ximiao He; Yong Liu; Chen Ye; Wei Lin; Jianfei Hu

    2003-01-01

    The E (envelope) protein is the smallest structural protein in all coronaviruses andis the only viral structural protein in which no variation has been detected. Weconducted genome sequencing and phylogenetic analyses of SARS-CoV. Based ongenome sequencing, we predicted the E protein is a transmembrane (TM) pro-tein characterized by a TM region with strong hydrophobicity and α-helix con-formation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in thecarboxyl-terminal region of the E protein that appears to form three disulfide bondswith another segment of corresponding cysteines in the carboxyl-terminus of the S(spike) protein. These bonds point to a possible structural association between theE and S proteins. Our phylogenetic analyses of the E protein sequences in all pub-lished coronaviruses place SARS-CoV in an independent group in Coronaviridaeand suggest a non-human animal origin.

  12. Combinatorial Synthetic Peptide Vaccine Strategy Protects against Hypervirulent CovR/S Mutant Streptococci.

    Science.gov (United States)

    Pandey, Manisha; Mortensen, Rasmus; Calcutt, Ainslie; Powell, Jessica; Batzloff, Michael R; Dietrich, Jes; Good, Michael F

    2016-04-15

    Cluster of virulence responder/sensor (CovR/S) mutant group A streptococci (GAS) are serious human pathogens of multiple M protein strains that upregulate expression of virulence factors, including the IL-8 proteaseStreptococcus pyogenescell envelope proteinase (SpyCEP), thus blunting neutrophil-mediated killing and enabling ingress of bacteria from a superficial wound to deep tissue. We previously showed that a combination vaccine incorporating J8-DT (conserved peptide vaccine from the M protein) and a recombinant SpyCEP fragment protects against CovR/S mutants. To enhance the vaccine's safety profile, we identified a minimal epitope (S2) that was the target for anti-SpyCEP Abs that could protect IL-8 from SpyCEP-mediated proteolysis. Abs from healthy humans and from mice experimentally infected with GAS also recognized S2, albeit at low titers. Native SpyCEP may be poorly immunogenic (cryptic or subdominant), and it would be to the organism's advantage if the host did not induce a strong Ab response against it. However, S2 conjugated to diphtheria toxoid is highly immunogenic and induces Abs that recognize and neutralize SpyCEP. Hence, we describe a two-component peptide vaccine that induces Abs (anti-S2) that protect IL-8 from proteolysis and other Abs (anti-J8) that cause strain-independent killing in the presence of neutrophils. We show that either component alone is ineffectual in preventing skin infection and bacteremia due to CovR/S mutants but that the combination induces complete protection. This protection correlated with a significant influx of neutrophils to the infection site. The data strongly suggest that the lack of natural immunity to hypervirulent GAS strains in humans could be rectified by this combination vaccine.

  13. Antibodies against MERS coronavirus in dromedary camels, United Arab Emirates, 2003 and 2013.

    Science.gov (United States)

    Meyer, Benjamin; Müller, Marcel A; Corman, Victor M; Reusken, Chantal B E M; Ritz, Daniel; Godeke, Gert-Jan; Lattwein, Erik; Kallies, Stephan; Siemens, Artem; van Beek, Janko; Drexler, Jan F; Muth, Doreen; Bosch, Berend-Jan; Wernery, Ulrich; Koopmans, Marion P G; Wernery, Renate; Drosten, Christian

    2014-04-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedary camels have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary camel serum samples from the United Arab Emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. Recombinant spike protein-specific immunofluorescence and virus neutralization tests enabled clear discrimination between MERS-CoV and bovine CoV infections. Most (632/651, 97.1%) camels had antibodies against MERS-CoV. This result included all 151 serum samples obtained in 2003. Most (389/651, 59.8%) serum samples had MERS-CoV-neutralizing antibody titers >1,280. Dromedary camels from the United Arab Emirates were infected at high rates with MERS-CoV or a closely related, probably conspecific, virus long before the first human MERS cases.

  14. Molecular and antigenic characterization of bovine Coronavirus circulating in Argentinean cattle during 1994-2010.

    Science.gov (United States)

    Bok, M; Miño, S; Rodriguez, D; Badaracco, A; Nuñes, I; Souza, S P; Bilbao, G; Louge Uriarte, E; Galarza, R; Vega, C; Odeon, A; Saif, L J; Parreño, V

    2015-12-31

    Bovine coronavirus (BCoV) is an important viral pathogen associated with neonatal calf diarrhea. Our aim was to investigate the incidence of BCoV in diarrhea outbreaks in beef and dairy herds from Argentina during 1994-2010. A total of 5.365 fecal samples from diarrheic calves were screened for BCoV diagnosis by ELISA. The virus was detected in 1.71% (92/5365) of the samples corresponding to 5.95% (63/1058) of the diarrhea cases in 239 beef and 324 dairy farms. The detection rate of BCoV was significantly higher in dairy than in beef herds: 12.13% (29/239) vs. 4.32% (14/324) respectively. Phylogenetic analysis of the hypervariable S1 region of seven representative samples (from different husbandry systems, farm locations and years of sampling) indicated that BCoV strains circulating in Argentinean beef and dairy herds formed a cluster distinct from other geographical regions. Interestingly, Argentinean strains are distantly related (at both the nucleotide and amino acid levels) with the Mebus historic reference BCoV strain included in the vaccines currently available in Argentina. However, Mebus-induced antibodies were capable of neutralizing the BCoV Arg95, a field strain adapted to grow in vitro, and vice versa, indicating that both strains belong to the same CoV serotype reported in cattle. This work represents the first large survey describing BCoV circulation in Argentinean cattle.

  15. Infection of cats with atypical feline coronaviruses harbouring a truncated form of the canine type I non-structural ORF3 gene.

    Science.gov (United States)

    Le Poder, Sophie; Pham-Hung d'Alexandry d'Orangiani, Anne-Laure; Duarte, Lidia; Fournier, Annie; Horhogea, Cristina; Pinhas, Carine; Vabret, Astrid; Eloit, Marc

    2013-12-01

    Feline and canine coronaviruses (FCoV and CCoV, respectively) are common pathogens of cats and dogs sometimes leading to lethal infections named feline infectious peritonitis (FIP) and canine pantropic coronavirus infection. FCoV and CCoV are each subdivided into two serotypes, FCoV-I/II and CCoV-I/II. A phylogenetic relationship is evident between, on one hand, CCoV-I/FCoV-I, and on the other hand, CCoV-II/FCoV-II, suggesting that interspecies transmission can occur. The aim of the present study was to evaluate the prevalence of coronavirus (CoV)-infected cats according to their contact with dogs and to genetically analyse the CoV strains infecting cats. From 2003 to 2009, we collected 88 faecal samples from healthy cats and 11 ascitic fluids from FIP cats. We investigated the possible contact with dog in the household and collected dogs samples if appropriate. Out of 99 cat samples, 26 were coronavirus positive, with six cats living with at least one dog, thus showing that contact with dogs does not appear as a predisposing factor for cats CoV infections. Molecular and phylogenetic analyses of FCoV strains were conducted using partial N and S sequences. Six divergent strains were identified with the N gene clustering with CCoV-I whereas the 3' end of S was related to FCoV-I. Further analysis on those six samples was attempted by researching the presence of the ORF3 gene, the latter being peculiar to CCoV-I to date. We succeeded to amplify the ORF3 gene in five samples out of six. Thus, our data strongly suggest the circulation of atypical FCoV strains harbouring the CCoV-I ORF3 gene among cats. Moreover, the ORF3 genes recovered from the feline strains exhibited shared deletions, never described before, suggesting that these deletions could be critical in the adaptation of these strains to the feline host. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  16. CovR and VicRK regulate cell surface biogenesis genes required for biofilm formation in Streptococcus mutans.

    Directory of Open Access Journals (Sweden)

    Rafael N Stipp

    Full Text Available The two-component system VicRK and the orphan regulator CovR of Streptococcus mutans co-regulate a group of virulence genes associated with the synthesis of and interaction with extracellular polysaccharides of the biofilm matrix. Knockout mutants of vicK and covR display abnormal cell division and morphology phenotypes, although the gene function defects involved are as yet unknown. Using transcriptomic comparisons between parent strain UA159 with vicK (UAvic or covR (UAcov deletion mutants together with electrophoretic motility shift assays (EMSA, we identified genes directly regulated by both VicR and CovR with putative functions in cell wall/surface biogenesis, including gbpB, wapE, smaA, SMU.2146c, and lysM. Deletion mutants of genes regulated by VicR and CovR (wapE, lysM, smaA, or regulated only by VicR (SMU.2146c or CovR (epsC promoted significant alterations in biofilm initiation, including increased fragility, defects in microcolony formation, and atypical cell morphology and/or chaining. Significant reductions in mureinolytic activity and/or increases in DNA release during growth were observed in knockout mutants of smaA, wapE, lysM, SMU.2146c and epsC, implying roles in cell wall biogenesis. WapE and lysM mutations also affected cell hydrophobicity and sensitivity to osmotic or oxidative stress. Finally, vicR, covR and VicRK/CovR-targets (gbpB, wapE, smaA, SMU.2146c, lysM, epsC are up-regulated in UA159 during biofilm initiation, in a sucrose-dependent manner. These data support a model in which VicRK and CovR coordinate cell division and surface biogenesis with the extracellular synthesis of polysaccharides, a process apparently required for formation of structurally stable biofilms in the presence of sucrose.

  17. Transmission characteristics of MERS and SARS in the healthcare setting: a comparative study.

    Science.gov (United States)

    Chowell, Gerardo; Abdirizak, Fatima; Lee, Sunmi; Lee, Jonggul; Jung, Eunok; Nishiura, Hiroshi; Viboud, Cécile

    2015-09-03

    The Middle East respiratory syndrome (MERS) coronavirus has caused recurrent outbreaks in the Arabian Peninsula since 2012. Although MERS has low overall human-to-human transmission potential, there is occasional amplification in the healthcare setting, a pattern reminiscent of the dynamics of the severe acute respiratory syndrome (SARS) outbreaks in 2003. Here we provide a head-to-head comparison of exposure patterns and transmission dynamics of large hospital clusters of MERS and SARS, including the most recent South Korean outbreak of MERS in 2015. To assess the unexpected nature of the recent South Korean nosocomial outbreak of MERS and estimate the probability of future large hospital clusters, we compared exposure and transmission patterns for previously reported hospital clusters of MERS and SARS, based on individual-level data and transmission tree information. We carried out simulations of nosocomial outbreaks of MERS and SARS using branching process models rooted in transmission tree data, and inferred the probability and characteristics of large outbreaks. A significant fraction of MERS cases were linked to the healthcare setting, ranging from 43.5 % for the nosocomial outbreak in Jeddah, Saudi Arabia, in 2014 to 100 % for both the outbreak in Al-Hasa, Saudi Arabia, in 2013 and the outbreak in South Korea in 2015. Both MERS and SARS nosocomial outbreaks are characterized by early nosocomial super-spreading events, with the reproduction number dropping below 1 within three to five disease generations. There was a systematic difference in the exposure patterns of MERS and SARS: a majority of MERS cases occurred among patients who sought care in the same facilities as the index case, whereas there was a greater concentration of SARS cases among healthcare workers throughout the outbreak. Exposure patterns differed slightly by disease generation, however, especially for SARS. Moreover, the distributions of secondary cases per single primary case varied

  18. MERS-coronavirus: From discovery to intervention

    NARCIS (Netherlands)

    W. Widagdo; N. Okba (Nisreen); V. Stalin Raj; B.L. Haagmans (Bart)

    2017-01-01

    textabstractMiddle East respiratory syndrome coronavirus (MERS-CoV) still causes outbreaks despite public awareness and implementation of health care measures, such as rapid viral diagnosis and patient quarantine. Here we describe the current epidemiological picture of MERS-CoV, focusing on humans a

  19. Coronavirus infection of polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Horzinek, M C; Rottier, P J

    1995-01-01

    Epithelial cells are the first host cells to be infected by incoming c oronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for

  20. Interactions of Rodent Coronaviruses with Cellular Receptors

    Science.gov (United States)

    2016-05-08

    eel to block binding of S to its receptor on various mouse cell lines and then challenged these cells with an HE expressing strain of MEV to...MAb-CCl an MEV iii strain expressing, HE could not infect mouse fibroblast cell lines or primary brain cells. Although murine coronavirus (MHV) and...Cell Cultures .. Virus Propagation and Purification ...............• Plaque assay .................... .... ............. . Hemagglutination Assay

  1. PepO, a CovRS-controlled endopeptidase, disrupts Streptococcus pyogenes quorum sensing.

    Science.gov (United States)

    Wilkening, Reid V; Chang, Jennifer C; Federle, Michael J

    2016-01-01

    Group A Streptococcus (GAS, Streptococcus pyogenes) is a human-restricted pathogen with a capacity to both colonize asymptomatically and cause illnesses ranging from pharyngitis to necrotizing fasciitis. An understanding of how and when GAS switches between genetic programs governing these different lifestyles has remained an enduring mystery and likely requires carefully tuned environmental sensors to activate and silence genetic schemes when appropriate. Herein, we describe the relationship between the Control of Virulence (CovRS, CsrRS) two-component system and the Rgg2/3 quorum-sensing pathway. We demonstrate that responses of CovRS to the stress signals Mg(2+) and a fragment of the antimicrobial peptide LL-37 result in modulated activity of pheromone signaling of the Rgg2/3 pathway through a means of proteolysis of SHP peptide pheromones. This degradation is mediated by the cytoplasmic endopeptidase PepO, which is the first identified enzymatic silencer of an RRNPP-type quorum-sensing pathway. These results suggest that under conditions in which the virulence potential of GAS is elevated (i.e. enhanced virulence gene expression), cellular responses mediated by the Rgg2/3 pathway are abrogated and allow individuals to escape from group behavior. These results also indicate that Rgg2/3 signaling is instead functional during non-virulent GAS lifestyles.

  2. Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type I and canine coronavirus

    NARCIS (Netherlands)

    Horzinek, M.C.; Herrewegh, A.A.; Rottier, P.J.M.; Groot, R.J. de

    1998-01-01

    Recent evidence suggests that the type II feline coronavirus (FCoV) strains 79-1146 and 79-1683 have arisen from a homologous RNA recombination event between FCoV type I and canine coronavirus (CCV). In both cases, the template switch apparently took place between the S and M genes, giving rise to r

  3. Modal parameter identification of flexible spacecraft using the covariance-driven stochastic subspace identification (SSI-COV) method

    Science.gov (United States)

    Xie, Yong; Liu, Pan; Cai, Guo-Ping

    2016-08-01

    In this paper, the on-orbit identification of modal parameters for a spacecraft is investigated. Firstly, the coupled dynamic equation of the system is established with the Lagrange method and the stochastic state-space model of the system is obtained. Then, the covariance-driven stochastic subspace identification (SSI-COV) algorithm is adopted to identify the modal parameters of the system. In this algorithm, it just needs the covariance of output data of the system under ambient excitation to construct a Toeplitz matrix, thus the system matrices are obtained by the singular value decomposition on the Toeplitz matrix and the modal parameters of the system can be found from the system matrices. Finally, numerical simulations are carried out to demonstrate the validity of the SSI-COV algorithm. Simulation results indicate that the SSI-COV algorithm is effective in identifying the modal parameters of the spacecraft only using the output data of the system under ambient excitation.

  4. Averaged cov-driven subspace identification for modal analysis of a modified troposkien blade with displacement measurement

    DEFF Research Database (Denmark)

    Najafi, Nadia; Panah, Mohammad Esmail Aryaee; Schmidt Paulsen, Uwe

    2015-01-01

    in the analysis are very short because of limitations in the image acquisition system. Short time series are not fully qualified for OMA and analyzing the data needs a proper method. Covariance driven Stochastic Subspace Identification method (SSI-cov) has been used for short time series like earthquakes....... In the SSI-cov method, a block Toeplitz matrix is formed which contains output correlation functions. 10 displacement time series have been recorded with 187 Hz sampling rate, and about 3 time series were chosen to be analyzed. The block Toeplitz matrix of 3 time series are averaged out and the procedure how...

  5. Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus.

    Science.gov (United States)

    Cavanagh, Dave

    2003-12-01

    Vaccines against infectious bronchitis of chickens (Gallus gallus domesticus) have arguably been the most successful, and certainly the most widely used, of vaccines for diseases caused by coronaviruses, the others being against bovine, canine, feline and porcine coronaviruses. Infectious bronchitis virus (IBV), together with the genetically related coronaviruses of turkey (Meleagris gallopovo) and ring-necked pheasant (Phasianus colchicus), is a group 3 coronavirus, severe acute respiratory syndrome (SARS) coronavirus being tentatively in group 4, the other known mammalian coronaviruses being in groups 1 and 2. IBV replicates not only in respiratory tissues (including the nose, trachea, lungs and airsacs, causing respiratory disease), but also in the kidney (associated with minor or major nephritis), oviduct, and in many parts of the alimentary tract--the oesophagus, proventriculus, duodenum, jejunum, bursa of Fabricius, caecal tonsils (near the distal end of the tract), rectum and cloaca (the common opening for release of eggs and faeces), usually without clinical effects. The virus can persist, being re-excreted at the onset of egg laying (4 to 5 months of age), believed to be a consequence of the stress of coming into lay. Genetic lines of chickens differ in the extent to which IBV causes mortality in chicks, and in respect of clearance of the virus after the acute phase. Live attenuated (by passage in chicken embryonated eggs) IBV strains were introduced as vaccines in the 1950s, followed a couple of decades later by inactivated vaccines for boosting protection in egg-laying birds. Live vaccines are usually applied to meat-type chickens at 1 day of age. In experimental situations this can result in sterile immunity when challenged by virulent homologous virus. Although 100% of chickens may be protected (against clinical signs and loss of ciliary activity in trachea), sometimes 10% of vaccinated chicks do not respond with a protective immune response

  6. Macro Domain from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Is an Efficient ADP-ribose Binding Module: CRYSTAL STRUCTURE AND BIOCHEMICAL STUDIES.

    Science.gov (United States)

    Cho, Chao-Cheng; Lin, Meng-Hsuan; Chuang, Chien-Ying; Hsu, Chun-Hua

    2016-03-01

    The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) encodes the conserved macro domain within non-structural protein 3. However, the precise biochemical function and structure of the macro domain is unclear. Using differential scanning fluorimetry and isothermal titration calorimetry, we characterized the MERS-CoV macro domain as a more efficient adenosine diphosphate (ADP)-ribose binding module than macro domains from other CoVs. Furthermore, the crystal structure of the MERS-CoV macro domain was determined at 1.43-Å resolution in complex with ADP-ribose. Comparison of macro domains from MERS-CoV and other human CoVs revealed structural differences in the α1 helix alters how the conserved Asp-20 interacts with ADP-ribose and may explain the efficient binding of the MERS-CoV macro domain to ADP-ribose. This study provides structural and biophysical bases to further evaluate the role of the MERS-CoV macro domain in the host response via ADP-ribose binding but also as a potential target for drug design.

  7. A chimeric virus-mouse model system for evaluating the function and inhibition of papain-like proteases of emerging coronaviruses.

    Science.gov (United States)

    Deng, Xufang; Agnihothram, Sudhakar; Mielech, Anna M; Nichols, Daniel B; Wilson, Michael W; StJohn, Sarah E; Larsen, Scott D; Mesecar, Andrew D; Lenschow, Deborah J; Baric, Ralph S; Baker, Susan C

    2014-10-01

    To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here, we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to coexpress PLpro and a substrate, murine interferon-stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of alpha/beta interferon receptor knockout (IFNAR(-/-)) mice with these chimeric viruses revealed that PLpro deISGylation activity removed ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect the mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric-virus platform to evaluate the papain-like protease/deISGylating activity of Middle East respiratory syndrome coronavirus (MERS-CoV) to provide a small-animal model to evaluate PLpro inhibitors of this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activities. Importance: Evaluating viral protease inhibitors in a small-animal model is a critical step in the path toward antiviral drug development. We modified a biosafety level 2 chimeric virus system to

  8. Potential Broad Spectrum Inhibitors of the Coronavirus 3CLpro: A Virtual Screening and Structure-Based Drug Design Study.

    Science.gov (United States)

    Berry, Michael; Fielding, Burtram C; Gamieldien, Junaid

    2015-12-15

    Human coronaviruses represent a significant disease burden; however, there is currently no antiviral strategy to combat infection. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) less than 10 years later demonstrates the potential of coronaviruses to cross species boundaries and further highlights the importance of identifying novel lead compounds with broad spectrum activity. The coronavirus 3CL(pro) provides a highly validated drug target and as there is a high degree of sequence homology and conservation in main chain architecture the design of broad spectrum inhibitors is viable. The ZINC drugs-now library was screened in a consensus high-throughput pharmacophore modeling and molecular docking approach by Vina, Glide, GOLD and MM-GBSA. Molecular dynamics further confirmed results obtained from structure-based techniques. A highly defined hit-list of 19 compounds was identified by the structure-based drug design methodologies. As these compounds were extensively validated by a consensus approach and by molecular dynamics, the likelihood that at least one of these compounds is bioactive is excellent. Additionally, the compounds segregate into 15 significantly dissimilar (p < 0.05) clusters based on shape and features, which represent valuable scaffolds that can be used as a basis for future anti-coronaviral inhibitor discovery experiments. Importantly though, the enriched subset of 19 compounds identified from the larger library has to be validated experimentally.

