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.

Cleavage of group 1 coronavirus spike proteins: how furin cleavage is traded off against heparan sulfate binding upon cell culture adaptation

NARCIS (Netherlands)

Haan, de C.A.M.; Haijema, B.J.; Schellen, P.; Wichgers Schreur, P.J.; Lintelo, te E.; Vennema, H.; Rottier, P.J.M.

2008-01-01

A longstanding enigmatic feature of the group 1 coronaviruses is the uncleaved phenotype of their spike protein, an exceptional property among class I fusion proteins. Here, however, we show that some group 1 coronavirus spike proteins carry a furin enzyme recognition motif and can actually be

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

Synthesis and processing of structural and intracellular proteins of two enteric coronaviruses

International Nuclear Information System (INIS)

Sardinia, L.M.

1985-01-01

The synthesis and processing of virus-specific proteins of two economically important enteric coronaviruses, bovine enteric coronavirus (BCV) and transmissible gastroenteritis virus (TGEV), were studied at the molecular level. To determine the time of appearance of virus-specific proteins, virus-infected cells were labeled with 35 S-methionine at various times during infection, immunoprecipitated with specific hyperimmune ascitic fluid, and analyzed by SDS-polyacrylamide gel electrophoresis. The peak of BCV protein synthesis was found to be at 12 hours postinfection (hpi). The appearance of all virus-specific protein was coordinated. In contrast, the peak of TGEV protein synthesis was at 8 hpi, but the nucleocapsid proteins was present as early as 4 hpi. Virus-infected cells were treated with tunicamycin to ascertain the types of glycosidic linkages of the glycoproteins. The peplomer proteins of both viruses were sensitive to inhibition by tunicamycin indicating that they possessed N-linked carbohydrates. The matrix protein of TGEV was similarly affected. The matrix protein of BCV, however, was resistant to tunicamycin treatment and, therefore, has O-linked carbohydrates. Only the nucleocapsid protein of both viruses is phosphorylated as detected by radiolabeling with 32 P-orthophosphate. Pulse-chase studies and comparison of intracellular and virion proteins were done to detect precursor-product relationships

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.

Characterization of an Immunodominant Epitope in the Endodomain of the Coronavirus Membrane Protein

Directory of Open Access Journals (Sweden)

Hui Dong

2016-12-01

Full Text Available The coronavirus membrane (M protein acts as a dominant immunogen and is a major player in virus assembly. In this study, we prepared two monoclonal antibodies (mAbs; 1C3 and 4C7 directed against the transmissible gastroenteritis virus (TGEV M protein. The 1C3 and 4C7 mAbs both reacted with the native TGEV M protein in western blotting and immunofluorescence (IFA assays. Two linear epitopes, 243YSTEART249 (1C3 and 243YSTEARTDNLSEQEKLLHMV262 (4C7, were identified in the endodomain of the TGEV M protein. The 1C3 mAb can be used for the detection of the TGEV M protein in different assays. An IFA method for the detection of TGEV M protein was optimized using mAb 1C3. Furthermore, the ability of the epitope identified in this study to stimulate antibody production was also evaluated. An immunodominant epitope in the TGEV membrane protein endodomain was identified. The results of this study have implications for further research on TGEV replication.

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

2009-12-01

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.

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

Structure of the C-terminal domain of nsp4 from feline coronavirus

International Nuclear Information System (INIS)

Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh; Snijder, Eric J.; Gorbalenya, Alexander E.; Berglind, Hanna; Nordlund, Pär; Coutard, Bruno; Tucker, Paul A.

2009-01-01

The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4 3 . The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions

Structure of the C-terminal domain of nsp4 from feline coronavirus

Energy Technology Data Exchange (ETDEWEB)

Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany); Snijder, Eric J.; Gorbalenya, Alexander E. [Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden (Netherlands); Berglind, Hanna; Nordlund, Pär [Division of Biophysics, Department of Medical Biochemistry and Biophysics, Scheeles väg 2, Karolinska Institute, SE-171 77 Stockholm (Sweden); Coutard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098, AFMB-CNRS-ESIL, Case 925, 163 Avenue de Luminy, 13288 Marseille (France); Tucker, Paul A., E-mail: tucker@embl-hamburg.de [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany)

2009-08-01

The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.

Cooperation of an RNA Packaging Signal and a Viral Envelope Protein in Coronavirus RNA Packaging

OpenAIRE

Narayanan, Krishna; Makino, Shinji

2001-01-01

Murine coronavirus mouse hepatitis virus (MHV) produces a genome-length mRNA, mRNA 1, and six or seven species of subgenomic mRNAs in infected cells. Among these mRNAs, only mRNA 1 is efficiently packaged into MHV particles. MHV N protein binds to all MHV mRNAs, whereas envelope M protein interacts only with mRNA 1. This M protein-mRNA 1 interaction most probably determines the selective packaging of mRNA 1 into MHV particles. A short cis-acting MHV RNA packaging signal is necessary and suffi...

Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in Gammacoronavirus.