  9. Potent and specific inhibition of SARS-CoV antigen expression by RNA interference

    Institute of Scientific and Technical Information of China (English)

    TAO Peng; ZHANG Jun; TANG Ni; ZHANG Bing-qiang; HE Tong-chuan; HUANG Ai-long

    2005-01-01

    Background Severe acute respiratory syndrome (SARS) is an infectious disease caused by SARS-CoV. There are no effective antiviral drugs for SARS although the epidemic of SARS was controlled. The aim of this study was to develop an RNAi (RNA interference) approach that specifically targeted the N gene sequence of severe acute respiratory syndrome associated coronavirus (SARS-CoV) by synthesizing short hairpin RNA (shRNA) in vivo, and to assess the inhibitory effect of this shRNA on SARS-CoV N antigen expression. Methods The eukaryotic expression plasmid pEGFP-C1-N, containing SARS-CoV N gene, was co-transfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV-C4. At 24, 48 and 72 hours post transfection, the green fluorescence was observed through a fluorescence microscope. The RNA levels of SARS-CoV N were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of Green Fluorescent Protein (GFP) and protein N were detected using Western blot.Results The vector, pshRNA-N expressing shRNA which targeted the N gene of SARS-CoV, was successfully constructed. The introduction of RNAi plasmid efficiently and specifically inhibited the synthesis of protein N. RT-PCR showed that RNAs of N gene were clearly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N, whereas the control vector did not exhibit inhibitory effect on N gene transcription.Conclusions Our results demonstrate that RNAi mediated silencing of SARS-CoV gene could effectively inhibit expression of SARS-CoV antigen, hence RNAi based strategy should be further explored as a more efficacious antiviral therapy of SARS-CoV infection.

  10. SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production.

    Science.gov (United States)

    Tseng, Ying-Tzu; Wang, Shiu-Mei; Huang, Kuo-Jung; Wang, Chin-Tien

    2014-04-27

    Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly. SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production. The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly.

  11. The emergence of human coronavirus EMC: how scared should we be?

    Science.gov (United States)

    Chan, Renee W Y; Poon, Leo L M

    2013-04-09

    A novel betacoronavirus, human coronavirus (HCoV-EMC), has recently been detected in humans with severe respiratory disease. Further characterization of HCoV-EMC suggests that this virus is different from severe acute respiratory syndrome coronavirus (SARS-CoV) because it is able to replicate in multiple mammalian cell lines and it does not use angiotensin-converting enzyme 2 as a receptor to achieve infection. Additional research is urgently needed to better understand the pathogenicity and tissue tropism of this virus in humans. In their recent study published in mBio, Kindler et al. shed some light on these important topics (E. Kindler, H. R. Jónsdóttir, M. Muth, O. J. Hamming, R. Hartmann, R. Rodriguez, R. Geffers, R. A. Fouchier, C. Drosten, M. A. Müller, R. Dijkman, and V. Thiel, mBio 4[1]:e00611-12, 2013). These authors report the use of differentiated pseudostratified human primary airway epithelial cells, an in vitro model with high physiological relevance to the human airway epithelium, to characterize the cellular tropism of HCoV-EMC. More importantly, the authors demonstrate the potential use of type I and type III interferons (IFNs) to control viral infection.

  12. MERS Coronaviruses in Dromedary Camels, Egypt

    OpenAIRE

    Chu, Daniel K. W.; Poon, Leo L.M.; Gomaa, Mokhtar M.; Shehata, Mahmoud M.; Perera, Ranawaka A. P. M.; Abu Zeid, Dina; El Rifay, Amira S.; Siu, Lewis Y.; Guan, Yi; Webby, Richard J; Mohamed A Ali; Peiris, Malik; Kayali, Ghazi

    2014-01-01

    We identified the near-full-genome sequence (29,908 nt, >99%) of Middle East respiratory syndrome coronavirus (MERS-CoV) from a nasal swab specimen from a dromedary camel in Egypt. We found that viruses genetically very similar to human MERS-CoV are infecting dromedaries beyond the Arabian Peninsula, where human MERS-CoV infections have not yet been detected.

  13. Unconventional magnetic phase separation in γ -CoV2O6

    Science.gov (United States)

    Shen, L.; Jellyman, E.; Forgan, E. M.; Blackburn, E.; Laver, M.; Canévet, E.; Schefer, J.; He, Z.; Itoh, M.

    2017-08-01

    We have explored the magnetism in the nongeometrically frustrated spin-chain system γ -CoV2O6 which possesses a complex magnetic exchange network. Our neutron diffraction patterns at low temperatures (T ≤TN=6.6 K) are best described by a model in which two magnetic phases coexist in a volume ratio 65(1) : 35(1), with each phase consisting of a single spin modulation. This model fits previous studies and our observations better than the model proposed by Lenertz et al. [J. Phys. Chem. C 118, 13981 (2014), 10.1021/jp503389c], which consisted of one phase with two spin modulations. By decreasing the temperature from TN, the minority phase of our model undergoes an incommensurate-commensurate lock-in transition at T*=5.6 K. Based on these results, we propose that phase separation is an alternative approach for degeneracy-lifting in frustrated magnets.

  14. Elastic Anomalies in Orbital-Degenerate Frustrated Spinel CoV2O4

    Science.gov (United States)

    Watanabe, Tadataka; Yamada, Shogo; Koborinai, Rui; Katsufuji, Takuro

    Ultrasound velocity measurements were performed on a single crystal of the orbital-degenerate frustrated spinel CoV2O4 in all the symmetrically-independent elastic moduli of the cubic crystal. The measurements of temperature dependence of the elastic moduli observed discontinuous elastic anomalies due to a ferrimagnetic transition at TC = 165 K and another phase transition at T* = 50 K. Additionally, the measurements observed anomalous temperature dependence of the elastic moduli, specifically, non-monotonic temperature dependence in the magnetically-ordered phase below TC, and magnetic-field-sensitive elastic softening with decreasing temperature in the paramagnetic phase above TC. These anomalous temperature variations below and above TC should be driven by the coupling of lattice to magnetic excitations.

  15. Structural transition and orbital glass physics in near-itinerant CoV2O4

    Science.gov (United States)

    Reig-i-Plessis, D.; Casavant, D.; Garlea, V. O.; Aczel, A. A.; Feygenson, M.; Neuefeind, J.; Zhou, H. D.; Nagler, S. E.; MacDougall, G. J.

    2016-01-01

    The ferrimagnetic spinel CoV2O4 has been a topic of intense recent interest, both as a frustrated insulator with unquenched orbital degeneracy and as a near-itinerant magnet which can be driven metallic with moderate applied pressure. Here, we report on our recent neutron diffraction and inelastic scattering measurements on powders with minimal cation site disorder. Our main new result is the identification of a weak (Δ/a a ˜10-4 ), first order structural phase transition at T*=90 K, the same temperature where spin canting was seen in recent single crystal measurements. This transition is characterized by a short-range distortion of oxygen octahedral positions, and inelastic data further establish a weak Δ ˜1.25 meV spin gap at low temperature. Together, these findings provide strong support for the local orbital picture and the existence of an orbital glass state at temperatures below T*.

  16. A Review of Novel Coronavirus, cause of Middle East Respiratory Syndrome

    Directory of Open Access Journals (Sweden)

    Katayoun Vahdat

    2014-01-01

    Full Text Available Abstract Background: Isolation of a novel virus belonging to coronavirdae family in September 2012, from patients in Middle East who had died of an acute respiratory illness & renal failure lead to researches on this new virus. Here, a brief review to summarize the events of scientific findings of this new emerging virus. Material and Methods: This review is based on a comprehensive search of three databases (Pubmed, Embase and Cochrane and WHO reports. The searched keywords were new coronavirus, Middle East, acute respieratory distress syndrom & Saudi Arabia. Results: Due to novelty of virus only limited papers exist on searched databases, so only 50 papers were identified which after omitting repeated case reports, papers related to SARS and updated WHO reports, 30 papers were finally reviewed. Conclusion: WHO recommendation is evaluation of all patients with acute respiratory illness and history of travel to Saudi Arabia or other countries where this novel virus is epidemic.

  17. covR Mediated Antibiofilm Activity of 3-Furancarboxaldehyde Increases the Virulence of Group A Streptococcus.

    Directory of Open Access Journals (Sweden)

    Ganapathy Ashwinkumar Subramenium

    Full Text Available Group A streptococcus (GAS, Streptococcus pyogenes, a multi-virulent, exclusive human pathogen responsible for various invasive and non-invasive diseases possesses biofilm forming phenomenon as one of its pathogenic armaments. Recently, antibiofilm agents have gained prime importance, since inhibiting the biofilm formation is expected to reduce development of antibiotic resistance and increase their susceptibility to the host immune cells.The current study demonstrates the antibiofilm activity of 3Furancarboxaldehyde (3FCA, a floral honey derived compound, against GAS biofilm, which was divulged using crystal violet assay, light microscopy, and confocal laser scanning microscopy. The report is extended to study its effect on various aspects of GAS (morphology, virulence, aggregation at its minimal biofilm inhibitory concentration (132μg/ml. 3FCA was found to alter the growth pattern of GAS in solid and liquid medium and increased the rate of auto-aggregation. Electron microscopy unveiled the increase in extra polymeric substances around cell. Gene expression studies showed down-regulation of covR gene, which is speculated to be the prime target for the antibiofilm activity. Increased hyaluronic acid production and down regulation of srtB gene is attributed to the enhanced rate of auto-aggregation. The virulence genes (srv, mga, luxS and hasA were also found to be over expressed, which was manifested with the increased susceptibility of the model organism Caenorhabditis elegans to 3FCA treated GAS. The toxicity of 3FCA was ruled out with no adverse effect on C. elegans.Though 3FCA possess antibiofilm activity against GAS, it was also found to increase the virulence of GAS. This study demonstrates that, covR mediated antibiofilm activity may increase the virulence of GAS. This also emphasizes the importance to analyse the acclimatization response and virulence of the pathogen in the presence of antibiofilm compounds prior to their clinical trials.

  18. Study of magnetic and magnetocaloric properties of monoclinic and triclinic spin chain CoV2O6

    Science.gov (United States)

    Nandi, Moumita; Mandal, Prabhat

    We have investigated magnetic and magnetocaloric properties of both monoclinic and triclinic phases of CoV2O6 from magnetization and heat capacity measurements. Conventional and inverse magnetocaloric effects have been observed in both phases of CoV2O6. For a field change from 0 to 7 T, maximum values of magnetic entropy change and adiabatic temperature change reach 11.8 J kg-1 K-1 and 9.5 K respectively for monoclinic CoV2O6 while the corresponding values reach 12.1 J kg-1 K-1 and 13.1 K for triclinic CoVO6. Particularly for triclinic CoVO6, the magnetocaloric parameters are quite large in low or moderate field range. Apart from this, we have constructed magnetic phase diagram of monoclinic CoV2O6 where field-induced complex magnetic phases appear below a certain critical temperature 6 K when external magnetic field is applied along crystallographic easy axis.

  19. Elastase-mediated activation of the severe acute respiratory syndrome coronavirus spike protein at discrete sites within the S2 domain.

    Science.gov (United States)

    Belouzard, Sandrine; Madu, Ikenna; Whittaker, Gary R

    2010-07-23

    Proteolytic priming is a common method of controlling the activation of membrane fusion mediated by viral glycoproteins. The severe acute respiratory syndrome coronavirus spike protein (SARS-CoV S) can be primed by a variety of host cell proteases, with proteolytic cleavage occurring both as the S1/S2 boundary and adjacent to a fusion peptide in the S2 domain. Here, we studied the priming of SARS-CoV S by elastase and show an important role for residue Thr(795) in the S2 domain. A series of alanine mutants were generated in the vicinity of the S2 cleavage site, with the goal of examining elastase-mediated cleavage within S2. Both proteolytic cleavage and fusion activation were modulated by altering the cleavage site position. We propose a novel mechanism whereby SARS-CoV fusion protein function can be controlled by spatial regulation of the proteolytic priming site, with important implications for viral pathogenesis.

  20. Crystal structure of the papain-like protease of MERS coronavirus reveals unusual, potentially druggable active-site features.

    Science.gov (United States)

    Lei, Jian; Mesters, Jeroen R; Drosten, Christian; Anemüller, Stefan; Ma, Qingjun; Hilgenfeld, Rolf

    2014-09-01

    The Middle-East Respiratory Syndrome coronavirus (MERS-CoV) causes severe acute pneumonia and renal failure. The MERS-CoV papain-like protease (PL(pro)) is a potential target for the development of antiviral drugs. To facilitate these efforts, we determined the three-dimensional structure of the enzyme by X-ray crystallography. The molecule consists of a ubiquitin-like domain and a catalytic core domain. The catalytic domain displays an extended right-hand fold with a zinc ribbon and embraces a solvent-exposed substrate-binding region. The overall structure of the MERS-CoV PL(pro) is similar to that of the corresponding SARS-CoV enzyme, but the architecture of the oxyanion hole and of the S3 as well as the S5 specificity sites differ from the latter. These differences are the likely reason for reduced in vitro peptide hydrolysis and deubiquitinating activities of the MERS-CoV PL(pro), compared to the homologous enzyme from the SARS coronavirus. Introduction of a side-chain capable of oxyanion stabilization through the Leu106Trp mutation greatly enhances the in vitro catalytic activity of the MERS-CoV PL(pro). The unique features observed in the crystal structure of the MERS-CoV PL(pro) should allow the design of antivirals that would not interfere with host ubiquitin-specific proteases.

  1. Characterisation of human coronavirus-NL63 nucleocapsid protein

    African Journals Online (AJOL)

    Michael

    2012-09-18

    Sep 18, 2012 ... Coronavirus N is a multifunctional protein that plays an essential role in enhancing the efficiency of .... HCoV-NL63 was shown to be most similar to the human ... evolution of these coronaviruses and gave rise to the.

  2. Yeast based small molecule screen for inhibitors of SARS-CoV.

    Directory of Open Access Journals (Sweden)

    Matthew Frieman

    Full Text Available Severe acute respiratory coronavirus (SARS-CoV emerged in 2002, resulting in roughly 8000 cases worldwide and 10% mortality. The animal reservoirs for SARS-CoV precursors still exist and the likelihood of future outbreaks in the human population is high. The SARS-CoV papain-like protease (PLP is an attractive target for pharmaceutical development because it is essential for virus replication and is conserved among human coronaviruses. A yeast-based assay was established for PLP activity that relies on the ability of PLP to induce a pronounced slow-growth phenotype when expressed in S. cerevisiae. Induction of the slow-growth phenotype was shown to take place over a 60-hour time course, providing the basis for conducting a screen for small molecules that restore growth by inhibiting the function of PLP. Five chemical suppressors of the slow-growth phenotype were identified from the 2000 member NIH Diversity Set library. One of these, NSC158362, potently inhibited SARS-CoV replication in cell culture without toxic effects on cells, and it specifically inhibited SARS-CoV replication but not influenza virus replication. The effect of NSC158362 on PLP protease, deubiquitinase and anti-interferon activities was investigated but the compound did not alter these activities. Another suppressor, NSC158011, demonstrated the ability to inhibit PLP protease activity in a cell-based assay. The identification of these inhibitors demonstrated a strong functional connection between the PLP-based yeast assay, the inhibitory compounds, and SARS-CoV biology. Furthermore the data with NSC158362 suggest a novel mechanism for inhibition of SARS-CoV replication that may involve an unknown activity of PLP, or alternatively a direct effect on a cellular target that modifies or bypasses PLP function in yeast and mammalian cells.

  3. Complement-mediated opsonization of invasive group A Streptococcus pyogenes strain AP53 is regulated by the bacterial two-component cluster of virulence responder/sensor (CovRS) system.

    Science.gov (United States)

    Agrahari, Garima; Liang, Zhong; Mayfield, Jeffrey A; Balsara, Rashna D; Ploplis, Victoria A; Castellino, Francis J

    2013-09-20

    Group A Streptococcus pyogenes (GAS) strain AP53 is a primary isolate from a patient with necrotizing fasciitis. These AP53 cells contain an inactivating mutation in the sensor component of the cluster of virulence (cov) responder (R)/sensor (S) two-component gene regulatory system (covRS), which enhances the virulence of the primary strain, AP53/covR(+)S(-). However, specific mechanisms by which the covRS system regulates the survival of GAS in humans are incomplete. Here, we show a key role for covRS in the regulation of opsonophagocytosis of AP53 by human neutrophils. AP53/covR(+)S(-) cells displayed potent binding of host complement inhibitors of C3 convertase, viz. Factor H (FH) and C4-binding protein (C4BP), which concomitantly led to minimal C3b deposition on AP53 cells, further showing that these plasma protein inhibitors are active on GAS cells. This resulted in weak killing of the bacteria by human neutrophils and a corresponding high death rate of mice after injection of these cells. After targeted allelic alteration of covS(-) to wild-type covS (covS(+)), a dramatic loss of FH and C4BP binding to the AP53/covR(+)S(+) cells was observed. This resulted in elevated C3b deposition on AP53/covR(+)S(+) cells, a high level of opsonophagocytosis by human neutrophils, and a very low death rate of mice infected with AP53/covR(+)S(+). We show that covRS is a critical transcriptional regulator of genes directing AP53 killing by neutrophils and regulates the levels of the receptors for FH and C4BP, which we identify as the products of the fba and enn genes, respectively.

  4. Could the coefficient of variation (COV) of the corneal endothelium be overestimated when a centre-dot method is used?

    Science.gov (United States)

    Doughty, Michael J

    2008-01-01

    Little has been published on the reliability of estimates of the coefficient of variation (COV) in cell area for human corneal endothelia. The present study compares two methods. A non-contact specular micrograph (Topcon SP-2000P) was obtained from the central region of the corneal endothelium of 20 healthy myopic white European subjects, aged from 32 to 53 years, half of whom were successful long-term soft contact lens wearers. The captured image file was either assessed using a machine-based algorithm, in which 25 cells in the middle of the image were marked and their areas reported (designated as 'centre-dot' method) or by a manual method, by which all the cells in the image were outlined on very high magnification prints of the endothelia and the cell areas measured by a manual digitiser in stream mode. The average cell area was used to calculate the endothelial cell density (ECD), while the COV was calculated from the standard deviation (SD) of the cell area measures. Identical mean cell area values were found (392 microm(2)) with the two methods, a marginally higher ECD estimate (2,594 versus 2,569) with the centre-dot method (p = NS) but a much higher COV with the centre-dot method (43.8 versus 29.0 per cent). This highly statistically significant difference in COV (p definition of a single large cell domain on any individual image. A centre-dot method can be reliably used to generate useful data on cell area and ECD but it should be used cautiously for estimates of polymegethism (COV).

  5. Surface vimentin is critical for the cell entry of SARS-CoV.

    Science.gov (United States)

    Yu, Yvonne Ting-Chun; Chien, Ssu-Chia; Chen, I-Yin; Lai, Chia-Tsen; Tsay, Yeou-Guang; Chang, Shin C; Chang, Ming-Fu

    2016-01-22

    Severe acute respiratory syndrome coronavirus (SARS-CoV) caused a global panic due to its high morbidity and mortality during 2002 and 2003. Soon after the deadly disease outbreak, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional cellular receptor in vitro and in vivo for SARS-CoV spike protein. However, ACE2 solely is not sufficient to allow host cells to become susceptible to SARS-CoV infection, and other host factors may be involved in SARS-CoV spike protein-ACE2 complex. A host intracellular filamentous cytoskeletal protein vimentin was identified by immunoprecipitation and LC-MS/MS analysis following chemical cross-linking on Vero E6 cells that were pre-incubated with the SARS-CoV spike protein. Moreover, flow cytometry data demonstrated an increase of the cell surface vimentin level by 16.5 % after SARS-CoV permissive Vero E6 cells were treated with SARS-CoV virus-like particles (VLPs). A direct interaction between SARS-CoV spike protein and host surface vimentin was further confirmed by far-Western blotting. In addition, antibody neutralization assay and shRNA knockdown experiments indicated a vital role of vimentin in cell binding and uptake of SARS-CoV VLPs and the viral spike protein. A direct interaction between vimentin and SARS-CoV spike protein during viral entry was observed. Vimentin is a putative anti-viral drug target for preventing/reducing the susceptibility to SARS-CoV infection.

  6. Severe acute respiratory syndrome (SARS) - an emerging infection of the 21st century.

    Science.gov (United States)

    Hsueh, Po-Ren; Yang, Pan-Chyr

    2003-12-01

    Severe acute respiratory syndrome (SARS) is an emerging infection caused by a novel coronavirus known as SARS-CoV. The disease has a high propensity to spread to household members and healthcare workers and may be associated with transmission and outbreaks in the community. Severe illness in immunocompromised patients, sophisticated hospital facilities and treatment procedures, particularly those that generate aerosols, and lack of adequate isolation and control measures, can amplify transmission and contribute to so-called "super-spreading" events. The presence of non-specific clinical manifestations at presentation and a lack of validated early diagnostic methods and effective management pose great difficulty for frontline physicians in the containment of this disease. The mortality of SARS is in the region of 10 to 15%; the presence of underlying disease, high initial C-reactive protein levels, and positive SARS-CoV in nasopharyngeal aspirate samples are associated with a higher risk of respiratory failure and mortality. Despite the disappearance of SARS cases worldwide, the potential evolution of SARS-CoV in animals suggests the disease may re-emerge in the future. Heightened levels of clinical suspicion, rapid case detection and isolation, and contact tracing are essential to effective management of future outbreaks. Further ongoing requirements for successful management include research on the immunopathogenesis of SARS and the development of timely and reliable diagnostic tests, effective antiviral and immunomodulatory agents, and vaccines for the disease.

  7. Antigen Production in Plant to Tackle Infectious Diseases Flare Up: the Case of SARS

    Directory of Open Access Journals (Sweden)

    Olivia C eDemurtas

    2016-02-01

    Full Text Available Severe Acute Respiratory Syndrome (SARS is a dangerous infection with pandemic potential. It emerged in 2002 and its aetiological agent, the SARS Coronavirus (SARS-CoV, crossed the species barrier to infect humans, showing high morbidity and mortality rates. No vaccines are currently licensed for SARS-CoV and important efforts have been performed during the first outbreak to develop diagnostic tools. Here we demonstrate the transient expression in Nicotiana benthamiana of two important antigenic determinants of the SARS-CoV, the nucleocapsid protein (N and the membrane protein (M using a virus-derived vector or agro-infiltration, respectively. For the M protein, this is the first description of production in plants, while for plant-derived N protein we demonstrate that it is recognized by sera of patients from the SARS outbreak in Hong Kong in 2003. The availability of recombinant N and M proteins from plants opens the way to further evaluation of their potential utility for the development of diagnostic and protection/therapy tools to be quickly manufactured, at low cost and with minimal risk, to face potential new highly infectious SARS-CoV outbreaks.

  8. Antigen Production in Plant to Tackle Infectious Diseases Flare Up: The Case of SARS.

    Science.gov (United States)

    Demurtas, Olivia C; Massa, Silvia; Illiano, Elena; De Martinis, Domenico; Chan, Paul K S; Di Bonito, Paola; Franconi, Rosella

    2016-01-01

    Severe acute respiratory syndrome (SARS) is a dangerous infection with pandemic potential. It emerged in 2002 and its aetiological agent, the SARS Coronavirus (SARS-CoV), crossed the species barrier to infect humans, showing high morbidity and mortality rates. No vaccines are currently licensed for SARS-CoV and important efforts have been performed during the first outbreak to develop diagnostic tools. Here we demonstrate the transient expression in Nicotiana benthamiana of two important antigenic determinants of the SARS-CoV, the nucleocapsid protein (N) and the membrane protein (M) using a virus-derived vector or agro-infiltration, respectively. For the M protein, this is the first description of production in plants, while for plant-derived N protein we demonstrate that it is recognized by sera of patients from the SARS outbreak in Hong Kong in 2003. The availability of recombinant N and M proteins from plants opens the way to further evaluation of their potential utility for the development of diagnostic and protection/therapy tools to be quickly manufactured, at low cost and with minimal risk, to face potential new highly infectious SARS-CoV outbreaks.

  9. The Multi-parameter Prediction of B-cell Epitopes in Amino Acid Sequence Fragments of Spike Glycoprotein of SARS Coronavirus%SARS病毒S蛋白部分片段B-细胞表位的多参数预测

    Institute of Scientific and Technical Information of China (English)

    曾桥; 万志刚; 肖建华; 吴移谋; 谭立志; 万艳平; 张文波; 杨秋林; 尹卫国; 刘双全; 胡四海

    2003-01-01

    目的预测SARS病毒S蛋白(spike glycoprotein)部分片段(aa No.400~600 & aa No.1 100~1 195)的B-细胞表位.方法应用多种参数和方法进行综合分析,包括跨膜分析、保守区分析、同源性比较、Hopp & Woods亲水性参数、抗原性参数、可及性参数、β-转角、万氏法等综合预测方法.结果显示B-细胞识别的表位可能在436~456和1 136~1 146残基或其附近,这两个被预测的表位均含有β-转角和不规则卷曲结构.结论本研究为应用合成肽抗原制备抗SARS病毒S蛋白抗体、诊断非典型肺炎(severe acute respiratory syndrome, SARS)提供了依据.

  10. Identification of residues of SARS-CoV nsp1 that differentially affect inhibition of gene expression and antiviral signaling.

    Science.gov (United States)

    Jauregui, Andrew R; Savalia, Dhruti; Lowry, Virginia K; Farrell, Cara M; Wathelet, Marc G

    2013-01-01

    An epidemic of Severe Acute Respiratory Syndrome (SARS) led to the identification of an associated coronavirus, SARS-CoV. This virus evades the host innate immune response in part through the expression of its non-structural protein (nsp) 1, which inhibits both host gene expression and virus- and interferon (IFN)-dependent signaling. Thus, nsp1 is a promising target for drugs, as inhibition of nsp1 would make SARS-CoV more susceptible to the host antiviral defenses. To gain a better understanding of nsp1 mode of action, we generated and analyzed 38 mutants of the SARS-CoV nsp1, targeting 62 solvent exposed residues out of the 180 amino acid protein. From this work, we identified six classes of mutants that abolished, attenuated or increased nsp1 inhibition of host gene expression and/or antiviral signaling. Each class of mutants clustered on SARS-CoV nsp1 surface and suggested nsp1 interacts with distinct host factors to exert its inhibitory activities. Identification of the nsp1 residues critical for its activities and the pathways involved in these activities should help in the design of drugs targeting nsp1. Significantly, several point mutants increased the inhibitory activity of nsp1, suggesting that coronaviruses could evolve a greater ability to evade the host response through mutations of such residues.

  11. Evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus

    Institute of Scientific and Technical Information of China (English)

    MO Hong-ying; XU Jun; REN Xiao-lan; ZENG Guang-qiao; TAN Ya-xia; CHEN Rong-chang; Moira Chan-Yeung; ZHONG Nan-shan

    2005-01-01

    Background Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerging virus that gives rise to SARS patients with high rates of infectivity and fatality. To study the humoral immune responses to SARS-CoV, the authors evaluated IgG and IgM specific antibodies in patients' sera.Methods Two methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent assay (IFA), were used to detect specific serum IgG and IgM against SARS-CoV in 98 SARS patients and 250 controls consisting of patients with pneumonia, health-care professionals and healthy subjects. The serum antibody profiles were investigated at different times over one and a half years in 18 of the SARS patients. Results The sensitivity and specificity of ELISA for detecting IgG against SARS-CoV were 100.0% and 97.2% and for IgM 89.8% and 97.6% respectively; the figures using IFA for IgG were 100.0% and 100.0% and for IgM 81.8% and 100.0% respectively. During the first seven days of the antibodies trace test, no IgG and IgM were detected, but on day 15, IgG response increased dramatically, reaching a peak on day 60, remaining high up to day 180 and decreasing gradually until day 540. On day 15, IgM was detected, rapidly reached a peak, then declined gradually until day 180 when IgM was undetectable. Conclusion The detection of antibodies against SARS virus is helpful in the clinical diagnosis of SARS.

  12. The C-Terminal Portion of the Nucleocapsid Protein Demonstrates SARS-CoV Antigenicity

    Institute of Scientific and Technical Information of China (English)

    Guozhen Liu; Bo You; Ye Yin; Shuting Li; Hao Wang; Yan Ren; Jia Ji; Xiaoqian Zhao; Yongqiao Sun; Xiaowei Zhang; Jianqiu Fang; Shaohui Hu; Jian Wang; Siqi Liu; Jun Yu; Heng Zhu; Huanming Yang; Yongwu Hu; Peng Chen; Jianning Yin; Jie Wen; Jingqiang Wang; Liang Lin; Jinxiu Liu

    2003-01-01

    In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC)gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter.Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls.The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

  13. Novel Coronaviruses and Astroviruses in Bats

    Institute of Scientific and Technical Information of China (English)

    Daniel K. W. Chu; J. S. Malik Peiris; Leo L. M. Poon

    2009-01-01

    Zoonotic transmissions of emerging pathogens from wildlife to human have shaped the history of mankind. These events have also highlighted our poor understanding of microorganisms circulated in wild animals. Coronaviruses and astroviruses, which can be found from a wide range of mammals, were recently detected in bats. Strikingly, these bat viruses are genetically highly diverse and these interesting findings might help to better understand the evolution and ecology of these viruses. The discoveries of these novel bats viruses not only suggested that bats are important hosts for these virus families, but also reiterated the role of bats as a reservoir of viruses that might pose a zoonotic threat to human health.

  14. Inhibition of SARS Pseudovirus Cell Entry by Lactoferrin Binding to Heparan Sulfate Proteoglycans

    Science.gov (United States)

    Lang, Jianshe; Yang, Ning; Deng, Jiejie; Liu, Kangtai; Yang, Peng; Zhang, Guigen; Jiang, Chengyu

    2011-01-01

    It has been reported that lactoferrin (LF) participates in the host immune response against Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) invasion by enhancing NK cell activity and stimulating neutrophil aggregation and adhesion. We further investigated the role of LF in the entry of SARS pseudovirus into HEK293E/ACE2-Myc cells. Our results reveal that LF inhibits SARS pseudovirus infection in a dose-dependent manner. Further analysis suggested that LF was able to block the binding of spike protein to host cells at 4°C, indicating that LF exerted its inhibitory function at the viral attachment stage. However, LF did not disrupt the interaction of spike protein with angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV. Previous studies have shown that LF colocalizes with the widely distributed cell-surface heparan sulfate proteoglycans (HSPGs). Our experiments have also confirmed this conclusion. Treatment of the cells with heparinase or exogenous heparin prevented binding of spike protein to host cells and inhibited SARS pseudovirus infection, demonstrating that HSPGs provide the binding sites for SARS-CoV invasion at the early attachment phase. Taken together, our results suggest that, in addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry. LF may play a protective role in host defense against SARS-CoV infection through binding to HSPGs and blocking the preliminary interaction between SARS-CoV and host cells. Our findings may provide further understanding of SARS-CoV pathogenesis and aid in treatment of this deadly disease. PMID:21887302

  15. Comparative pathology of rhesus macaque and common marmoset animal models with Middle East respiratory syndrome coronavirus

    Science.gov (United States)

    Yu, Pin; Xu, Yanfeng; Deng, Wei; Bao, Linlin; Huang, Lan; Xu, Yuhuan; Yao, Yanfeng; Qin, Chuan

    2017-01-01

    Middle East respiratory syndrome (MERS), which is caused by a newly discovered coronavirus (CoV), has recently emerged. It causes severe viral pneumonia and is associated with a high fatality rate. However, the pathogenesis, comparative pathology and inflammatory cell response of rhesus macaques and common marmosets experimentally infected with MERS-CoV are unknown. We describe the histopathological, immunohistochemical, and ultrastructural findings from rhesus macaque and common marmoset animal models of MERS-CoV infection. The main histopathological findings in the lungs of rhesus macaques and common marmosets were varying degrees of pulmonary lesions, including pneumonia, pulmonary oedema, haemorrhage, degeneration and necrosis of the pneumocytes and bronchial epithelial cells, and inflammatory cell infiltration. The characteristic inflammatory cells in the lungs of rhesus macaques and common marmosets were eosinophils and neutrophils, respectively. Based on these observations, the lungs of rhesus macaques and common marmosets appeared to develop chronic and acute pneumonia, respectively. MERS-CoV antigens and viral RNA were identified in type I and II pneumocytes, alveolar macrophages and bronchial epithelial cells, and ultrastructural observations showed that viral protein was found in type II pneumocytes and inflammatory cells in both species. Correspondingly, the entry receptor DDP4 was found in type I and II pneumocytes, bronchial epithelial cells, and alveolar macrophages. The rhesus macaque and common marmoset animal models of MERS-CoV can be used as a tool to mimic the oncome of MERS-CoV infections in humans. These models can help to provide a better understanding of the pathogenic process of this virus and to develop effective medications and prophylactic treatments. PMID:28234937

  16. Isolation of Coronavirus NL63 from Blood from Children in Rural Haiti: Phylogenetic Similarities with Recent Isolates from Malaysia.

    Science.gov (United States)

    Beau De Rochars, Valery Madsen; Lednicky, John; White, Sarah; Loeb, Julia; Elbadry, Maha A; Telisma, Taina; Chavannes, Sonese; Anilis, Marie Gina; Cella, Eleonora; Ciccozzi, Massimo; Okech, Bernard A; Salemi, Marco; Morris, J Glenn

    2017-01-11

    Human coronavirus (HCoV) NL63 is recognized as a common cause of upper respiratory infections and influenza-like illness. In screening children with acute undifferentiated febrile illness in a school cohort in rural Haiti, we identified HCoV-NL63 in blood samples from four children. Cases clustered over an 11-day period; children did not have respiratory symptoms, but two had gastrointestinal complaints. On phylogenetic analysis, the Haitian HCoV-NL63 strains cluster together in a highly supported monophyletic clade linked most closely with recently reported strains from Malaysia; two respiratory HCoV-NL63 strains identified in north Florida in the same general period form a separate clade, albeit again with close linkages with the Malaysian strains. Our data highlight the variety of presentations that may be seen with HCoV-NL63, and underscore the apparent ease with which CoV strains move among countries, with our data consistent with recurrent introduction of strains into the Caribbean (Haiti and Florida) from Asia. © The American Society of Tropical Medicine and Hygiene.

  17. A complete sequence and comparative analysis of a SARS-associated virus (Isolate BJ01)

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-associated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs. We report the complete genome sequence and comparative analysis of an isolate (BJ01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 2 ORFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS- associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymousmutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host. Two amino acid changes have been detected in the M protein, which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).

  18. Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor.

    Science.gov (United States)

    Kim, Yunjeong; Liu, Hongwei; Galasiti Kankanamalage, Anushka C; Weerasekara, Sahani; Hua, Duy H; Groutas, William C; Chang, Kyeong-Ok; Pedersen, Niels C

    2016-03-01

    Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further

  19. Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor

    Science.gov (United States)

    Kim, Yunjeong; Liu, Hongwei; Galasiti Kankanamalage, Anushka C.; Weerasekara, Sahani; Hua, Duy H.; Groutas, William C.; Chang, Kyeong-Ok; Pedersen, Niels C.

    2016-01-01

    Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further

  20. Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor.

    Directory of Open Access Journals (Sweden)

    Yunjeong Kim

    2016-03-01

    Full Text Available Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP, can arise through mutation of FECV to FIP virus (FIPV. The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for

  1. Diagnostic Methods for Feline Coronavirus: A Review

    Directory of Open Access Journals (Sweden)

    Saeed Sharif

    2010-01-01

    Full Text Available Feline coronaviruses (FCoVs are found throughout the world. Infection with FCoV can result in a diverse range of signs from clinically inapparent infections to a highly fatal disease called feline infectious peritonitis (FIP. FIP is one of the most serious viral diseases of cats. While there is neither an effective vaccine, nor a curative treatment for FIP, a diagnostic protocol for FCoV would greatly assist in the management and control of the virus. Clinical findings in FIP are non-specific and not helpful in making a differential diagnosis. Haematological and biochemical abnormalities in FIP cases are also non-specific. The currently available serological tests have low specificity and sensitivity for detection of active infection and cross-react with FCoV strains of low pathogenicity, the feline enteric coronaviruses (FECV. Reverse transcriptase polymerase chain reaction (RT-PCR has been used to detect FCoV and is rapid and sensitive, but results must be interpreted in the context of clinical findings. At present, a definitive diagnosis of FIP can be established only by histopathological examination of biopsies. This paper describes and compares diagnostic methods for FCoVs and includes a brief account of the virus biology, epidemiology, and pathogenesis.

  2. Feline Coronaviruses: Pathogenesis of Feline Infectious Peritonitis.

    Science.gov (United States)

    Tekes, G; Thiel, H-J

    2016-01-01

    Feline infectious peritonitis (FIP) belongs to the few animal virus diseases in which, in the course of a generally harmless persistent infection, a virus acquires a small number of mutations that fundamentally change its pathogenicity, invariably resulting in a fatal outcome. The causative agent of this deadly disease, feline infectious peritonitis virus (FIPV), arises from feline enteric coronavirus (FECV). The review summarizes our current knowledge of the genome and proteome of feline coronaviruses (FCoVs), focusing on the viral surface (spike) protein S and the five accessory proteins. We also review the current classification of FCoVs into distinct serotypes and biotypes, cellular receptors of FCoVs and their presumed role in viral virulence, and discuss other aspects of FIPV-induced pathogenesis. Our current knowledge of genetic differences between FECVs and FIPVs has been mainly based on comparative sequence analyses that revealed "discriminatory" mutations that are present in FIPVs but not in FECVs. Most of these mutations result in amino acid substitutions in the S protein and these may have a critical role in the switch from FECV to FIPV. In most cases, the precise roles of these mutations in the molecular pathogenesis of FIP have not been tested experimentally in the natural host, mainly due to the lack of suitable experimental tools including genetically engineered virus mutants. We discuss the recent progress in the development of FCoV reverse genetics systems suitable to generate recombinant field viruses containing appropriate mutations for in vivo studies.

  3. Modal parameter identification of flexible spacecraft using the covariance-driven stochastic subspace identification (SSI-COV) method

    Institute of Scientific and Technical Information of China (English)

    Yong Xie; Pan Liu; Guo-Ping Cai

    2016-01-01

    In this paper, the on-orbit identification of modal parameters for a spacecraft is investigated. Firstly, the cou-pled dynamic equation of the system is established with the Lagrange method and the stochastic state-space model of the system is obtained. Then, the covariance-driven stochas-tic subspace identification (SSI-COV) algorithm is adopted to identify the modal parameters of the system. In this algo-rithm, it just needs the covariance of output data of the system under ambient excitation to construct a Toeplitz matrix, thus the system matrices are obtained by the singular value decom-position on the Toeplitz matrix and the modal parameters of the system can be found from the system matrices. Finally, numerical simulations are carried out to demonstrate the validity of the SSI-COV algorithm. Simulation results indi-cate that the SSI-COV algorithm is effective in identifying the modal parameters of the spacecraft only using the output data of the system under ambient excitation.

  4. Regulation of Stress Responses and Translational Control by Coronavirus

    Science.gov (United States)

    Fung, To Sing; Liao, Ying; Liu, Ding Xiang

    2016-01-01

    Similar to other viruses, coronavirus infection triggers cellular stress responses in infected host cells. The close association of coronavirus replication with the endoplasmic reticulum (ER) results in the ER stress responses, which impose a challenge to the viruses. Viruses, in turn, have come up with various mechanisms to block or subvert these responses. One of the ER stress responses is inhibition of the global protein synthesis to reduce the amount of unfolded proteins inside the ER lumen. Viruses have evolved the capacity to overcome the protein translation shutoff to ensure viral protein production. Here, we review the strategies exploited by coronavirus to modulate cellular stress response pathways. The involvement of coronavirus-induced stress responses and translational control in viral pathogenesis will also be briefly discussed. PMID:27384577

  5. Regulation of Stress Responses and Translational Control by Coronavirus

    Directory of Open Access Journals (Sweden)

    To Sing Fung

    2016-07-01

    Full Text Available Similar to other viruses, coronavirus infection triggers cellular stress responses in infected host cells. The close association of coronavirus replication with the endoplasmic reticulum (ER results in the ER stress responses, which impose a challenge to the viruses. Viruses, in turn, have come up with various mechanisms to block or subvert these responses. One of the ER stress responses is inhibition of the global protein synthesis to reduce the amount of unfolded proteins inside the ER lumen. Viruses have evolved the capacity to overcome the protein translation shutoff to ensure viral protein production. Here, we review the strategies exploited by coronavirus to modulate cellular stress response pathways. The involvement of coronavirus-induced stress responses and translational control in viral pathogenesis will also be briefly discussed.

  6. A coronavirus detected in the vampire bat Desmodus rotundus

    Directory of Open Access Journals (Sweden)

    Paulo Eduardo Brandão

    Full Text Available This article reports on the identification of a group 2 coronavirus (BatCoV DR/2007 in a Desmodus rotundus vampire bat in Brazil. Phylogenetic analysis of ORF1b revealed that BatCoV DR/2007 originates from a unique lineage in the archetypical group 2 coronaviruses, as described for bat species elsewhere with putative importance in Public Health.

  7. Health Communication during SARS

    Science.gov (United States)

    Navin, Ava W.; Steele, Stefanie F.; Weld, Leisa H.; Kozarsky, Phyllis E.

    2004-01-01

    During the severe acute respiratory syndrome (SARS) outbreak, electronic media made it possible to disseminate prevention messages rapidly. The Centers for Disease Control and Prevention’s Travelers’ Health Web site was frequently visited in the first half of 2003; more than 2.6 million visits were made to travel alerts, advisories, and other SARS-related documents. PMID:15030717

  8. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis.

    Directory of Open Access Journals (Sweden)

    Jose L Nieto-Torres

    2014-05-01

    Full Text Available Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV envelope (E gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS

  9. SAR: Stroke Authorship Recognition

    KAUST Repository

    Shaheen, Sara

    2015-10-15

    Are simple strokes unique to the artist or designer who renders them? If so, can this idea be used to identify authorship or to classify artistic drawings? Also, could training methods be devised to develop particular styles? To answer these questions, we propose the Stroke Authorship Recognition (SAR) approach, a novel method that distinguishes the authorship of 2D digitized drawings. SAR converts a drawing into a histogram of stroke attributes that is discriminative of authorship. We provide extensive classification experiments on a large variety of data sets, which validate SAR\\'s ability to distinguish unique authorship of artists and designers. We also demonstrate the usefulness of SAR in several applications including the detection of fraudulent sketches, the training and monitoring of artists in learning a particular new style and the first quantitative way to measure the quality of automatic sketch synthesis tools. © 2015 The Eurographics Association and John Wiley & Sons Ltd.