Science.gov (United States)

Woo, Patrick C Y; Lau, Susanna K P; Lam, Carol S F; Tsang, Alan K L; Hui, Suk-Wai; Fan, Rachel Y Y; Martelli, Paolo; Yuen, Kwok-Yung

2014-01-01

While gammacoronaviruses mainly comprise infectious bronchitis virus (IBV) and its closely related bird coronaviruses (CoVs), the only mammalian gammacoronavirus was discovered from a white beluga whale (beluga whale CoV [BWCoV] SW1) in 2008. In this study, we discovered a novel gammacoronavirus from fecal samples from three Indo-Pacific bottlenose dolphins (Tursiops aduncus), which we named bottlenose dolphin CoV (BdCoV) HKU22. All the three BdCoV HKU22-positive samples were collected on the same date, suggesting a cluster of infection, with viral loads of 1 × 10(3) to 1 × 10(5) copies per ml. Clearance of virus was associated with a specific antibody response against the nucleocapsid of BdCoV HKU22. Complete genome sequencing and comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 have similar genome characteristics and structures. Their genome size is about 32,000 nucleotides, the largest among all CoVs, as a result of multiple unique open reading frames (NS5a, NS5b, NS5c, NS6, NS7, NS8, NS9, and NS10) between their membrane (M) and nucleocapsid (N) protein genes. Although comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 should belong to the same species, a major difference was observed in the proteins encoded by their spike (S) genes, which showed only 74.3 to 74.7% amino acid identities. The high ratios of the number of synonymous substitutions per synonymous site (Ks) to the number of nonsynonymous substitutions per nonsynonymous site (Ka) in multiple regions of the genome, especially the S gene (Ka/Ks ratio, 2.5), indicated that BdCoV HKU22 may be evolving rapidly, supporting a recent transmission event to the bottlenose dolphins. We propose a distinct species, Cetacean coronavirus, in Gammacoronavirus, to include BdCoV HKU22 and BWCoV SW1, whereas IBV and its closely related bird CoVs represent another species, Avian coronavirus, in Gammacoronavirus.

Characterization of the expression and immunogenicity of the ns4b protein of human coronavirus 229E

DEFF Research Database (Denmark)

Chagnon, F; Lamarre, A; Lachance, C

1998-01-01

to demonstrate the expression of ns4b in HCV-229E-infected cells using flow cytometry. Given a previously reported contiguous five amino acid shared region between ns4b and myelin basic protein, a purified recombinant histidine-tagged ns4b protein and (or) human myelin basic protein were injected into mice......Sequencing of complementary DNAs prepared from various coronaviruses has revealed open reading frames encoding putative proteins that are yet to be characterized and are so far only described as nonstructural (ns). As a first step in the elucidation of its function, we characterized the expression...... and immunogenicity of the ns4b gene product from strain 229E of human coronavirus (HCV-229E), a respiratory virus with a neurotropic potential. The gene was cloned and expressed in bacteria. A fusion protein of ns4b with maltose-binding protein was injected into rabbits to generate specific antibodies that were used...

Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

Directory of Open Access Journals (Sweden)

Jaume Torres

2015-06-01

Full Text Available Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i the envelope protein in coronaviruses and (ii the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity.

Discovery of Seven Novel Mammalian and Avian Coronaviruses in the Genus Deltacoronavirus Supports Bat Coronaviruses as the Gene Source of Alphacoronavirus and Betacoronavirus and Avian Coronaviruses as the Gene Source of Gammacoronavirus and Deltacoronavirus

OpenAIRE

Woo, Patrick C. Y.; Lau, Susanna K. P.; Lam, Carol S. F.; Lau, Candy C. Y.; Tsang, Alan K. L.; Lau, John H. N.; Bai, Ru; Teng, Jade L. L.; Tsang, Chris C. C.; Wang, Ming; Zheng, Bo-Jian; Chan, Kwok-Hung; Yuen, Kwok-Yung

2012-01-01

Recently, we reported the discovery of three novel coronaviruses, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13, which were identified as representatives of a novel genus, Deltacoronavirus, in the subfamily Coronavirinae. In this territory-wide molecular epidemiology study involving 3,137 mammals and 3,298 birds, we discovered seven additional novel deltacoronaviruses in pigs and birds, which we named porcine coronavirus HKU15, white-eye coronavirus HKU16, sp...

Identification of a new human coronavirus

NARCIS (Netherlands)

van der Hoek, Lia; Pyrc, Krzysztof; Jebbink, Maarten F.; Vermeulen-Oost, Wilma; Berkhout, Ron J. M.; Wolthers, Katja C.; Wertheim-van Dillen, Pauline M. E.; Kaandorp, Jos; Spaargaren, Joke; Berkhout, Ben

2004-01-01

Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). Here we report the identification of a fourth human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was

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.

Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

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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.

Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus.