  10. Nuclear Magnetic Resonance Structure Shows that the Severe Acute Respiratory Syndrome Coronavirus-Unique Domain Contains a Macrodomain Fold▿

    Science.gov (United States)

    Chatterjee, Amarnath; Johnson, Margaret A.; Serrano, Pedro; Pedrini, Bill; Joseph, Jeremiah S.; Neuman, Benjamin W.; Saikatendu, Kumar; Buchmeier, Michael J.; Kuhn, Peter; Wüthrich, Kurt

    2009-01-01

    The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for “middle of the SARS-unique domain”) in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1"-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection. PMID:19052085

  11. PIKA Provides an Adjuvant Effect to Induce Strong Mucosal and Systemic Humoral Immunity Against SARS-CoV

    Institute of Scientific and Technical Information of China (English)

    Wei-wei Gai; Yan Zhang; Di-han Zhou; Yao-qing Chen; Jing-yi Yang; Hui-min Yan

    2011-01-01

    Severe Acute Respiratory Syndrome(SARS)is a deadly infectious disease caused by SARS Coronavirus(SARS-CoV).Inactivated SARS-CoV has been explored as a vaccine against SARS-CoV.However,safe and potent adjuvants,especially with more efficient and economical needle-free vaccination are always needed more urgently in a pandemic.The development of a safe and effective mucosal adjuvant and vaccine for prevention of emergent infectious diseases such as SARS will be an important advancement.PIKA,a stabilized derivative of Poly(I:C),was previously reported to be safe and potent as adjuvant in mouse models.In the present study,we demonstrated that the intraperitoneal and intranasal co-administration of inactivated SARS-CoV vaccine together with this improved Poly(I:C)derivative induced strong anti-SARS-CoV mucosal and systemic humoral immune responses with neutralizing activity against pseudotyped virus.Although intraperitoneal immunization of inactivated SARS-CoV vaccine alone could induce a certain level of neutralizing activity in serum as well as in mucosal sites,co-administration of inactivated SARS-CoV vaccine with PIKA as adjuvant could induce a much higher neutralizing activity.When intranasal immunization was used,PIKA was obligatorily for inducing neutralizing activity in serum as well as in mucosal sites and was correlated with both mucosal IgA and mucosal IgG response.Overall,PIKA could be a good mucosal adjuvant candidate for inactivated SARS-CoV vaccine for use in possible future pandemic.

  12. European Surveillance for Pantropic Canine Coronavirus

    Science.gov (United States)

    Cordonnier, Nathalie; Demeter, Zoltan; Egberink, Herman; Elia, Gabriella; Grellet, Aurélien; Le Poder, Sophie; Mari, Viviana; Martella, Vito; Ntafis, Vasileios; von Reitzenstein, Marcela; Rottier, Peter J.; Rusvai, Miklos; Shields, Shelly; Xylouri, Eftychia; Xu, Zach; Buonavoglia, Canio

    2013-01-01

    Highly virulent pantropic canine coronavirus (CCoV) strains belonging to subtype IIa were recently identified in dogs. To assess the distribution of such strains in Europe, tissue samples were collected from 354 dogs that had died after displaying systemic disease in France (n = 92), Hungary (n = 75), Italy (n = 69), Greece (n = 87), The Netherlands (n = 27), Belgium (n = 4), and Bulgaria (n = 1). A total of 124 animals tested positive for CCoV, with 33 of them displaying the virus in extraintestinal tissues. Twenty-four CCoV strains (19.35% of the CCoV-positive dogs) detected in internal organs were characterized as subtype IIa and consequently assumed to be pantropic CCoVs. Sequence and phylogenetic analyses of the 5′ end of the spike protein gene showed that pantropic CCoV strains are closely related to each other, with the exception of two divergent French viruses that clustered with enteric strains. PMID:23100349

  13. [New coronavirus infection: new challenges, new legacies].

    Science.gov (United States)

    Cabrera-Gaytán, David Alejandro; Vargas-Valerio, Alfredo; Grajales-Muñiz, Concepción

    2014-01-01

    Introducción: emergió una nueva enfermedad por coronavirus. Su historia natural y sus determinantes todavía se están investigando. Se carece de una publicación que estudie todos los casos identificados en el mundo, por lo que el objetivo de este artículo estriba en describir los casos y defunciones por el nuevo coronavirus. Métodos: se revisaron las publicaciones en línea de la Organización Mundial de la Salud, del Centro Europeo para el Control y Prevención de Enfermedades y de la Eurosurveillance. Se realizó un análisis descriptivo de los casos, se calcularon los límites para proporciones con un alfa del 0.05 por prueba de Wilson y una prueba t de Student para diferencia de medias. Resultados: son 17 casos confirmados y 11 defunciones en varios países de Asia y Europa; predominaron los pacientes masculinos. La tasa de letalidad fue de 64.70 %; los que fallecieron se hospitalizaron cinco días después de los primeros síntomas. Se carece de publicaciones que describan la historia natural de la enfermedad; sin embargo, lo descrito en las publicaciones de Europa coincide con los resultados de este estudio. Conclusión: es necesario continuar con la vigilancia epidemiológica y la realización de nuevos estudios para evaluar el impacto de esta enfermedad en la salud pública internacional.

  14. Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2.

    Science.gov (United States)

    Reinke, Lennart Michel; Spiegel, Martin; Plegge, Teresa; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael; Pöhlmann, Stefan

    2017-01-01

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.

  15. Human LINE1 endonuclease domain as a putative target of SARS-associated autoantibodies involved in the pathogenesis of severe acute respiratory syndrome

    Institute of Scientific and Technical Information of China (English)

    HE Wei-ping; SHU Cui-li; LI Bo-an; ZHAO Jun; CHENG Yun

    2008-01-01

    Background Severe acute respiratory syndrome(SARS)is a disease with a mortality of 9.56%.Although SARS is etiologically linked to a new coronavirus(SARS-CoV)and functional cell receptor has been identified,the pathogenesis of the virus infection is largely unclear.Methods The clinical specimens were processed and analyzed using an indirect enzyme-linked immunosorbent assay (ELISA) in-house.Further investigations of target antigen included reviews of phage display technique,rapid amplification of cDNA ends(RACE)technique,protein expression and purification,Western blotting validation,serological and immunohistochemical staining in postmortem tissue.Results A type of medium or low titer anti-lung tissue antibodies were found in the sera of SARS patients at the early stage of the disease.Human long interspersed nuclear element 1(LINE1)gene endonuclease(EN)domain protein was one of the target autoantigens and it was aberrantly expressed in the lung tissue of SARS patients.Anti-EN antibody was positive in the sera of 40.9% of SARS patients.Conclusions Human LINE1 endonuclease domain was identified as a putative target of SARS-associated autoantibodies,which were presented in the serum of SARS patients and may be involved in the pathogenesis of SARS.

  16. Identification of Aminopeptidase N as a Cellular Receptor for Human Coronavirus-229E

    Science.gov (United States)

    1992-05-12

    feline enteric coronav irus feline infectious peritonitis virus hUman adult intestine hUman aminopeptidase N human aminopeptidase with 39 amino...coronavirus (TCV), rat coronavirus (RCV), cat feline infectious peritonitis virus (FIPV), and the hUman coronaviruses. These include the slow, patchy...While the cat, dog and pig serve as natural hosts for the other coronavirus group 1 viruses, feline infectious peritonitis virus (FIPV), canine

  17. Variation analysis of the severe acute respiratory syndrome coronavirus putative non-structural protein 2 gene and construction of three-dimensional model

    Institute of Scientific and Technical Information of China (English)

    LU Jia-hai; CHEN Wei-qing; LING Wen-hua; YU Xin-bing; ZHONG Nan-shan; ZHANG Ding-mei; WANG Guo-ling; GUO Zhong-min; ZHANG Chuan-hai; TAN Bing-yan; OUYANG Li-ping; LIN Li; LIU Yi-min

    2005-01-01

    Background The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CLpro, following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain.Methods The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein.Results The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7

  18. ONERA airborne SAR facilities

    Energy Technology Data Exchange (ETDEWEB)

    Boutry, J.M. [Office National d`Etudes et de Recherches Aerospatiales (ONERA), Chatillon (France)

    1996-11-01

    ONERA has developed and operates the RAMSES experimental SAR on board a TRANSALL C160 aircraft. This system has been designed in order to analyze the effect of various parameters, such as frequency, polarization, incidence, resolution,... in the field of air-to-ground radar applications. These applications include SAR imaging for ground radar applications. These applications include SAR imaging for various purposes such as map-matching for navigation update, battlefield surveillance, reconnaissance, treaty applications... It consists of several radar sections operating over a wide range of frequency bands (L, S, C, X, Ku, Ka, W). 7 figs., 3 tabs.

  19. Raman scattering investigations of the interaction of a COV with pure and acid doped ice particles

    Science.gov (United States)

    Facq, S.; Oancea, A.; Focsa, C.; Chazallon, B.

    2009-04-01

    Ice present in polar stratosphere is as well a common component of the troposphere, particularly in cirrus clouds widespread in tropopause and upper troposphere region. With water droplets, ice constitutes the condensed matter that can interact with atmospheric trace gases via many different trapping processes (co-deposition i.e; incorporation during growing ice conditions, adsorption, freezing etc). The incorporation of trace gases in ice surface/volume can both affect the atmospheric chemistry and the ice structure and reactivity. This can therefore modify the nature and composition of the incorporated species in ice, or in the gas phase. Recently, field measurements have demonstrated the presence of nitric acid in ice particles from cirrus clouds(1,2) (concentration between 0.63 wt% and 2.5 wt %). Moreover, laboratory experiments have shown that the uptake of atmospheric trace gases can be enhanced up to 1 or 2 orders of magnitude in these doped ice particles. Among trace gases capable to interact with atmospheric condensed matter figure volatile organic compounds such as aldehydes, ketones and alcohols (ex: ethanol and methanol). They play an important role in the upper troposphere (3,4) and snowpack chemistry (5) as they can be easily photolysed, producing free radicals and so influence the oxidizing capacity and the ozone-budget of the atmosphere (3,4). The temperature range at which these physico-chemical processes occur extents between ~ 190 K and 273K. Interaction between ice and trace gases are therefore largely dependent on the ice surface properties as well as on the phase formation dynamic (crystalline or not). This study aims to examine and characterize the incorporation of a COV (ex: ethanol), at the surface or in the volume of ice formed by different growth mechanisms (vapour deposition or droplets freezing). Vibrational spectra of water OH and ethanol CH-spectral regions are analysed using confocal micro-Raman spectroscopy at different temperatures

  20. Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate.

    Science.gov (United States)

    Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

    2014-01-01

    Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318-510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318-536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.

  1. The R Protein of SARS-CoV: Analyses of Structure and Function Based on Four Complete Genome Sequences of Isolates BJ01-BJ04

    Institute of Scientific and Technical Information of China (English)

    Zuyuan Xu; Zizhang Zhang; Jing Xu; Wei Wei; Jingui Zhu; Haiyan Sun; Xiaowei Zhang; Jun Zhou; Songgang Li; Jun Wang; Jian Wang; Haiqing Zhang; Shengli Bi; Huanming Yang; Xiangjun Tian; Jia Ji; Wei Li; Yan Li; Wei Tian; Yujun Han; Lili Wang

    2003-01-01

    The R (replicase) protein is the uniquely defined non-structural protein (NSP)responsible for RNA replication, mutation rate or fidelity, regulation of transcrip-tion in coronaviruses and many other ssRNA viruses. Based on our completegenome sequences of four isolates (BJ01-BJ04) of SARS-CoV from Beijing, China,we analyzed the structure and predicted functions of the R protein in comparisonwith 13 other isolates of SARS-CoV and 6 other coronaviruses. The entire ORF(open-reading frame) encodes for two major enzyme activities, RNA-dependentRNA polymerase (RdRp) and proteinase activities. The R polyprotein under-goes a complex proteolytic process to produce 15 function-related peptides. Ahydrophobic domain (HOD) and a hydrophilic domain (HID) are newly identifiedwithin NSP1. The substitution rate of the R protein is close to the average ofthe SARS-CoV genome. The functional domains in all NSPs of the R proteingive different phylogenetic results that suggest their different mutation rate underselective pressure. Eleven highly conserved regions in RdRp and twelve cleavagesites by 3CLP (chymotrypsin-like protein) have been identified as potential drugtargets. Findings suggest that it is possible to obtain information about the phy-logeny of SARS-CoV, as well as potential tools for drug design, genotyping anddiagnostics of SARS.

  2. Exacerbated Innate Host Response to SARS-CoV in Aged Non-Human Primates

    Science.gov (United States)

    Smits, Saskia L.; de Lang, Anna; van den Brand, Judith M. A.; Leijten, Lonneke M.; van IJcken, Wilfred F.; Eijkemans, Marinus J. C.; van Amerongen, Geert; Kuiken, Thijs; Andeweg, Arno C.; Osterhaus, Albert D. M. E.; Haagmans, Bart L.

    2010-01-01

    The emergence of viral respiratory pathogens with pandemic potential, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza A H5N1, urges the need for deciphering their pathogenesis to develop new intervention strategies. SARS-CoV infection causes acute lung injury (ALI) that may develop into life-threatening acute respiratory distress syndrome (ARDS) with advanced age correlating positively with adverse disease outcome. The molecular pathways, however, that cause virus-induced ALI/ARDS in aged individuals are ill-defined. Here, we show that SARS-CoV-infected aged macaques develop more severe pathology than young adult animals, even though viral replication levels are similar. Comprehensive genomic analyses indicate that aged macaques have a stronger host response to virus infection than young adult macaques, with an increase in differential expression of genes associated with inflammation, with NF-κB as central player, whereas expression of type I interferon (IFN)-β is reduced. Therapeutic treatment of SARS-CoV-infected aged macaques with type I IFN reduces pathology and diminishes pro-inflammatory gene expression, including interleukin-8 (IL-8) levels, without affecting virus replication in the lungs. Thus, ALI in SARS-CoV-infected aged macaques developed as a result of an exacerbated innate host response. The anti-inflammatory action of type I IFN reveals a potential intervention strategy for virus-induced ALI. PMID:20140198

  3. Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV.

    Science.gov (United States)

    Park, Ji-Young; Ko, Jin-A; Kim, Dae Wook; Kim, Young Min; Kwon, Hyung-Jun; Jeong, Hyung Jae; Kim, Cha Young; Park, Ki Hun; Lee, Woo Song; Ryu, Young Bae

    2016-01-01

    Two viral proteases of severe acute respiratory syndrome coronavirus (SARS-CoV), a chymotrypsin-like protease (3CL(pro)) and a papain-like protease (PL(pro)) are attractive targets for the development of anti-SARS drugs. In this study, nine alkylated chalcones (1-9) and four coumarins (10-13) were isolated from Angelica keiskei, and the inhibitory activities of these constituents against SARS-CoV proteases (3CL(pro) and PL(pro)) were determined (cell-free/based). Of the isolated alkylated chalcones, chalcone 6, containing the perhydroxyl group, exhibited the most potent 3CL(pro) and PL(pro) inhibitory activity with IC50 values of 11.4 and 1.2 µM. Our detailed protein-inhibitor mechanistic analysis of these species indicated that the chalcones exhibited competitive inhibition characteristics to the SARS-CoV 3CL(pro), whereas noncompetitive inhibition was observed with the SARS-CoV PL(pro).

  4. An immunosuppressed Syrian golden hamster model for SARS-CoV infection.

    Science.gov (United States)

    Schaecher, Scott R; Stabenow, Jennifer; Oberle, Christina; Schriewer, Jill; Buller, R Mark; Sagartz, John E; Pekosz, Andrew

    2008-10-25

    Several small animal models have been developed for the study of severe acute respiratory syndrome coronavirus (SARS-CoV) replication and pathogenesis. Syrian golden hamsters are among the best small animal models, though little clinical illness and no mortality are observed after virus infection. Cyclophosphamide was used to immunosuppress hamsters leading to a prolonged disease course and higher mortality after SARS-CoV infection. In addition, there was a significant weight loss, expanded tissue tropism, and increased viral pathology in the lung, heart, kidney, and nasal turbinate tissues. Infection with recombinant SARS-CoV viruses bearing disruptions in the gene 7 coding region showed no significant change in replication kinetics, tissue tropism, morbidity, or mortality suggesting that the ORF7a (7a) and ORF7b (7b) proteins are not required for virus replication in immunosuppressed hamsters. This modified hamster model may provide a useful tool for SARS-CoV pathogenesis studies, evaluation of antiviral therapy, and analysis of additional SARS-CoV mutants.

  5. Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

    Directory of Open Access Journals (Sweden)

    Mark W. Jackwood

    2011-09-01

    Full Text Available Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus.

  6. SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

    Science.gov (United States)

    Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P C; Ovaa, Huib; Drag, Marcin; Lima, Christopher D; Huang, Tony T

    2015-06-01

    Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

  7. Identification of an HLA-A* 0201-restricted CD8+ T-cell epitope SSp-1 of SARS-CoV spike protein

    Institute of Scientific and Technical Information of China (English)

    Wang B; Yu Y; Wang X; Yang R; Cao X; Chen H; Jiang X; Zhang M; Wan T; Li N; Zhou X; Wu Y; Yang F

    2004-01-01

    A novel coronavirus, severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), has been identified as the causal agent of SARS. Spike (S) protein is a major structural glycoprotein of the SARS virus and a potential target for SARS-specific cell-mediated immune responses. A panel of S protein-derived peptides was tested for their binding affinity to HLA-A * 0201 molecules. Peptides with high affinity for HLA-A * 0201 were then assessed for their capacity to elicit specific immune responses mediated by cytotoxic T lymphocytes (CTLs) both in vivo, in HLA-A2. 1/Kb transgenic mice, and in vitro, from peripheral blood lymphocytes (PBLs) harvested from healthy HLA-A2.1 + donors. SARS-CoV protein-derived peptide-1 (SSp-1 RLNEVAKNL), induced peptide-specific CTLs both in vivo (transgenic mice) and in vitro (human PBLs), which specifically released interferon-gamma (IFN-gamma) upon stimulation with SSp-1-pulsed autologous dendritic cells (DCs) or T2 cells. SSp-1-specific CTLs also lysed major histocompatibility complex (MHC)-matched tumor cell lines engineered to express S proteins. HLA-A * 0201-SSp-1 tetramer staining revealed the presence of significant populations of SSp-1-specific CTLs in SSp-1-induced CD8+ T cells. We propose that the newly identified epitope SSp-1 will help in the characterization of virus control mechanisms and immunopathology in SARS-CoV infection, and may be relevant to the development of immunotherapeutic approaches for SARS.

  8. Middle East Respiratory Syndrome Coronavirus: A Review

    Directory of Open Access Journals (Sweden)

    Leila Sarparast

    2015-01-01

    Full Text Available Context: Middle East Respiratory Syndrome Coronavirus (MERS-CoV infection is an emerging human disease that has been reported from the Arabian Peninsula and Middle East countries since 2012. Although zoonotic transmission was postulated, virological and serological finding suggest that the dromedary camels act as the potential reservoirs of MERS-CoV infection to humans. As October 2014, a totally 855 confirmed cases with 333 related deaths were reported to WHO. All cases occurred in or epidemiologically linked to affected countries. The virus ability to induce a pandemic attack is limited. The clinical presentations vary and range from asymptomatic infection to severe respiratory disease and death. However, most severe disease occurs in elderly and in those with underlying conditions. Infection prevention and control measures are critical to prevent the possible spread of MERS-CoV infection is health care facilities and in the community. The WHO encourages all member states to perform surveillance of patients with acute severe respiratory infection and to carefully monitor any unusual patterns. This paper aims to review the current key characteristics of MERS-CoV infection in human and update the WHO recommendations about this illness.

  9. Genotyping coronaviruses associated with feline infectious peritonitis.

    Science.gov (United States)

    Lewis, Catherine S; Porter, Emily; Matthews, David; Kipar, Anja; Tasker, Séverine; Helps, Christopher R; Siddell, Stuart G

    2015-06-01

    Feline coronavirus (FCoV) infections are endemic among cats worldwide. The majority of infections are asymptomatic or result in only mild enteric disease. However, approximately 5 % of cases develop feline infectious peritonitis (FIP), a systemic disease that is a frequent cause of death in young cats. In this study, we report the complete coding genome sequences of six FCoVs: three from faecal samples from healthy cats and three from tissue lesion samples from cats with confirmed FIP. The six samples were obtained over a period of 8 weeks at a single-site cat rescue and rehoming centre in the UK. We found amino acid differences located at 44 positions across an alignment of the six virus translatomes and, at 21 of these positions, the differences fully or partially discriminated between the genomes derived from the faecal samples and the genomes derived from the tissue lesion samples. In this study, two amino acid differences fully discriminated the two classes of genomes: these were both located in the S2 domain of the virus surface glycoprotein gene. We also identified deletions in the 3c protein ORF of genomes from two of the FIP samples. Our results support previous studies that implicate S protein mutations in the pathogenesis of FIP.

  10. Coronaviruses in brain tissue from patients with multiple sclerosis

    DEFF Research Database (Denmark)

    Dessau, R B; Lisby, G; Frederiksen, J L

    2001-01-01

    Brain tissue from 25 patients with clinically definite multiple sclerosis (MS) and as controls brain tissue from 36 patients without neurological disease was tested for the presence of human coronaviral RNA. Four PCR assays with primers specific for N-protein of human coronavirus strain 229E...... in the proportion of positive signals from the MS patients compared to controls. Evidence for a chronic infection with the human coronaviruses strain 229E or OC43 in brain tissue from patients with MS or controls has not been found in this study....

  11. Functional genomics highlights differential induction of antiviral pathways in the lungs of SARS-CoV-infected macaques.

    Directory of Open Access Journals (Sweden)

    Anna de Lang

    2007-08-01

    Full Text Available The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV is likely mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. Using functional genomics, we analyzed early host responses to SARS-CoV infection in the lungs of adolescent cynomolgus macaques (Macaca fascicularis that show lung pathology similar to that observed in human adults with SARS. Analysis of gene signatures revealed induction of a strong innate immune response characterized by the stimulation of various cytokine and chemokine genes, including interleukin (IL-6, IL-8, and IP-10, which corresponds to the host response seen in acute respiratory distress syndrome. As opposed to many in vitro experiments, SARS-CoV induced a wide range of type I interferons (IFNs and nuclear translocation of phosphorylated signal transducer and activator of transcription 1 in the lungs of macaques. Using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. Our studies emphasize that the induction of early IFN signaling may be critical to confer protection against SARS-CoV infection and highlight the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of SARS.

  12. The SARS coronavirus spike glycoprotein is selectively recognized by lung surfactant protein D and activates macrophages

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Zhong, Fei; Chow, Vincent T K;

    2007-01-01

    Da glycosylated protein. It was not secreted in the presence of tunicamycin and was detected as a 130 kDa protein in the cell lysate. The purified S-protein bound to Vero but not 293T cells and was itself recognized by lung surfactant protein D (SP-D), a collectin found in the lung alveoli. The binding required...

  13. SARS transmission pattern in Singapore reassessed by viral sequence variation analysis.

    Directory of Open Access Journals (Sweden)

    Jianjun Liu

    2005-02-01

    Full Text Available BACKGROUND: Epidemiological investigations of infectious disease are mainly dependent on indirect contact information and only occasionally assisted by characterization of pathogen sequence variation from clinical isolates. Direct sequence analysis of the pathogen, particularly at a population level, is generally thought to be too cumbersome, technically difficult, and expensive. We present here a novel application of mass spectrometry (MS-based technology in characterizing viral sequence variations that overcomes these problems, and we apply it retrospectively to the severe acute respiratory syndrome (SARS outbreak in Singapore. METHODS AND FINDINGS: The success rate of the MS-based analysis for detecting SARS coronavirus (SARS-CoV sequence variations was determined to be 95% with 75 copies of viral RNA per reaction, which is sufficient to directly analyze both clinical and cultured samples. Analysis of 13 SARS-CoV isolates from the different stages of the Singapore outbreak identified nine sequence variations that could define the molecular relationship between them and pointed to a new, previously unidentified, primary route of introduction of SARS-CoV into the Singapore population. Our direct determination of viral sequence variation from a clinical sample also clarified an unresolved epidemiological link regarding the acquisition of SARS in a German patient. We were also able to detect heterogeneous viral sequences in primary lung tissues, suggesting a possible coevolution of quasispecies of virus within a single host. CONCLUSION: This study has further demonstrated the importance of improving clinical and epidemiological studies of pathogen transmission through the use of genetic analysis and has revealed the MS-based analysis to be a sensitive and accurate method for characterizing SARS-CoV genetic variations in clinical samples. We suggest that this approach should be used routinely during outbreaks of a wide variety of agents, in order

  14. A 3D model of SARS_CoV 3CL proteinase and its inhibitors design by virtual screening

    Institute of Scientific and Technical Information of China (English)

    LUOCheng; CHENJing; LUOHai-Bin; CHENLi-Li; LIGuo-Wei; SUNTao; YUChang-Ying; YUELi-Duo; SHENJian-Hua; JIANGHua-Liang; XIONGBin; GUIChun-Shan; XUXiao-Ying; DUANWen-Hu; SHENJing-Kang; QINLei; SHITi-Liu; LIYi-Xue; CHENKai-Xian; LUOXiao-Min; SHENXu

    2003-01-01

    AIM:To constructed a three-dimensional (3D) model for the 3C like (3CL) proteinase of SARS coronavirus (SARS_CoV), and to design inhibitors of the 3CL proteinase based on the 3D model. METHODS: Bioinformatics analyses were performed to search the homologous proteins of the SARS_CoV 3CL proteinase from the GenBank and PDB database. A 3D model of the proteinase was constructed by using homology modeling technique. Targeting to the 3D model and its X-ray crystal structure of the main proteinase (Mpro) of transmissible gastroenteritis virus(TGEV), virtual screening was performed employing molecular docking method to identify possible 3CL proteinase inhibitors from small molecular databases. RESULTS:Sequence alignment indicated that the SARS_CoV 3CL proteinase was extremely homologous to TGEV Mpro, especially the substrate-binding pocket (active site). Accordingly, a 3D model for the SARS_CoV 3CL proteinase was constructed based on the crystal structure of TGEV Mpro. The 3D model adopts a similar fold of the TGEV mpro, its structure and binding pocket feature are almost as same as that of TGEV Mpro. The tested virtual screening indicated that 73 available proteinase inhibitors in the MDDR database might dock into both the binding pockets of the TGEV Mpro and the SARS_CoV 3CL proteinase. CONCLUSIONS:Either the 3D model of the SARS_CoV 3CL proteinase or the X-ray crystal stucture of the TGEV Mpro may be used as a starting point for design anti-SARS drugs. Screening the known proteinase inhibitors may be an appreciated shortcut to discover anti-SARS drugs.

  15. Crop Classification by Polarimetric SAR

    DEFF Research Database (Denmark)

    Skriver, Henning; Svendsen, Morten Thougaard; Nielsen, Flemming;

    1999-01-01

    Polarimetric SAR-data of agricultural fields have been acquired by the Danish polarimetric L- and C-band SAR (EMISAR) during a number of missions at the Danish agricultural test site Foulum during 1995. The data are used to study the classification potential of polarimetric SAR data using...

  16. Case characteristics among Middle East respiratory syndrome coronavirus outbreak and non-outbreak cases in Saudi Arabia from 2012 to 2015

    Science.gov (United States)

    Alhamlan, F S; Majumder, M S; Brownstein, J S; Hawkins, J; Al-Abdely, H M; Alzahrani, A; Obaid, D A; Al-Ahdal, M N; BinSaeed, A

    2017-01-01

    Objectives As of 1 November 2015, the Saudi Ministry of Health had reported 1273 cases of Middle East respiratory syndrome (MERS); among these cases, which included 9 outbreaks at several hospitals, 717 (56%) patients recovered, 14 (1%) remain hospitalised and 543 (43%) died. This study aimed to determine the epidemiological, demographic and clinical characteristics that distinguished cases of MERS contracted during outbreaks from those contracted sporadically (ie, non-outbreak) between 2012 and 2015 in Saudi Arabia. Design Data from the Saudi Ministry of Health of confirmed outbreak and non-outbreak cases of MERS coronavirus (CoV) infections from September 2012 through October 2015 were abstracted and analysed. Univariate and descriptive statistical analyses were conducted, and the time between disease onset and confirmation, onset and notification and onset and death were examined. Results A total of 1250 patients (aged 0–109 years; mean, 50.825 years) were reported infected with MERS-CoV. Approximately two-thirds of all MERS cases were diagnosed in men for outbreak and non-outbreak cases. Healthcare workers comprised 22% of all MERS cases for outbreak and non-outbreak cases. Nosocomial infections comprised one-third of all Saudi MERS cases; however, nosocomial infections occurred more frequently in outbreak than non-outbreak cases (pnosocomial infections have fuelled MERS outbreaks. Given that the Kingdom of Saudi Arabia is a worldwide religious travel destination, localised outbreaks may have massive global implications and effective outbreak preventive measures are needed. PMID:28082362

  17. Utility of feline coronavirus antibody tests.

    Science.gov (United States)

    Addie, Diane D; le Poder, Sophie; Burr, Paul; Decaro, Nicola; Graham, Elizabeth; Hofmann-Lehmann, Regina; Jarrett, Oswald; McDonald, Michael; Meli, Marina L

    2015-02-01

    Eight different tests for antibodies to feline coronavirus (FCoV) were evaluated for attributes that are important in situations in veterinary practice. We compared four indirect immunofluorescent antibody tests (IFAT), one enzyme-linked immunosorbent assay (ELISA) (FCoV Immunocomb; Biogal) and three rapid immunochromatographic (RIM) tests against a panel of samples designated by consensus as positive or negative. Specificity was 100% for all but the two IFATs based on transmissible gastroenteritis virus (TGEV), at 83.3% and 97.5%. The IFAT and ELISA tests were best for obtaining an antibody titre and for working in the presence of virus. The RIM tests were the best for obtaining a result quickly (10-15 mins); of these, the Speed F-Corona was the most sensitive, at 92.4%, followed by FASTest feline infectious peritonitis (FIP; 84.6%) and Anigen Rapid FCoV antibody test (64.1%). Sensitivity was 100% for the ELISA, one FCoV IFAT and one TGEV IFAT; and 98.2% for a second TGEV IFA and 96.1% for a second FCoV IFAT. All tests worked with effusions, even when only blood products were stipulated in the instruction manual. The ELISA and Anigen RIM tests were best for small quantities of sample. The most appropriate FCoV antibody test to use depends on the reason for testing: in excluding a diagnosis of FIP, sensitivity, specificity, small sample quantity, rapidity and ability to work in the presence of virus all matter. For FCoV screening, speed and sensitivity are important, and for FCoV elimination antibody titre is essential. © ISFM and AAFP 2014.

  18. Structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease.

    Science.gov (United States)

    Chou, Chi-Yuan; Lai, Hsing-Yi; Chen, Hung-Yi; Cheng, Shu-Chun; Cheng, Kai-Wen; Chou, Ya-Wen

    2014-02-01

    Papain-like protease (PLpro) is one of two cysteine proteases involved in the proteolytic processing of the polyproteins of Severe acute respiratory syndrome coronavirus (SARS-CoV). PLpro also shows significant in vitro deubiquitinating and de-ISGylating activities, although the detailed mechanism is still unclear. Here, the crystal structure of SARS-CoV PLpro C112S mutant in complex with ubiquitin (Ub) is reported at 1.4 Å resolution. The Ub core makes mostly hydrophilic interactions with PLpro, while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PLpro, mimicking the P4-P1 residues and providing the first atomic insights into its catalysis. One of the O atoms of the C-terminal Gly residue of Ub is located in the oxyanion hole consisting of the main-chain amides of residues 112 and 113. Mutations of residues in the PLpro-Ub interface lead to reduced catalytic activity, confirming their importance for Ub binding and/or catalysis. The structure also revealed an N-cyclohexyl-2-aminethanesulfonic acid molecule near the catalytic triad, and kinetic studies suggest that this binding site is also used by other PLpro inhibitors. Overall, the structure provides a foundation for understanding the molecular basis of coronaviral PLpro catalysis.

  19. Geographic distribution of MERS coronavirus among dromedary camels, Africa

    NARCIS (Netherlands)

    Reusken, Chantal B E M; Messadi, Lilia; Feyisa, Ashenafi; Ularamu, Hussaini; Godeke, Gert Jan; Danmarwa, Agom; Dawo, Fufa; Jemli, Mohamed; Melaku, Simenew; Shamaki, David; Woma, Yusuf; Wungak, Yiltawe; Gebremedhin, Endrias Zewdu; Zutt, Ilse; Bosch, Berend Jan; Haagmans, Bart L.; Koopmans, Marion P G

    2014-01-01

    We found serologic evidence for the circulation of Middle East respiratory syndrome coronavirus among dromedary camels in Nigeria, Tunisia, and Ethiopia. Circulation of the virus among dromedaries across broad areas of Africa may indicate that this disease is currently underdiagnosed in humans outsi

  20. MERS Coronavirus in Dromedary Camel Herd Saudi Arabia

    OpenAIRE

    Hemida, Maged G.; Chu, Daniel K. W.; Poon, Leo L.M.; Perera, Ranawaka A. P. M.; Alhammadi, Mohammad A.; Ng, Hoi-yee; Siu, Lewis Y.; Guan, Yi; Alnaeem, Abdelmohsen; Peiris, Malik

    2014-01-01

    A prospective study of a dromedary camel herd during the 2013–14 calving season showed Middle East respiratory syndrome coronavirus infection of calves and adults. Virus was isolated from the nose and feces but more frequently from the nose. Preexisting neutralizing antibody did not appear to protect against infection.

  1. Transmission of MERS-coronavirus in household contacts

    NARCIS (Netherlands)

    Drosten, Christian; Meyer, Benjamin; Müller, Marcel A; Corman, Victor M; Al-Masri, Malak; Hossain, Raheela; Madani, Hosam; Sieberg, Andrea; Bosch, Berend Jan; Lattwein, Erik; Alhakeem, Raafat F; Assiri, Abdullah M; Hajomar, Waleed; Albarrak, Ali M; Al-Tawfiq, Jaffar A; Zumla, Alimuddin I; Memish, Ziad A

    2014-01-01

    BACKGROUND: Strategies to contain the Middle East respiratory syndrome coronavirus (MERS-CoV) depend on knowledge of the rate of human-to-human transmission, including subclinical infections. A lack of serologic tools has hindered targeted studies of transmission. METHODS: We studied 26 index patien

  2. [Nosocomial infections due to human coronaviruses in the newborn].

    Science.gov (United States)

    Gagneur, A; Legrand, M C; Picard, B; Baron, R; Talbot, P J; de Parscau, L; Sizun, J

    2002-01-01

    Human coronaviruses, with two known serogroups named 229-E and OC-43, are enveloped positive-stranded RNA viruses. The large RNA is surrounded by a nucleoprotein (protein N). The envelop contains 2 or 3 glycoproteins: spike protein (or protein S), matrix protein (or protein M) and a hemagglutinin (or protein HE). Their pathogen role remains unclear because their isolation is difficult. Reliable and rapid methods as immunofluorescence with monoclonal antibodies and reverse transcription-polymerase chain reaction allow new researches on epidemiology. Human coronaviruses can survive for as long as 6 days in suspension and 3 hours after drying on surfaces, suggesting that they could be a source of hospital-acquired infections. Two prospective studies conducted in a neonatal and paediatric intensive care unit demonstrated a significant association of coronavirus-positive nasopharyngal samples with respiratory illness in hospitalised preterm neonates. Positive samples from staff suggested either a patient-to-staff or a staff-to-patient transmission. No cross-infection were observed from community-acquired respiratory-syncitial virus or influenza-infected children to neonates. Universal precautions with hand washing and surface desinfection could be proposed to prevent coronavirus transmission.

  3. Infidelity of SARS-CoV Nsp14-Exonuclease Mutant Virus Replication Is Revealed by Complete Genome Sequencing

    Science.gov (United States)

    Eckerle, Lance D.; Becker, Michelle M.; Halpin, Rebecca A.; Li, Kelvin; Venter, Eli; Lu, Xiaotao; Scherbakova, Sana; Graham, Rachel L.; Baric, Ralph S.; Stockwell, Timothy B.; Spiro, David J.; Denison, Mark R.

    2010-01-01

    Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and

  4. SARS: just another viral acronym?

    Science.gov (United States)

    Broxmeyer, L

    2003-08-01

    Recent observations and experimental evidence have purported that a virus causes SARS, but such viruses have been isolated in only less than half of SARS patients in some studies and virologist Vincent Plummer of Winnipeg's National Microbiology Laboratory found that indeed 1 in 5 perfectly healthy Canadians with a history of recent travel to Asia had the virus. Therefore SARS microbiologic origins remain unclear. Outbreaks of multi-drug resistant (MDR) tuberculosis and the atypical mycobacteria simulate SARS on clinical, radiologic, epidemiologic, and diagnostic laboratory grounds and it is only logical then to include them in the differential to find a definitive cause and cure for SARS.

  5. Multichannel FMCW SAR

    NARCIS (Netherlands)

    Rossum, W.L. van; Otten, M.P.G.; Dorp, Ph. van

    2012-01-01

    A light weight SAR, suitable for use on short range tactical UAV, has been designed and built. The system consists of a fully digital receive array, and a very compact active transmit antenna. The approximate weight of the complete system is 6 kg, with power consumption below 75 W, depending on the

  6. Bistatic SAR: Proof of Concept.

    Energy Technology Data Exchange (ETDEWEB)

    Yocky, David A.; Doren, Neall E.; Bacon, Terry A.; Wahl, Daniel E.; Eichel, Paul H.; Jakowatz, Charles V,; Delaplain, Gilbert G.; Dubbert, Dale F.; Tise, Bertice L.; White, Kyle R.

    2014-10-01

    Typical synthetic aperture RADAR (SAR) imaging employs a co-located RADAR transmitter and receiver. Bistatic SAR imaging separates the transmitter and receiver locations. A bistatic SAR configuration allows for the transmitter and receiver(s) to be in a variety of geometric alignments. Sandia National Laboratories (SNL) / New Mexico proposed the deployment of a ground-based RADAR receiver. This RADAR receiver was coupled with the capability of digitizing and recording the signal collected. SNL proposed the possibility of creating an image of targets the illuminating SAR observes. This document describes the developed hardware, software, bistatic SAR configuration, and its deployment to test the concept of a ground-based bistatic SAR. In the proof-of-concept experiments herein, the RADAR transmitter will be a commercial SAR satellite and the RADAR receiver will be deployed at ground level, observing and capturing RADAR ground/targets illuminated by the satellite system.

  7. SARS (SEVERE ACUTE RESPIRATORY SYNDROME – A NEW CHALLENGE FOR THE MANKIND

    Directory of Open Access Journals (Sweden)

    Andrej Trampuž

    2003-07-01

    Full Text Available Background. SARS (severe acute respiratory syndrome is a recently recognized new infectious respiratory illness, which first appeared in southern China in November 2002, and has since then within months spread to 29 countries. In total, 8437 cases and 813 deaths occurred (situation as of August 1, 2003. SARS is caused by a novel coronavirus that is primarily spread by large droplet transmission, less commonly by surface contamination or by air (airborne. Around half of the infected were health care workers; the majority of cases acquired the infection in the hospital.Conclusions. Incubation period of SARS is 2 to 10 days. Early manifestations include fever, myalgia, and headache, followed 2 to 4 days later by cough, shortness of breath, and diarrhea. In 10–20% of patients, tracheal intubation and mechanical ventilation is required. Case-fatality is approximately 15%, in patients aged 60 years or older may be higher than 40%. There is no specific therapy or vaccine, and management consists of supportive care. This article summarizes updated information regarding epidemiology, clinical features, etiologic agent, modes of transmission of the disease, and infection control measures to contain SARS.

  8. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

    Energy Technology Data Exchange (ETDEWEB)

    Ratia, Kiira; Pegan, Scott; Takayama, Jun; Sleeman, Katrina; Coughlin, Melissa; Baliji, Surendranath; Chaudhuri, Rima; Fu, Wentao; Prabhakar, Bellur S.; Johnson, Michael E.; Baker, Susan C.; Ghosh, Arun K.; Mesecar, Andrew D. (Loyola); (Purdue); (UIC)

    2008-10-27

    We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC{sub 50} value of 20 {mu}M, which was improved to 600 nM via synthetic optimization. The resulting compound, GRL0617, inhibited SARS-CoV viral replication in Vero E6 cells with an EC{sub 50} of 15 {mu}M and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs.

  9. Coronavirus-like particles in laboratory rabbits with different syndromes in The Netherlands (Coronavirus-like particles in rabbits).

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); J.S. Teppema; G. van Steenis (Bert)

    1982-01-01

    textabstractVirus-like particles were identified from the plasma of rabbits which developed pleural effusion disease after inoculation with different strains of Treponema pallidum. These particles were considered coronavirus-like on the basis of their size, morphology, and buoyant density. Clinical

  10. Coronavirus-like particles in laboratory rabbits with different syndromes in The Netherlands (Coronavirus-like particles in rabbits).

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); J.S. Teppema; G. van Steenis (Bert)

    1982-01-01

    textabstractVirus-like particles were identified from the plasma of rabbits which developed pleural effusion disease after inoculation with different strains of Treponema pallidum. These particles were considered coronavirus-like on the basis of their size, morphology, and buoyant density. Clinical

  11. Discovery of unsymmetrical aromatic disulfides as novel inhibitors of SARS-CoV main protease: Chemical synthesis, biological evaluation, molecular docking and 3D-QSAR study.

    Science.gov (United States)

    Wang, Li; Bao, Bo-Bo; Song, Guo-Qing; Chen, Cheng; Zhang, Xu-Meng; Lu, Wei; Wang, Zefang; Cai, Yan; Li, Shuang; Fu, Sheng; Song, Fu-Hang; Yang, Haitao; Wang, Jian-Guo

    2017-09-08

    The worldwide outbreak of severe acute respiratory syndrome (SARS) in 2003 had caused a high rate of mortality. Main protease (M(pro)) of SARS-associated coronavirus (SARS-CoV) is an important target to discover pharmaceutical compounds for the therapy of this life-threatening disease. During the course of screening new anti-SARS agents, we have identified that a series of unsymmetrical aromatic disulfides inhibited SARS-CoV M(pro) significantly for the first time. Herein, 40 novel unsymmetrical aromatic disulfides were synthesized chemically and their biological activities were evaluated in vitro against SARS-CoV M(pro). These novel compounds displayed excellent IC50 data in the range of 0.516-5.954 μM. Preliminary studies indicated that these disulfides are reversible and mpetitive inhibitors. A possible binding mode was generated via molecular docking simulation and a comparative field analysis (CoMFA) model was constructed to understand the structure-activity relationships. The present research therefore has provided some meaningful guidance to design and identify anti-SARS drugs with totally new chemical structures. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. Successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus.

    Science.gov (United States)

    Sheahan, Timothy; Whitmore, Alan; Long, Kristin; Ferris, Martin; Rockx, Barry; Funkhouser, William; Donaldson, Eric; Gralinski, Lisa; Collier, Martha; Heise, Mark; Davis, Nancy; Johnston, Robert; Baric, Ralph S

    2011-01-01

    Newly emerging viruses often circulate as a heterogeneous swarm in wild animal reservoirs prior to their emergence in humans, and their antigenic identities are often unknown until an outbreak situation. The newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV) and reemerging influenza virus cause disproportionate disease in the aged, who are also notoriously difficult to successfully vaccinate, likely due to immunosenescence. To protect against future emerging strains, vaccine platforms should induce broad cross-reactive immunity that is sufficient to protect from homologous and heterologous challenge in all ages. From initial studies, we hypothesized that attenuated Venezuelan equine encephalitis virus (VEE) replicon particle (VRP) vaccine glycoproteins mediated vaccine failure in the aged. We then compared the efficacies of vaccines bearing attenuated (VRP(3014)) or wild-type VEE glycoproteins (VRP(3000)) in young and aged mice within novel models of severe SARS-CoV pathogenesis. Aged animals receiving VRP(3000)-based vaccines were protected from SARS-CoV disease, while animals receiving the VRP(3014)-based vaccines were not. The superior protection for the aged observed with VRP(3000)-based vaccines was confirmed in a lethal influenza virus challenge model. While the VRP(3000) vaccine's immune responses in the aged were sufficient to protect against lethal homologous and heterologous challenge, our data suggest that innate defects within the VRP(3014) platform mediate vaccine failure. Exploration into the mechanism(s) of successful vaccination in the immunosenescent should aid in the development of successful vaccine strategies for other viral diseases disproportionately affecting the elderly, like West Nile virus, influenza virus, norovirus, or other emerging viruses of the future.

  13. Characterization of human coronavirus etiology in Chinese adults with acute upper respiratory tract infection by real-time RT-PCR assays.

    Directory of Open Access Journals (Sweden)

    Roujian Lu

    Full Text Available BACKGROUND: In addition to SARS associated coronaviruses, 4 non-SARS related human coronaviruses (HCoVs are recognized as common respiratory pathogens. The etiology and clinical impact of HCoVs in Chinese adults with acute upper respiratory tract infection (URTI needs to be characterized systematically by molecular detection with excellent sensitivity. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we detected 4 non-SARS related HCoV species by real-time RT-PCR in 981 nasopharyngeal swabs collected from March 2009 to February 2011. All specimens were also tested for the presence of other common respiratory viruses and newly identified viruses, human metapneumovirus (hMPV and human bocavirus (HBoV. 157 of the 981 (16.0% nasopharyngeal swabs were positive for HCoVs. The species detected were 229E (96 cases, 9.8%, OC43 (42 cases, 4.3%, HKU1 (16 cases, 1.6% and NL63 (11 cases, 1.1%. HCoV-229E was circulated in 21 of the 24 months of surveillance. The detection rates for both OC43 and NL63 were showed significantly year-to-year variation between 2009/10 and 2010/11, respectively (P<0.001 and P = 0.003, and there was a higher detection frequency of HKU1 in patients aged over 60 years (P = 0.03. 48 of 157(30.57% HCoV positive patients were co-infected. Undifferentiated human rhinoviruses and influenza (Flu A were the most common viruses detected (more than 35% in HCoV co-infections. Respiratory syncytial virus (RSV, human parainfluenza virus (PIV and HBoV were detected in very low rate (less than 1% among adult patients with URTI. CONCLUSIONS/SIGNIFICANCE: All 4 non-SARS-associated HCoVs were more frequently detected by real-time RT-PCR assay in adults with URTI in Beijing and HCoV-229E led to the most prevalent infection. Our study also suggested that all non-SARS-associated HCoVs contribute significantly to URTI in adult patients in China.

  14. Bistatic sAR data processing algorithms

    CERN Document Server

    Qiu, Xiaolan; Hu, Donghui

    2013-01-01

    Synthetic Aperture Radar (SAR) is critical for remote sensing. It works day and night, in good weather or bad. Bistatic SAR is a new kind of SAR system, where the transmitter and receiver are placed on two separate platforms. Bistatic SAR is one of the most important trends in SAR development, as the technology renders SAR more flexible and safer when used in military environments. Imaging is one of the most difficult and important aspects of bistatic SAR data processing. Although traditional SAR signal processing is fully developed, bistatic SAR has a more complex system structure, so sign

  15. SAR++: A Multi-Channel Scalable and Reconfigurable SAR System

    DEFF Research Database (Denmark)

    Høeg, Flemming; Christensen, Erik Lintz

    2002-01-01

    SAR++ is a technology program aiming at developing know-how and technology needed to design the next generation civilian SAR systems. Technology has reached a state, which allows major parts of the digital subsystem to be built using custom-off-the-shelf (COTS) components. A design goal is to des......SAR++ is a technology program aiming at developing know-how and technology needed to design the next generation civilian SAR systems. Technology has reached a state, which allows major parts of the digital subsystem to be built using custom-off-the-shelf (COTS) components. A design goal...... is to design a modular, scalable and reconfigurable SAR system using such components, in order to ensure maximum flexibility for the users of the actual system and for future system updates. Having these aspects in mind the SAR++ system is presented with focus on the digital subsystem architecture...... and the analog to digital interface....

  16. Human Coronavirus-Associated Influenza-Like Illness in the Community Setting in Peru.

    Science.gov (United States)

    Razuri, Hugo; Malecki, Monika; Tinoco, Yeny; Ortiz, Ernesto; Guezala, M Claudia; Romero, Candice; Estela, Abel; Breña, Patricia; Morales, Maria-Luisa; Reaves, Erik J; Gomez, Jorge; Uyeki, Timothy M; Widdowson, Marc-Alain; Azziz-Baumgartner, Eduardo; Bausch, Daniel G; Schildgen, Verena; Schildgen, Oliver; Montgomery, Joel M

    2015-11-01

    We present findings describing the epidemiology of non-severe acute respiratory syndrome human coronavirus-associated influenza-like illness from a population-based active follow-up study in four different regions of Peru. In 2010, the prevalence of infections by human coronaviruses 229E, OC43, NL63, or HKU1 was 6.4% in participants with influenza-like illness who tested negative for influenza viruses. Ten of 11 human coronavirus infections were identified in the fall-winter season. Human coronaviruses are present in different regions of Peru and are relatively frequently associated with influenza-like illness in Peru.

  17. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang (Cornell); (UMM-MED); (Colorado)

    2011-09-28

    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

  18. Zn(2+ inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture.

    Directory of Open Access Journals (Sweden)

    Aartjan J W te Velthuis

    Full Text Available Increasing the intracellular Zn(2+ concentration with zinc-ionophores like pyrithione (PT can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn(2+ and PT at low concentrations (2 µM Zn(2+ and 2 µM PT inhibits the replication of SARS-coronavirus (SARS-CoV and equine arteritis virus (EAV in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp, which is the core enzyme of their multiprotein replication and transcription complex (RTC. Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV--thus eliminating the need for PT to transport Zn(2+ across the plasma membrane--we show that Zn(2+ efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9 purified from E. coli subsequently revealed that Zn(2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn(2+ was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn(2+ with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription.

  19. Association of SARS susceptibility with single nucleic acid polymorphisms of OAS1 and MxA genes: a case-control study

    Directory of Open Access Journals (Sweden)

    Yang Hong

    2006-07-01

    Full Text Available Abstract Background Host genetic factors may play a role in susceptibility and resistance to SARS associated coronavirus (SARS-CoV infection. The study was carried out to investigate the association between the genetic polymorphisms of 2',5'-oligoadenylate synthetase 1 (OAS1 gene as well as myxovirus resistance 1 (MxA gene and susceptibility to SARS in Chinese Han population. Methods A hospital-based case-control study was conducted. A collective of 66 SARS cases and 64 close contact uninfected controls were enrolled in this study. End point real time polymerase chain reaction (PCR and PCR-based Restriction Fragment Length Polymorphism (RFLP analysis were used to detect the single nucleic polymorphisms (SNPs in OAS1 and MxA genes. Information on other factors associated with SARS infection was collected using a pre-tested questionnaire. Univariate and multivariate logistic analyses were conducted. Results One polymorphism in the 3'-untranslated region (3'-UTR of the OAS1 gene was associated with SARS infection. Compared to AA genotype, AG and GG genotypes were found associated with a protective effect on SARS infection with ORs (95% CI of 0.42 (0.20~0.89 and 0.30 (0.09~0.97, respectively. Also, a GT genotype at position 88 in the MxA gene promoter was associated with increased susceptibility to SARS infection compared to a GG genotype (OR = 3.06, 95% CI: 1.25~7.50. The associations of AG genotype in OAS1 and GT genotype in MxA remained significant in multivariate analyses after adjusting for SARS protective measures (OR = 0.38, 95% CI: 0.14~0.98 and OR = 3.22, 95% CI: 1.13~9.18, respectively. Conclusion SNPs in the OAS1 3'-UTR and MxA promoter region appear associated with host susceptibility to SARS in Chinese Han population.

  20. Elastase-mediated Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein at Discrete Sites within the S2 Domain*

    Science.gov (United States)

    Belouzard, Sandrine; Madu, Ikenna; Whittaker, Gary R.

    2010-01-01

    Proteolytic priming is a common method of controlling the activation of membrane fusion mediated by viral glycoproteins. The severe acute respiratory syndrome coronavirus spike protein (SARS-CoV S) can be primed by a variety of host cell proteases, with proteolytic cleavage occurring both as the S1/S2 boundary and adjacent to a fusion peptide in the S2 domain. Here, we studied the priming of SARS-CoV S by elastase and show an important role for residue Thr795 in the S2 domain. A series of alanine mutants were generated in the vicinity of the S2 cleavage site, with the goal of examining elastase-mediated cleavage within S2. Both proteolytic cleavage and fusion activation were modulated by altering the cleavage site position. We propose a novel mechanism whereby SARS-CoV fusion protein function can be controlled by spatial regulation of the proteolytic priming site, with important implications for viral pathogenesis. PMID:20507992

  1. Síntese de látices com baixa concentração de compostos orgânicos voláteis (COVs: efeito das técnicas de redução dos COVs nas propriedades dos látexes e das tintas

    Directory of Open Access Journals (Sweden)

    Maurício Pinheiro de Oliveira

    2014-08-01

    Full Text Available A redução dos compostos orgânicos voláteis (COVs nos látices produzidos via polimerização em emulsão é uma opção viável, mas em algumas situações pode comprometer a qualidade do látex. Diferentes técnicas de redução da concentração dos monômeros e dos COVs foram estudadas com o objetivo de entender o efeito destas técnicas e da concentração dos COVs nas propriedades de aplicação dos látices e das tintas. Os látices de estireno com acrilato de 2-etil hexila funcionalizados com ácido acrílico e acrilamida foram produzidos via polimerização em emulsão, seguido por remoção química, física e a combinação de ambas as técnicas de redução dos monômeros e dos COVs. Os parâmetros relacionados à técnica de redução dos COVs, ao tipo de iniciador, ao agente de redução e à introdução de nitrogênio saturado com vapor de água foram estudados e correlacionados com as propriedades de aplicação das tintas. A combinação da técnica química com a técnica física foi mais eficiente na redução dos monômeros e dos COVs nos látices. As técnicas utilizadas na redução dos COVs tiveram influência negativa nas propriedades de aplicação dos látices. A resistência à abrasão dos filmes de tinta foi dependente da técnica empregada e da concentração dos COVs.

  2. Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding.

    Science.gov (United States)

    Gui, Miao; Song, Wenfei; Zhou, Haixia; Xu, Jingwei; Chen, Silian; Xiang, Ye; Wang, Xinquan

    2017-01-01

    The global outbreak of SARS in 2002-2003 was caused by the infection of a new human coronavirus SARS-CoV. The infection of SARS-CoV is mediated mainly through the viral surface glycoproteins, which consist of S1 and S2 subunits and form trimer spikes on the envelope of the virions. Here we report the ectodomain structures of the SARS-CoV surface spike trimer in different conformational states determined by single-particle cryo-electron microscopy. The conformation 1 determined at 4.3 Å resolution is three-fold symmetric and has all the three receptor-binding C-terminal domain 1 (CTD1s) of the S1 subunits in "down" positions. The binding of the "down" CTD1s to the SARS-CoV receptor ACE2 is not possible due to steric clashes, suggesting that the conformation 1 represents a receptor-binding inactive state. Conformations 2-4 determined at 7.3, 5.7 and 6.8 Å resolutions are all asymmetric, in which one RBD rotates away from the "down" position by different angles to an "up" position. The "up" CTD1 exposes the receptor-binding site for ACE2 engagement, suggesting that the conformations 2-4 represent a receptor-binding active state. This conformational change is also required for the binding of SARS-CoV neutralizing antibodies targeting the CTD1. This phenomenon could be extended to other betacoronaviruses utilizing CTD1 of the S1 subunit for receptor binding, which provides new insights into the intermediate states of coronavirus pre-fusion spike trimer during infection.

  3. Novel Polarimetric SAR Interferometry Algorithms Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Polarimetric SAR interferometry (PolInSAR) is a recently developed synthetic aperture radar (SAR) imaging mode that combines the capabilities of radar polarimetry...

  4. Wetland InSAR

    Science.gov (United States)

    Wdowinski, S.; Kim, S.; Amelung, F.; Dixon, T.

    2006-12-01

    Wetlands are transition zones where the flow of water, the nutrient cycling, and the sun energy meet to produce a unique and very productive ecosystem. They provide critical habitat for a wide variety of plant and animal species, including the larval stages of many ocean fish. Wetlands also have a valuable economical importance, as they filter nutrients and pollutants from fresh water used by human and provide aquatic habitats for outdoor recreation, tourism, and fishing. Globally, many such regions are under severe environmental stress, mainly from urban development, pollution, and rising sea level. However, there is increasing recognition of the importance of these habitats, and mitigation and restoration activities have begun in a few regions. A key element in wetlands conservation, management, and restoration involves monitoring its hydrologic system, as the entire ecosystem depends on its water supply. Heretofore, hydrologic monitoring of wetlands are conducted by stage (water level) stations, which provide good temporal resolution, but suffer from poor spatial resolution, as stage station are typically distributed several, or even tens of kilometers, from one another. Wetland application of InSAR provides the needed high spatial resolution hydrological observations, complementing the high temporal resolution terrestrial observations. Although conventional wisdom suggests that interferometry does not work in vegetated areas, several studies have shown that both L- and C-band interferograms with short acquisition intervals (1-105 days) can maintain excellent coherence over wetlands. In this study we explore the usage of InSAR for detecting water level changes in various wetland environments around the world, including the Everglades (south Florida), Louisiana Coast (southern US), Chesapeake Bay (eastern US), Pantanal (Brazil), Okavango Delta (Botswana), and Lena Delta (Siberia). Our main study area is the Everglades wetland (south Florida), which is covered by

  5. Isolation of MERS Coronavirus from a Dromedary Camel, Qatar, 2014

    Science.gov (United States)

    Raj, V. Stalin; Farag, Elmoubasher A.B.A.; Reusken, Chantal B.E.M.; Lamers, Mart M.; Pas, Suzan D.; Voermans, Jolanda; Smits, Saskia L.; Osterhaus, Albert D.M.E.; Al-Mawlawi, Naema; Al-Romaihi, Hamad E.; El-Sayed, Ahmed M.; Mohran, Khaled A.; Ghobashy, Hazem; Alhajri, Farhoud; Al-Thani, Mohamed; Al-Marri, Salih A.; El-Maghraby, Mamdouh M.; Koopmans, Marion P.G.

    2014-01-01

    We obtained the full genome of Middle East respiratory syndrome coronavirus (MERS-CoV) from a camel in Qatar. This virus is highly similar to the human England/Qatar 1 virus isolated in 2012. The MERS-CoV from the camel efficiently replicated in human cells, providing further evidence for the zoonotic potential of MERS-CoV from camels. PMID:25075761

  6. Coronavirus infection in mink (Mustela vison). Serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus

    DEFF Research Database (Denmark)

    Have, P; Moving, V; Svansson, V

    1992-01-01

    Antibodies to a transmissible gastroenteritis virus (TGEV)-related coronavirus have been demonstrated in mink sera by indirect immunofluorescence, peroxidase-linked antibody assays and immunoblotting. This is the first serological evidence of a specific coronavirus infection in mink. The putative......-reacted with N and M polypeptides of porcine epidemic diarrhea virus (PEDV). Thus MCV may occupy an intermediate position between the TGEV group of coronaviruses and PEDV. The possibility that MCV may be associated with syndromes of acute enteritis in preweaning mink is discussed....

  7. Prevalence of Korean cats with natural feline coronavirus infections

    Directory of Open Access Journals (Sweden)

    Lee Myoung-Heon

    2011-09-01

    Full Text Available Abstract Background Feline coronavirus is comprised of two pathogenic biotypes consisting of feline infectious peritonitis virus (FIPV and feline enteric coronavirus (FECV, which are both divided into two serotypes. To examine the prevalence of Korean cats infected with feline coronavirus (FCoV type I and II, fecal samples were obtained from 212 cats (107 pet and 105 feral in 2009. Results Fourteen cats were FCoV-positive, including infections with type I FCoV (n = 8, type II FCoV (n = 4, and types I and II co-infection (n = 2. Low seroprevalences (13.7%, 29/212 of FCoV were identified in chronically ill cats (19.3%, 16/83 and healthy cats (10.1%, 13/129. Conclusions Although the prevalence of FCoV infection was not high in comparison to other countries, there was a higher prevalence of type I FCoV in Korean felines. The prevalence of FCoV antigen and antibody in Korean cats are expected to gradually increase due to the rising numbers of stray and companion cats.

  8. Magnetic and magnetocaloric properties of quasi-one-dimensional Ising spin chain CoV2O6

    Science.gov (United States)

    Nandi, M.; Mandal, P.

    2016-04-01

    We have investigated the magnetic and magnetocaloric properties of antiferromagnetic Ising spin chain CoV2O6 by magnetization and heat capacity measurements. Both monoclinic α-CoV2O6 and triclinic γ-CoV2O6 exhibit field-induced metamagnetic transitions from antiferromagnetic to ferromagnetic state via an intermediate ferrimagnetic state with 1/3 magnetization plateau. Due to the field-induced metamagnetic transitions, these systems show large conventional as well as inverse magnetocaloric effects. In α-CoV2O6, we observe field-induced complex magnetic phases and multiple magnetization plateaus below 6 K when the field is applied along c axis. Several critical temperatures and fields have been identified from the temperature and field dependence of magnetization, magnetic entropy change, and heat capacity to construct the H-T phase diagram. As compared to α-CoV2O6, γ-CoV2O6 displays a relatively simple magnetic phase diagram. Due to the large magnetic entropy change and adiabatic temperature change at low or moderate applied magnetic field, γ-CoV2O6 may be considered as a magnetic refrigerant in the low-temperature region below 20 K.

  9. NParCov3: A SAS/IML Macro for Nonparametric Randomization-Based Analysis of Covariance

    Directory of Open Access Journals (Sweden)

    Richard C. Zink

    2012-07-01

    Full Text Available Analysis of covariance serves two important purposes in a randomized clinical trial. First, there is a reduction of variance for the treatment effect which provides more powerful statistical tests and more precise confidence intervals. Second, it provides estimates of the treatment effect which are adjusted for random imbalances of covariates between the treatment groups. The nonparametric analysis of covariance method of Koch, Tangen, Jung, and Amara (1998 defines a very general methodology using weighted least-squares to generate covariate-adjusted treatment effects with minimal assumptions. This methodology is general in its applicability to a variety of outcomes, whether continuous, binary, ordinal, incidence density or time-to-event. Further, its use has been illustrated in many clinical trial settings, such as multi-center, dose-response and non-inferiority trials.NParCov3 is a SAS/IML macro written to conduct the nonparametric randomization-based covariance analyses of Koch et al. (1998. The software can analyze a variety of outcomes and can account for stratification. Data from multiple clinical trials will be used for illustration.

  10. Genomic organization and expression of the 3' end of the canine and feline enteric coronaviruses

    NARCIS (Netherlands)

    Vennema, H; Rossen, J W; Wesseling, J; Horzinek, M C; Rottier, P J

    1993-01-01

    The genomic organization at the 3' end of canine coronavirus (CCV) and feline enteric coronavirus (FECV) was determined by sequence analysis and compared to that of feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) of swine. Comparison of the latter two has pr

  11. Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer

    NARCIS (Netherlands)

    Walls, Alexandra C; Tortorici, M Alejandra; Bosch, Berend-Jan; Frenz, Brandon; Rottier, Peter J M; DiMaio, Frank; Rey, Félix A; Veesler, David

    2016-01-01

    The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion f

  12. Recovering Seasat SAR Data

    Science.gov (United States)

    Logan, T. A.; Arko, S. A.; Rosen, P. A.

    2013-12-01

    To demonstrate the feasibility of orbital remote sensing for global ocean observations, NASA launched Seasat on June 27th, 1978. Being the first space borne SAR mission, Seasat produced the most detailed SAR images of Earth from space ever seen to that point in time. While much of the data collected in the USA was processed optically, a mere 150 scenes had been digitally processed by March 1980. In fact, only an estimated 3% of Seasat data was ever digitally processed. Thus, for over three decades, the majority of the SAR data from this historic mission has been dormant, virtually unavailable to scientists in the 21st century. Over the last year, researchers at the Alaska Satellite Facility (ASF) Distributed Active Archive Center (DAAC) have processed the Seasat SAR archives into imagery products. A telemetry decoding system was created and the data were filtered into readily processable signal files. Due to nearly 35 years of bit rot, the bit error rate (BER) for the ASF DAAC Seasat archives was on the order of 1 out of 100 to 1 out of 100,000. This extremely high BER initially seemed to make much of the data undecodable - because the minor frame numbers are just 7 bits and no range line numbers exist in the telemetry even the 'simple' tasks of tracking the minor frame number or locating the start of each range line proved difficult. Eventually, using 5 frame numbers in sequence and a handful of heuristics, the data were successfully decoded into full range lines. Concurrently, all metadata were stored into external files. Recovery of this metadata was also problematic, the BER making the information highly suspect and, initially at least, unusable in any sort of automated fashion. Because of the BER, all of the single bit metadata fields proved unreliable. Even fields that should be constant for a data take (e.g. receiving station, day of the year) showed high variability, each requiring a median filter to be usable. The most challenging, however, were the

  13. Isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14, from domestic rabbits.

    Science.gov (United States)

    Lau, Susanna K P; Woo, Patrick C Y; Yip, Cyril C Y; Fan, Rachel Y Y; Huang, Yi; Wang, Ming; Guo, Rongtong; Lam, Carol S F; Tsang, Alan K L; Lai, Kenneth K Y; Chan, Kwok-Hung; Che, Xiao-Yan; Zheng, Bo-Jian; Yuen, Kwok-Yung

    2012-05-01

    We describe the isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14 (RbCoV HKU14), from domestic rabbits. The virus was detected in 11 (8.1%) of 136 rabbit fecal samples by reverse transcriptase PCR (RT-PCR), with a viral load of up to 10(8) copies/ml. RbCoV HKU14 was able to replicate in HRT-18G and RK13 cells with cytopathic effects. Northern blotting confirmed the production of subgenomic mRNAs coding for the HE, S, NS5a, E, M, and N proteins. Subgenomic mRNA analysis revealed a transcription regulatory sequence, 5'-UCUAAAC-3'. Phylogenetic analysis showed that RbCoV HKU14 formed a distinct branch among Betacoronavirus subgroup A coronaviruses, being most closely related to but separate from the species Betacoronavirus 1. A comparison of the conserved replicase domains showed that RbCoV HKU14 possessed rabbit sera tested by an N-protein-based Western blot assay, whereas neutralizing antibody was detected in 1 of these 20 rabbits.

  14. Dysregulated Type I Interferon and Inflammatory Monocyte-Macrophage Responses Cause Lethal Pneumonia in SARS-CoV-Infected Mice.

    Science.gov (United States)

    Channappanavar, Rudragouda; Fehr, Anthony R; Vijay, Rahul; Mack, Matthias; Zhao, Jincun; Meyerholz, David K; Perlman, Stanley

    2016-02-10

    Highly pathogenic human respiratory coronaviruses cause acute lethal disease characterized by exuberant inflammatory responses and lung damage. However, the factors leading to lung pathology are not well understood. Using mice infected with SARS (severe acute respiratory syndrome)-CoV, we show that robust virus replication accompanied by delayed type I interferon (IFN-I) signaling orchestrates inflammatory responses and lung immunopathology with diminished survival. IFN-I remains detectable until after virus titers peak, but early IFN-I administration ameliorates immunopathology. This delayed IFN-I signaling promotes the accumulation of pathogenic inflammatory monocyte-macrophages (IMMs), resulting in elevated lung cytokine/chemokine levels, vascular leakage, and impaired virus-specific T cell responses. Genetic ablation of the IFN-αβ receptor (IFNAR) or IMM depletion protects mice from lethal infection, without affecting viral load. These results demonstrate that IFN-I and IMM promote lethal SARS-CoV infection and identify IFN-I and IMMs as potential therapeutic targets in patients infected with pathogenic coronavirus and perhaps other respiratory viruses. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Analytical SAR-GMTI principles

    Science.gov (United States)

    Soumekh, Mehrdad; Majumder, Uttam K.; Barnes, Christopher; Sobota, David; Minardi, Michael

    2016-05-01

    This paper provides analytical principles to relate the signature of a moving target to parameters in a SAR system. Our objective is to establish analytical tools that could predict the shift and smearing of a moving target in a subaperture SAR image. Hence, a user could identify the system parameters such as the coherent processing interval for a subaperture that is suitable to localize the signature of a moving target for detection, tracking and geolocating the moving target. The paper begins by outlining two well-known SAR data collection methods to detect moving targets. One uses a scanning beam in the azimuth domain with a relatively high PRF to separate the moving targets and the stationary background (clutter); this is also known as Doppler Beam Sharpening. The other scheme uses two receivers along the track to null the clutter and, thus, provide GMTI. We also present results on implementing our SAR-GMTI analytical principles for the anticipated shift and smearing of a moving target in a simulated code. The code would provide a tool for the user to change the SAR system and moving target parameters, and predict the properties of a moving target signature in a subaperture SAR image for a scene that is composed of both stationary and moving targets. Hence, the SAR simulation and imaging code could be used to demonstrate the validity and accuracy of the above analytical principles to predict the properties of a moving target signature in a subaperture SAR image.

  16. Inactivation of the CovR/S virulence regulator impairs infection in an improved murine model of Streptococcus pyogenes naso-pharyngeal infection.

    Science.gov (United States)

    Alam, Faraz M; Turner, Claire E; Smith, Ken; Wiles, Siouxsie; Sriskandan, Shiranee

    2013-01-01

    Streptococcus pyogenes is a leading cause of pharyngeal infection, with an estimated 616 million cases per year. The human nasopharynx represents the major reservoir for all S. pyogenes infection, including severe invasive disease. To investigate bacterial and host factors that influence S. pyogenes infection, we have devised an improved murine model of nasopharyngeal colonization, with an optimized dosing volume to avoid fulminant infections and a sensitive host strain. In addition we have utilized a refined technique for longitudinal monitoring of bacterial burden that is non-invasive thereby reducing the numbers of animals required. The model was used to demonstrate that the two component regulatory system, CovR/S, is required for optimum infection and transmission from the nasopharynx. There is a fitness cost conferred by covR/S mutation that is specific to the nasopharynx. This may explain why S. pyogenes with altered covR/S have not become prevalent in community infections despite possessing a selective advantage in invasive infection.

  17. Acetylene Black Induced Heterogeneous Growth of Macroporous CoV2O6 Nanosheet for High-Rate Pseudocapacitive Lithium-Ion Battery Anode.

    Science.gov (United States)

    Zhang, Lei; Zhao, Kangning; Luo, Yanzhu; Dong, Yifan; Xu, Wangwang; Yan, Mengyu; Ren, Wenhao; Zhou, Liang; Qu, Longbing; Mai, Liqiang

    2016-03-23

    Metal vanadates suffer from fast capacity fading in lithium-ion batteries especially at a high rate. Pseudocapacitance, which is associated with surface or near-surface redox reactions, can provide fast charge/discharge capacity free from diffusion-controlled intercalation processes and is able to address the above issue. In this work, we report the synthesis of macroporous CoV2O6 nanosheets through a facile one-pot method via acetylene black induced heterogeneous growth. When applied as lithium-ion battery anode, the macroporous CoV2O6 nanosheets show typical features of pseudocapacitive behavior: (1) currents that are mostly linearly dependent on sweep rate and (2) redox peaks whose potentials do not shift significantly with sweep rate. The macroporous CoV2O6 nanosheets display a high reversible capacity of 702 mAh g(-1) at 200 mA g(-1), excellent cyclability with a capacity retention of 89% (against the second cycle) after 500 cycles at 500 mA g(-1), and high rate capability of 453 mAh g(-1) at 5000 mA g(-1). We believe that the introduction of pseudocapacitive properties in lithium battery is a promising direction for developing electrode materials with high-rate capability.

  18. Prophylactic and therapeutic intranasal administration with an immunomodulator, Hiltonol(®) (Poly IC:LC), in a lethal SARS-CoV-infected BALB/c mouse model.

    Science.gov (United States)

    Kumaki, Yohichi; Salazar, Andres M; Wandersee, Miles K; Barnard, Dale L

    2017-03-01

    Hiltonol(®), (Poly IC:LC), a potent immunomodulator, is a synthetic, double-stranded polyriboinosinic-polyribocytidylic acid (poly IC) stabilized with Poly-L-lysine and carboxymethyl cellulose (LC). Hiltonol(®) was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. Hiltonol(®) at 5, 1, 0.5 or 0.25 mg/kg/day by intranasal (i.n.) route resulted in significant survival benefit when administered at selected times 24 h prior to challenge with a lethal dose of mouse-adapted severe acute respiratory syndrome coronavirus (SARS-CoV). The infected BALB/c mice receiving the Hiltonol(®) treatments were also significantly effective in protecting mice against weight loss due to infection (p SARS-CoV infection in mice leads to substantial prophylactic and therapeutic effects and could be used for treatment of other virus disease such as those caused by MERS-CoV a related coronavirus. These properties might be therapeutically advantageous if Hiltonol(®) is considered for possible clinical use. Published by Elsevier B.V.

  19. Bistatic SAR: Imagery & Image Products.

    Energy Technology Data Exchange (ETDEWEB)

    Yocky, David A.; Wahl, Daniel E.; Jakowatz, Charles V,

    2014-10-01

    While typical SAR imaging employs a co-located (monostatic) RADAR transmitter and receiver, bistatic SAR imaging separates the transmitter and receiver locations. The transmitter and receiver geometry determines if the scattered signal is back scatter, forward scatter, or side scatter. The monostatic SAR image is backscatter. Therefore, depending on the transmitter/receiver collection geometry, the captured imagery may be quite different that that sensed at the monostatic SAR. This document presents imagery and image products formed from captured signals during the validation stage of the bistatic SAR research. Image quality and image characteristics are discussed first. Then image products such as two-color multi-view (2CMV) and coherent change detection (CCD) are presented.

  20. SAR Altimetry Applications over Water

    CERN Document Server

    Martin-Puig, C; Ruffini, G; Raney, R K; Benveniste, J

    2008-01-01

    The application of Synthetic Aperture Radar (SAR) techniques to classical radar altimetry offers the potential for greatly improved Earth surface mapping. This paper provides an overview of the progress of SAMOSA, Development of SAR Altimetry Studies and Applications over Ocean, Coastal zones and Inland waters, an on-going ESA-funded project. The main objective of SAMOSA is to better quantify the improvement of SAR altimetry over conventional altimetry on water surfaces. More specifically, one of the tasks focuses on the reduction of SAR mode data to pulse-limited altimeter data, and a theoretical modelling to characterize the expected gain between high Pulse Repetition Frequency (PRF) reduced SAR mode data and low PRF classical Low-Resolution Mode (LRM) data. To this end, theoretical modelling using the Cramer-Rao bound (CRB) will be used and the results will be compared to previous theoretical estimates [7], using an analysis akin to that in [8].

  1. Coronavirus bovino: Infecciones neumoentéricas (Bovine coronavirus:Neumoenteric infections

    Directory of Open Access Journals (Sweden)

    Betancourt, Martell, Alexander|

    2006-12-01

    Full Text Available Coronavirus bovino (BCoV es reconocido como un importante agente patógeno del ganado bovino, el cual está asociado a tres síndromes clínicos diferentes, Síndrome diarreico neonatal del ternero, caracterizado en terneros recién nacidos por diarreas líquidas profusas, en ocasiones hemorrágicas, anorexia, deshidratación y frecuentemente la muerte; Disentería de Invierno, la cual ocurre primariamente en bovinos adultos y cursa con severas diarreas, algunas veces con restos de sangre y mucus, decrecimiento de laproducción láctea, depresión, anorexia y descargas nasolagrimales; y finalmente como causa de infecciones respiratorias en vacas, incluida la Fiebre de Embarque. En todos los casos el diagnóstico requiere deensayos de laboratorio para la confirmación de BCoV, debido que resulta imposible su reconocimiento basado en elementos clínicos y anatomopatológicos por su similitud con otras enfermedades. Hasta elmomento todos los aislados de BCoV, tanto de cuadros entéricos como respiratorios pertenecen a un solo serotipo, pero con dos o tres subtipos identificados por seroneutralización empleando anticuerposmonoclonales. En adición, diferencias genéticas (por mutaciones puntuales, no delecciones han sido detectadas en el gen S, diferenciando entre aislados entéricos y respiratorios. No obstante, numerosos experimentos han demostrado la protección cruzada experimentada por terneros recién nacidos, privados de calostro ygnotobióticos, inoculados con aislados de BCoV obtenidos a partir de cuadros entéricos y respiratorios de terneros y bovinos adultos, los cuales resultaron protegidos al desafío subsiguiente con cepas de BCoV asociadas a diarrea.

  2. MicroRNome analysis unravels the molecular basis of SARS infection in bronchoalveolar stem cells.

    Directory of Open Access Journals (Sweden)

    Bibekanand Mallick

    Full Text Available Severe acute respiratory syndrome (SARS, caused by the coronavirus SARS-CoV, is an acute infectious disease with significant mortality. A typical clinical feature associated with SARS is pulmonary fibrosis and associated lung failure. In the aftermath of the SARS epidemic, although significant progress towards understanding the underlying molecular mechanism of the infection has been made, a large gap still remains in our knowledge regarding how SARS-CoV interacts with the host cell at the onset of infection. The rapidly changing viral genome adds another variable to this equation. We have focused on a novel concept of microRNA (miRNA-mediated host-virus interactions in bronchoalveolar stem cells (BASCs at the onset of infection by correlating the "BASC-microRNome" with their targets within BASCs and viral genome. This work encompasses miRNA array data analysis, target prediction, and miRNA-mRNA enrichment analysis and develops a complex interaction map among disease-related factors, miRNAs, and BASCs in SARS pathway, which will provide some clues for diagnostic markers to view an overall interplay leading to disease progression. Our observation reveals the BASCs (Sca-1+ CD34+ CD45- Pecam-, a subset of Oct-4+ ACE2+ epithelial colony cells at the broncho-alveolar duct junction, to be the prime target cells of SARS-CoV infection. Upregulated BASC miRNAs-17*, -574-5p, and -214 are co-opted by SARS-CoV to suppress its own replication and evade immune elimination until successful transmission takes place. Viral Nucleocapsid and Spike protein targets seem to co-opt downregulated miR-223 and miR-98 respectively within BASCs to control the various stages of BASC differentiation, activation of inflammatory chemokines, and downregulation of ACE2. All these effectively accounts for a successful viral transmission and replication within BASCs causing continued deterioration of lung tissues and apparent loss of capacity for lung repair. Overall, this

  3. Anatomy of a SAR impulse response.

    Energy Technology Data Exchange (ETDEWEB)

    Doerry, Armin Walter

    2007-08-01

    A principal measure of Synthetic Aperture Radar (SAR) image quality is the manifestation in the SAR image of a spatial impulse, that is, the SAR's Impulse Response (IPR). IPR requirements direct certain design decisions in a SAR. Anomalies in the IPR can point to specific anomalous behavior in the radar's hardware and/or software.

  4. Roads as sources of heavy metals in urban areas. The Covões Catchment experiment, Coimbra, Portugal

    Science.gov (United States)

    Ferreira, António J. D.; Soares, Daniel; Ferreira, Carla S. S.; Walsh, Rory P. D.

    2015-04-01

    Cities are the home to 50% of the human specie [UN 2011 Ramalho & Hobbs 2012], whose wellbeing, way of life and exposure to hazard situations are directly related to the built environment. Cities are often seen as ecological systems just a short step away from collapse [Newman 2006]. Being a human construction, cities disrupt the natural cycles and the patterns of temporal and spatial distribution of environmental and ecological processes. Urbanization produces ruptures in biota, water, energy and nutrients connectivity that can lead to an enhanced exposure to disruptive events that hamper the wellbeing and the resilience of urban communities in a global change context. A major issue in what concerns the threats to human and ecosystem health in urban areas is the presence of heavy metals, and the related processes that govern their source, transport and fade r uptake by the vegetation. In this work, we present an analysis of heavy metal sources and transport processes at various types of roads within the Ribeira dos Covões peri-urban experimental catchment in central Portugal. The surveyed heavy metals (Cadmium, Lead, Coper, and Zinc) show significant differences as a result of the type of rainfall event, the length of the antecedent dry spell, the traffic volume and the heavy metals sources. For some locations, namely for the roads with heavy traffic volume, the heavy metal concentrations exceed the limits established by law, which has severe implications to the downstream ecosystems and to the possible use of the water from roads to close the resources loop in urban areas, namely in what concerns their use to water the urban green infrastructure or to irrigate the urban agriculture fields.

  5. State-of-art of Geosynchronous SAR

    Institute of Scientific and Technical Information of China (English)

    MAO Er-ke; LONG Teng; ZENG Tao; HU Cheng; TIAN Ye

    2012-01-01

    Geosynchronous Earth Orbit Synthetic Aperture Radar (GEO SAR) runs in the height of 360000Km geosynchronous earth orbit,compared with traditional Low Earth Orbit (LEO) SAR (orbit height under 1000Km),GEO SAR has advantages of shorter repeat period,wider swath and so on.Firstly,the basic principle and state-of-art of GEO SAR in domestic and overseas are introduced.Secondly,coverage characteristic of GEO SAR is analyzed.Thirdly,the key problems of yaw steering and imaging on curved trajectory in GEO SAR are discussed in detail,and the corresponding primary solutions are presented in order to promote future research on GEO SAR.

  6. High Resolution Processing with an Active Phased Array SAR

    NARCIS (Netherlands)

    Nijenboer, F.J.; Otten, M.P.G.

    1999-01-01

    The Dutch PHARUS system is a polarimetric active phased array SAR capable of performing advanced SAR modes. Advanced SAR modes that are being investigated are: spotlight SAR, sliding spotlight SAR, stepped frequency SAR and interferometric SAR. The flight experiments and automatic beam steering

  7. DEM FROM SAR:PRINCIPLE AND APPLICATION

    Institute of Scientific and Technical Information of China (English)

    Li Deren; Yang Jie

    2003-01-01

    The paper gives an overview of the principle and application of generating DEM from SAR, including the principle and processing flow of generating DEM from single SAR and SAR interferometry. Afterwards, the application fields of InSAR for terrain surveying, volcanic terrain surveying and D-InSAR for monitoring ground subsiding are listed and described as well.The problem and prospect of application are also pointed out in the last part of this paper.

  8. sar Ades

    Directory of Open Access Journals (Sweden)

    Aparecida Angélica Zoqui Paulovic Sabadini

    Full Text Available Este artigo é uma homenagem ao ilustre professor César Ades (1943-2012. Etólogo, Especialista em comportamento animal, Ades foi professor titular do Instituto de Psicologia da Universidade de São Paulo (IPUSP, atuando como docente do Departamento de Psicologia Experimental. O artigo descreve parte de sua rica vida acadêmica e profissional e apresenta, de forma resumida, sua trajetória na Universidade de São Paulo, como aluno, professor, pesquisador e orientador e sua atuação como administrador no Instituto de Psicologia e no Instituto de Estudos Avançados, além de sua atuação na Academia Paulista de Psicologia e em sociedades científicas. São destacados a importância de suas contribuições para a área de Psicologia e seu respeito pela vida, pelas pessoas e pelos animais.

  9. Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants.

    Directory of Open Access Journals (Sweden)

    Damon Deming

    2006-12-01

    Full Text Available In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV was identified as the etiological agent of severe acute respiratory syndrome, a disease characterized by severe pneumonia that sometimes results in death. SARS-CoV is a zoonotic virus that crossed the species barrier, most likely originating from bats or from other species including civets, raccoon dogs, domestic cats, swine, and rodents. A SARS-CoV vaccine should confer long-term protection, especially in vulnerable senescent populations, against both the 2003 epidemic strains and zoonotic strains that may yet emerge from animal reservoirs. We report the comprehensive investigation of SARS vaccine efficacy in young and senescent mice following homologous and heterologous challenge.Using Venezuelan equine encephalitis virus replicon particles (VRP expressing the 2003 epidemic Urbani SARS-CoV strain spike (S glycoprotein (VRP-S or the nucleocapsid (N protein from the same strain (VRP-N, we demonstrate that VRP-S, but not VRP-N vaccines provide complete short- and long-term protection against homologous strain challenge in young and senescent mice. To test VRP vaccine efficacy against a heterologous SARS-CoV, we used phylogenetic analyses, synthetic biology, and reverse genetics to construct a chimeric virus (icGDO3-S encoding a synthetic S glycoprotein gene of the most genetically divergent human strain, GDO3, which clusters among the zoonotic SARS-CoV. icGD03-S replicated efficiently in human airway epithelial cells and in the lungs of young and senescent mice, and was highly resistant to neutralization with antisera directed against the Urbani strain. Although VRP-S vaccines provided complete short-term protection against heterologous icGD03-S challenge in young mice, only limited protection was seen in vaccinated senescent animals. VRP-N vaccines not only failed to protect from homologous or heterologous challenge, but resulted in enhanced immunopathology with eosinophilic

  10. Structural Characterization of Human Coronavirus NL63 N Protein.

    Science.gov (United States)

    Szelazek, Bozena; Kabala, Wojciech; Kus, Krzysztof; Zdzalik, Michal; Twarda-Clapa, Aleksandra; Golik, Przemyslaw; Burmistrz, Michal; Florek, Dominik; Wladyka, Benedykt; Pyrc, Krzysztof; Dubin, Grzegorz

    2017-06-01

    Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections.IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of

  11. The Paradox of Feline Coronavirus Pathogenesis: A Review

    Directory of Open Access Journals (Sweden)

    Luciana Wanderley Myrrha

    2011-01-01

    Full Text Available Feline coronavirus (FCoV is an enveloped single-stranded RNA virus, of the family Coronaviridae and the order Nidovirales. FCoV is an important pathogen of wild and domestic cats and can cause a mild or apparently symptomless enteric infection, especially in kittens. FCoV is also associated with a lethal, systemic disease known as feline infectious peritonitis (FIP. Although the precise cause of FIP pathogenesis remains unclear, some hypotheses have been suggested. In this review we present results from different FCoV studies and attempt to elucidate existing theories on the pathogenesis of FCoV infection.

  12. 人卵巢颗粒肿瘤细胞COV434中抗苗勒氏管激素抑制干细胞生长因子表达的实验研究%Inhibition of stem cell factor expression by anti-müllerian hormone in human ovarian granulosa cell tumor COV434 cell line: an experimental study

    Institute of Scientific and Technical Information of China (English)

    王飞苗; 宋梦玲; 芦瑞萍; 马会明; 郑梦雪; 胡蓉

    2015-01-01

    目的 探讨人卵巢颗粒肿瘤细胞COV434中AMH对SCF表达的抑制作用.方法 用课题组前期已构建及鉴定成功转染AMH真核表达载体pIRES2-EGFP-AMH的COV434细胞系作为实验组,将空质粒载体转染的COV434细胞系作为阴性对照组;未经转染的COV434系作为空白对照组,分别采用Western blot和免疫荧光法检测不同干预组SCF蛋白的表达水平.结果 ①Western blot实验结果显示转染AMH真核表达载体pIRES2-EGFP-AMH的COV434中AMH表达较空白对照组和阴性对照组明显增强(P<0.05),阴性对照组和空白对照组无统计学差异(P>0.05).②Western blot实验结果显示SCF在实验组表达较空白对照组和阴性对照组显著降低(P<0.05).③免疫荧光实验结果显示SCF在实验组的平均光密度值较阴性对照组及空白对照组的表达显著升高(P<0.05).结论 人卵巢颗粒肿瘤细胞COV434中AMH能有效抑制SCF蛋白的表达,为颗粒细胞中AMH调控SCF机制的研究提供科学基础.

  13. Severe acute respiratory syndrome (SARS)

    Science.gov (United States)

    ... include: Arterial blood tests Blood clotting tests Blood chemistry tests Chest x-ray or chest CT scan ... The death rate from SARS was 9 to 12% of those diagnosed. In people over age 65, the death ...

  14. TerraSAR-X mission

    Science.gov (United States)

    Werninghaus, Rolf

    2004-01-01

    The TerraSAR-X is a German national SAR- satellite system for scientific and commercial applications. It is the continuation of the scientifically and technologically successful radar missions X-SAR (1994) and SRTM (2000) and will bring the national technology developments DESA and TOPAS into operational use. The space segment of TerraSAR-X is an advanced high-resolution X-Band radar satellite. The system design is based on a sound market analysis performed by Infoterra. The TerraSAR-X features an advanced high-resolution X-Band Synthetic Aperture Radar based on the active phased array technology which allows the operation in Spotlight-, Stripmap- and ScanSAR Mode with various polarizations. It combines the ability to acquire high resolution images for detailed analysis as well as wide swath images for overview applications. In addition, experimental modes like the Dual Receive Antenna Mode allow for full-polarimetric imaging as well as along track interferometry, i.e. moving target identification. The Ground Segment is optimized for flexible response to (scientific and commercial) User requests and fast image product turn-around times. The TerraSAR-X mission will serve two main goals. The first goal is to provide the strongly supportive scientific community with multi-mode X-Band SAR data. The broad spectrum of scientific application areas include Hydrology, Geology, Climatology, Oceanography, Environmental Monitoring and Disaster Monitoring as well as Cartography (DEM Generation) and Interferometry. The second goal is the establishment of a commercial EO-market in Europe which is driven by Infoterra. The commercial goal is the development of a sustainable EO-business so that the e.g. follow-on systems can be completely financed by industry from the profit. Due to its commercial potential, the TerraSAR-X project will be implemented based on a public-private partnership with the Astrium GmbH. This paper will describe first the mission objectives as well as the

  15. Focusing of bistatic SAR data

    Science.gov (United States)

    Bia, Pietro; Ricci, Nicola; Zonno, Mariantonietta; Nico, Giovanni; Catalao, Joao; Tesauro, Manlio

    2014-10-01

    The problems of simulation of bistatic SAR raw data and focusing are studied. A discrete target simulator is described. The simulator introduces the scene topography and compute the integration time of general bistatic configurations providing a means to derived maps of the range and azimuth spatial resolutions. The problem of focusing of bistatic SAR data acquired in a translational-invariant bistatic configuration is studied by deriving the bistatic Point Target Reference spectrum and presenting an analytical solution for its stationary points.

  16. Antibody binding site mapping of SARS-CoV spike protein receptor-binding domain by a combination of yeast surface display and phage peptide library screening.

    Science.gov (United States)

    Zhang, Xiaoping; Wang, Jingxue; Wen, Kun; Mou, Zhirong; Zou, Liyun; Che, Xiaoyan; Ni, Bing; Wu, Yuzhang

    2009-12-01

    The receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein plays an important role in viral infection, and is a potential major neutralizing determinant. In this study, three hybridoma cell lines secreting specific monoclonal antibodies against the RBD of the S protein were generated and their exact binding sites were identified. Using yeast surface display, the binding sites of these antibodies were defined to two linear regions on the RBD: S(337-360) and S(380-399). Using these monoclonal antibodies in phage peptide library screening identified 10 distinct mimotopes 12 amino acids in length. Sequence comparison between native epitopes and these mimotopes further confirmed the binding sites, and revealed key amino acid residues involved in antibody binding. None of these antibodies could neutralize the murine leukemia virus pseudotyped expressing the SARS-CoV spike protein (MLV/SARS-CoV). However, these mAbs could be useful in the diagnosis of SARS-CoV due to their exclusive reactivity with SARS-CoV. Furthermore, this study established a feasible platform for epitope mapping. Yeast surface display combined with phage peptide library screening provides a convenient strategy for the identification of epitope peptides from certain antigenic proteins.

  17. Three-Dimensional Human Bronchial-Tracheal Epithelial Tissue-Like Assemblies (TLAs) as Hosts For Severe Acute Respiratory Syndrome (SARS)-CoV Infection

    Science.gov (United States)

    Suderman, M. T.; McCarthy, M.; Mossell, E.; Watts, D. M.; Peters, C. J.; Shope, R.; Goodwin, T. J.

    2006-01-01

    A three-dimensional (3-D) tissue-like assembly (TLA) of human bronchial-tracheal mesenchymal (HBTC) cells with an overlay of human bronchial epithelial (BEAS-2B) cells was constructed using a NASA Bioreactor to survey the infectivity of SARS-CoV. This TLA was inoculated with a low passage number Urbani strain of SARS-CoV. At selected intervals over a 10-day period, media and cell aliquots of the 3-D TLA were harvested for viral titer assay and for light and electron microscopy examination. All viral titer assays were negative in both BEAS-2B two-dimensional monolayer and TLA. Light microscopy immunohistochemistry demonstrated antigen-antibody reactivity with anti-SARS-CoV polyclonal antibody to spike and nuclear proteins on cell membranes and cytoplasm. Coronavirus Group 2 cross-reactivity was demonstrated by positive reaction to anti-FIPV 1 and anti-FIPV 1 and 2 antibodies. TLA examination by transmission electron microscopy indicated increasing cytoplasmic vacuolation with numerous electron-dense bodies measuring 45 to 270 nm from days 4 through 10. There was no evidence of membrane blebbing, membrane duplication, or fragmentation of organelles in the TLAs. However, progressive disruption of endoplasmic reticulum was observed throughout the cells. Antibody response to SARS-CoV specific spike and nucleocapsid glycoproteins, cross-reactivity with FIPV antibodies, and the cytoplasmic pathology suggests this HBTE TLA model is permissive to SARS-CoV infection.

  18. Middle East respiratory syndrome coronavirus: epidemiology and disease control measures

    Directory of Open Access Journals (Sweden)

    Al-Tawfiq JA

    2014-11-01

    Full Text Available Jaffar A Al-Tawfiq,1,2 Ziad A Memish3,4 1Johns Hopkins Aramco Healthcare, Dhahran, Saudi Arabia; 2Indiana University School of Medicine, Indianapolis, IN, USA; 3Ministry of Health, 4Alfaisal University, Riyadh, Saudi Arabia Abstract: The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV infection in 2012 resulted in an increased concern of the spread of the infection globally. MERS-CoV infection had previously caused multiple health-care-associated outbreaks and resulted in transmission of the virus within families. Community onset MERS-CoV cases continue to occur. Dromedary camels are currently the most likely animal to be linked to human MERS-CoV cases. Serologic tests showed significant infection in adult camels compared to juvenile camels. The control of MERS-CoV infection relies on prompt identification of cases within health care facilities, with institutions applying appropriate infection control measures. In addition, determining the exact route of transmission from camels to humans would further add to the control measures of MERS-CoV infection. Keywords: MERS, Middle East respiratory syndrome coronavirus, epidemiology, control measures, transmission, Saudi Arabia

  19. Pathogenic characteristics of persistent feline enteric coronavirus infection in cats.

    Science.gov (United States)

    Vogel, Liesbeth; Van der Lubben, Mariken; te Lintelo, Eddie G; Bekker, Cornelis P J; Geerts, Tamara; Schuijff, Leontine S; Grinwis, Guy C M; Egberink, Herman F; Rottier, Peter J M

    2010-01-01

    Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed.

  20. Traditional Chinese medicine herbal extracts of Cibotium barometz, Gentiana scabra, Dioscorea batatas, Cassia tora, and Taxillus chinensis inhibit SARS-CoV replication.

    Science.gov (United States)

    Wen, Chih-Chun; Shyur, Lie-Fen; Jan, Jia-Tsrong; Liang, Po-Huang; Kuo, Chih-Jung; Arulselvan, Palanisamy; Wu, Jin-Bin; Kuo, Sheng-Chu; Yang, Ning-Sun

    2011-10-01

    Development of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV) agents is pivotal to prevent the reemergence of the life-threatening disease, SARS. In this study, more than 200 extracts from Chinese medicinal herbs were evaluated for anti-SARS-CoV activities using a cell-based assay that measured SARS-CoV-induced cytopathogenic effect (CPE) in vitro on Vero E6 cells. Six herbal extracts, one each from Gentianae Radix ( lóng dǎn; the dried rhizome of Gentiana scabra), Dioscoreae Rhizoma ( shān yào; the tuber of Dioscorea batatas), Cassiae Semen ( jué míng zǐ; the dried seed of Cassia tora) and Loranthi Ramus ( sāng jì shēng; the dried stem, with leaf of Taxillus chinensis) (designated as GSH, DBM, CTH and TCH, respectively), and two from Rhizoma Cibotii ( gǒu jǐ; the dried rhizome of Cibotium barometz) (designated as CBE and CBM), were found to be potent inhibitors of SARS-CoV at concentrations between 25 and 200 μg/ml. The concentrations of the six extracts needed to inhibit 50% of Vero E6 cell proliferation (CC50) and 50% of viral replication (EC50) were determined. The resulting selective index values (SI = CC50/EC50) of the most effective extracts CBE, GSH, DBM, CTH and TCH were > 59.4, > 57.5, > 62.1, > 59.4, and > 92.9, respectively. Among these extracts, CBM and DBM also showed significant inhibition of SARS-CoV 3CL protease activity with IC50 values of 39 μg/ml and 44 μg/ml, respectively. Our findings suggest that these six herbal extracts may have potential as candidates for future development of anti-SARS therapeutics.AbbreviationsSARS,severe acute respiratory syndromeCoV,coronavirusCPE,cytopathogenic effectTCM,traditional Chinese medicine.

  1. Comparison of two next-generation sequencing kits for diagnosis of epileptic disorders with a user-friendly tool for displaying gene coverage, DeCovA

    Directory of Open Access Journals (Sweden)

    Sarra Dimassi

    2015-12-01

    Full Text Available In recent years, molecular genetics has been playing an increasing role in the diagnostic process of monogenic epilepsies. Knowing the genetic basis of one patient's epilepsy provides accurate genetic counseling and may guide therapeutic options. Genetic diagnosis of epilepsy syndromes has long been based on Sanger sequencing and search for large rearrangements using MLPA or DNA arrays (array-CGH or SNP-array. Recently, next-generation sequencing (NGS was demonstrated to be a powerful approach to overcome the wide clinical and genetic heterogeneity of epileptic disorders. Coverage is critical for assessing the quality and accuracy of results from NGS. However, it is often a difficult parameter to display in practice. The aim of the study was to compare two library-building methods (Haloplex, Agilent and SeqCap EZ, Roche for a targeted panel of 41 genes causing monogenic epileptic disorders. We included 24 patients, 20 of whom had known disease-causing mutations. For each patient both libraries were built in parallel and sequenced on an Ion Torrent Personal Genome Machine (PGM. To compare coverage and depth, we developed a simple homemade tool, named DeCovA (Depth and Coverage Analysis. DeCovA displays the sequencing depth of each base and the coverage of target genes for each genomic position. The fraction of each gene covered at different thresholds could be easily estimated. None of the two methods used, namely NextGene and Ion Reporter, were able to identify all the known mutations/CNVs displayed by the 20 patients. Variant detection rate was globally similar for the two techniques and DeCovA showed that failure to detect a mutation was mainly related to insufficient coverage.

  2. The structure of a rigorously conserved RNA element within the SARS virus genome.

    Directory of Open Access Journals (Sweden)

    Michael P Robertson

    2005-01-01

    Full Text Available We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m RNA element of the SARS virus genome to 2.7-A resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3' end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90 degrees kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold.

  3. Photodissociation of ketene: CH{sub 2}CO {yields} CH{sub 2}(a{sup 1}A{sub 1}) + CO(v=1) rates and dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Wade, E.A.

    1996-12-01

    The rotational energy release in the dissociation of ketene (CH{sub 2}CO) along its singlet potential energy surface is observed and compared with several statistical and dynamical theories. Rotational distributions for the product, CO(X{sup 1}{Sigma}+)(v=1), are measured from the threshold for production of CH{sub 2}(a {sup 1}A{sub 1}) (0,0,0) + CO(X{sup 1}{Sigma}+)(v=1) to 1720 cm{sup -1} above. Near threshold (E{le} 200 cm{sup -1} over threshold), phase space theory (PST) matches the observed distributions. At 357 and 490 cm{sup -1}, PST constrained by the measured state distributions of the methylene fragment, provides a good fit to these CO(v=1) rotational distributions. For E > 490 cm{sup -1}, the constrained PST matches the average rotational energy observed but predicts distributions which are broader than observed. This contrasts to the rotational distributions of the {sup 1}CH{sub 2} fragment which become shifted to lower rotational states than PST as energy increases from 200 cm{sup -1} above threshold. Dynamical models, the impulsive model and Franck-Condon mapping, do not account for the product rotational state distributions. The CO(v=1) rotational distributions for E > 200 cm{sup -1} contain no measurable product from triplet channel fragmentation. Therefore, they can be compared with the previously determined CO(v=0) rotational distributions in order to partition the CO(v=0) yield between singlet and triplet channels and recalculate the singlet yield. This new yield is found to be at the upper limits of the range previously reported. Rate constants and quantum yields have been determined for the photodissociation of ketene to produce CH{sub 2}(a {sup 1}A{sub 1}) (0,0,0) + CO(X {sup 1}{Sigma}+)(v=1). At 57, 110, 200, 357, and 490 cm{sup -1} above this product threshold, vibrational branching ratios for the singlet products were measured and compared to phase space theory (PST), separate statistical ensembles (SSE), and variational RRKM (var. RRKM).

  4. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) origin and animal reservoir

    OpenAIRE

    Mohd, Hamzah A.; Al-Tawfiq, Jaffar A; Memish, Ziad A.

    2016-01-01

    Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans. Though not confirmed yet, multiple surveillance and phylogenetic studies suggest a bat origin. The disease is heavily endemic in dromedary camel populations of East Africa and the Middle East. It is unclear as to when the virus was introduced to dromedary camels, but data from studies that investigated stored dromedary camel sera and ge...

  5. TMPRSS2: A potential target for treatment of influenza virus and coronavirus infections.

    Science.gov (United States)

    Shen, Li Wen; Mao, Hui Juan; Wu, Yan Ling; Tanaka, Yoshimasa; Zhang, Wen

    2017-08-01

    Influenza virus and coronavirus epidemics or pandemics have occurred in succession worldwide throughout the early 21st century. These epidemics or pandemics pose a major threat to human health. Here, we outline a critical role of the host cell protease TMPRSS2 in influenza virus and coronavirus infections and highlight an antiviral therapeutic strategy targeting TMPRSS2. Copyright © 2017. Published by Elsevier B.V.

  6. Fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency.

    Science.gov (United States)

    Szczawinska-Poplonyk, Aleksandra; Jonczyk-Potoczna, Katarzyna; Breborowicz, Anna; Bartkowska-Sniatkowska, Alicja; Figlerowicz, Magdalena

    2013-09-01

    Coronaviruses have been demonstrated to contribute substantially to respiratory tract infections among the child population. Though infected children commonly present mild upper airway symptoms, in high-risk patients with underlying conditions, particularly in immunocompromised children these pathogens may lead to severe lung infection and extrapulmonary disorders. In this paper, we provide the first report of the case of a 15-month-old child with severe combined immunodeficiency and coronavirus HKU1-related pneumonia with fatal respiratory distress syndrome.

  7. Discovery of a novel coronavirus, China Rattus coronavirus HKU24, from Norway rats supports murine origin of Betacoronavirus 1 and has implications for the ancestor of Betacoronavirus lineage A

    OpenAIRE

    Susanna K. P. Lau; Woo, Patrick C.Y.; Li, Kenneth S. M.; Tsang, Alan K. L.; Fan, Rachel Y. Y.; Luk, Hayes K. H.; Cai, Jian-Piao; Chan, Kwok-Hung; Zheng, Bo-Jian; Wang, Ming; Yuen, Kwok-Yung

    2015-01-01

    We discovered a novel Betacoronavirus lineage A coronavirus, China Rattus coronavirus (ChRCoV) HKU24, from Norway rats in China. ChRCoV HKU24 occupied a deep branch at the root of members of Betacoronavirus 1, being distinct from murine coronavirus and human coronavirus HKU1. Its unique putative cleavage sites between nonstructural proteins 1 and 2 and in the spike (S) protein and low sequence identities to other lineage A betacoronaviruses (βCoVs) in conserved replicase domains support ChRCo...

  8. Severe acute respiratory syndrome: 'SARS' or 'not SARS'.

    Science.gov (United States)

    Li, A M; Hon, K L E; Cheng, W T; Ng, P C; Chan, F Y; Li, C K; Leung, T F; Fok, T F

    2004-01-01

    Accurate clinical diagnosis of severe acute respiratory syndrome (SARS) based on the current World Health Organization definition is difficult and at times impossible at the early stage of the disease. Both false positive and false negative cases are commonly encountered and this could have far-reaching detrimental effects on the patients, their family and the clinicians alike. Contact history is particularly important in diagnosing SARS in children as their presenting features are often non-specific. The difficulty in making a correct diagnosis is further compounded by the lack of a sensitive rapid diagnostic test. Serology is not particularly helpful in the initial triaging of patients as it takes at least 3 weeks to become positive. Co-infection and other treatable conditions should not be missed and conventional antibiotics should remain as part of the first-line treatment regimen. We report five cases to illustrate the difficulties and dilemmas faced by clinicians in diagnosing SARS in children.

  9. Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus.

    Science.gov (United States)

    Castilla, J; Sola, I; Pintado, B; Sánchez-Morgado, J M; Enjuanes, L

    1998-01-01

    Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection

  10. Wave directional spectrum from SAR imagery

    Digital Repository Service at National Institute of Oceanography (India)

    Fernandes, A.A.; Sarma, Y.V.B.; Menon, H.B.; Vethamony, P.

    Gaussian smoothed SAR image spectra have been evaluated from 512 x 512 pixel sub- scenes of image mode ERS-1 SAR scenes off Goa, Visakhapatnam, Paradeep and Portugal. The two recently acquired scenes off Portugal showed the signature of swell...

  11. SAR Image Enhancement using Particle Filters

    Data.gov (United States)

    National Aeronautics and Space Administration — In this paper, we propose a novel approach to reduce the noise in Synthetic Aperture Radar (SAR) images using particle filters. Interpretation of SAR images is a...

  12. Novel Polarimetric SAR Interferometry Algorithms Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Polarimetric radar interferometry (PolInSAR) is a new SAR imaging mode that is rapidly becoming an important technique for bare earth topographic mapping, tree...

  13. SARS Patients and Their Close Contacts

    Science.gov (United States)

    ... Links Clinician Registry Travelers' Health Fact Sheet for SARS Patients and Their Close Contacts Format: Select one ... of the World Health Organization (WHO) . Symptoms of SARS The illness usually begins with a fever (measured ...

  14. Wave directional spectrum from SAR imagery

    Digital Repository Service at National Institute of Oceanography (India)

    Fernandes, A.A.; Sarma, Y.V.B.; Menon, H.B.; Vethamony, P.

    Gaussian smoothed SAR image spectra have been evaluated from 512 x 512 pixel subscenes of image mode ERS-1 SAR scenes off Goa, Visakhapatnam, Paradeep and Portugal. The two recently acquired scenes off Portugal showed the signature of swell...

  15. Accelerated Scientific InSAR Processing Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Neva Ridge Technologies proposes to develop a suite of software tools for the analysis of SAR and InSAR data, focused on having a robust and adopted capability well...

  16. Inhibitor recognition specificity of MERS-CoV papain-like protease may differ from that of SARS-CoV.

    Science.gov (United States)

    Lee, Hyun; Lei, Hao; Santarsiero, Bernard D; Gatuz, Joseph L; Cao, Shuyi; Rice, Amy J; Patel, Kavankumar; Szypulinski, Michael Z; Ojeda, Isabel; Ghosh, Arun K; Johnson, Michael E

    2015-06-19

    The Middle East Respiratory Syndrome coronavirus (MERS-CoV) papain-like protease (PLpro) blocking loop 2 (BL2) structure differs significantly from that of SARS-CoV PLpro, where it has been proven to play a crucial role in SARS-CoV PLpro inhibitor binding. Four SARS-CoV PLpro lead inhibitors were tested against MERS-CoV PLpro, none of which were effective against MERS-CoV PLpro. Structure and sequence alignments revealed that two residues, Y269 and Q270, responsible for inhibitor binding to SARS-CoV PLpro, were replaced by T274 and A275 in MERS-CoV PLpro, making critical binding interactions difficult to form for similar types of inhibitors. High-throughput screening (HTS) of 25 000 compounds against both PLpro enzymes identified a small fragment-like noncovalent dual inhibitor. Mode of inhibition studies by