Science.gov (United States)

Woo, Patrick C Y; Lau, Susanna K P; Lam, Carol S F; Lau, Candy C Y; Tsang, Alan K L; Lau, John H N; Bai, Ru; Teng, Jade L L; Tsang, Chris C C; Wang, Ming; Zheng, Bo-Jian; Chan, Kwok-Hung; Yuen, Kwok-Yung

2012-04-01

Recently, we reported the discovery of three novel coronaviruses, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13, which were identified as representatives of a novel genus, Deltacoronavirus, in the subfamily Coronavirinae. In this territory-wide molecular epidemiology study involving 3,137 mammals and 3,298 birds, we discovered seven additional novel deltacoronaviruses in pigs and birds, which we named porcine coronavirus HKU15, white-eye coronavirus HKU16, sparrow coronavirus HKU17, magpie robin coronavirus HKU18, night heron coronavirus HKU19, wigeon coronavirus HKU20, and common moorhen coronavirus HKU21. Complete genome sequencing and comparative genome analysis showed that the avian and mammalian deltacoronaviruses have similar genome characteristics and structures. They all have relatively small genomes (25.421 to 26.674 kb), the smallest among all coronaviruses. They all have a single papain-like protease domain in the nsp3 gene; an accessory gene, NS6 open reading frame (ORF), located between the M and N genes; and a variable number of accessory genes (up to four) downstream of the N gene. Moreover, they all have the same putative transcription regulatory sequence of ACACCA. Molecular clock analysis showed that the most recent common ancestor of all coronaviruses was estimated at approximately 8100 BC, and those of Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus were at approximately 2400 BC, 3300 BC, 2800 BC, and 3000 BC, respectively. From our studies, it appears that bats and birds, the warm blooded flying vertebrates, are ideal hosts for the coronavirus gene source, bats for Alphacoronavirus and Betacoronavirus and birds for Gammacoronavirus and Deltacoronavirus, to fuel coronavirus evolution and dissemination.

About Coronavirus

Science.gov (United States)

... Coronaviruses Symptoms and Diagnosis Transmission Prevention and Treatment Human Coronavirus Types SARS-CoV MERS-CoV Resources and References About Coronaviruses Recommend on Facebook Tweet Share Compartir Symptoms and Diagnosis Lists illnesses ...

Induction of Apoptosis by the Severe Acute Respiratory Syndrome Coronavirus 7a Protein Is Dependent on Its Interaction with the Bcl-XL Protein▿

Science.gov (United States)

Tan, Ying-Xim; Tan, Timothy H. P.; Lee, Marvin J.-R.; Tham, Puay-Yoke; Gunalan, Vithiagaran; Druce, Julian; Birch, Chris; Catton, Mike; Fu, Nai Yang; Yu, Victor C.; Tan, Yee-Joo

2007-01-01

The severe acute respiratory syndrome coronavirus (SARS-CoV) 7a protein, which is not expressed by other known coronaviruses, can induce apoptosis in various cell lines. In this study, we show that the overexpression of Bcl-XL, a prosurvival member of the Bcl-2 family, blocks 7a-induced apoptosis, suggesting that the mechanism for apoptosis induction by 7a is at the level of or upstream from the Bcl-2 family. Coimmunoprecipitation experiments showed that 7a interacts with Bcl-XL and other prosurvival proteins (Bcl-2, Bcl-w, Mcl-1, and A1) but not with the proapoptotic proteins (Bax, Bak, Bad, and Bid). A good correlation between the abilities of 7a deletion mutants to induce apoptosis and to interact with Bcl-XL was observed, suggesting that 7a triggers apoptosis by interfering directly with the prosurvival function of Bcl-XL. Interestingly, amino acids 224 and 225 within the C-terminal transmembrane domain of Bcl-XL are essential for the interaction with the 7a protein, although the BH3 domain of Bcl-XL also contributes to this interaction. In addition, fractionation experiments showed that 7a colocalized with Bcl-XL at the endoplasmic reticulum as well as the mitochondria, suggesting that they may form complexes in different membranous compartments. PMID:17428862

Sialic Acid Binding Properties of Soluble Coronavirus Spike (S1 Proteins: Differences between Infectious Bronchitis Virus and Transmissible Gastroenteritis Virus

Directory of Open Access Journals (Sweden)

Christine Winter

2013-07-01

Full Text Available The spike proteins of a number of coronaviruses are able to bind to sialic acids present on the cell surface. The importance of this sialic acid binding ability during infection is, however, quite different. We compared the spike protein of transmissible gastroenteritis virus (TGEV and the spike protein of infectious bronchitis virus (IBV. Whereas sialic acid is the only receptor determinant known so far for IBV, TGEV requires interaction with its receptor aminopeptidase N to initiate infection of cells. Binding tests with soluble spike proteins carrying an IgG Fc-tag revealed pronounced differences between these two viral proteins. Binding of the IBV spike protein to host cells was in all experiments sialic acid dependent, whereas the soluble TGEV spike showed binding to APN but had no detectable sialic acid binding activity. Our results underline the different ways in which binding to sialoglycoconjugates is mediated by coronavirus spike proteins.

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

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.

Development of Broad-Spectrum Halomethyl Ketone Inhibitors Against Coronavirus Main Protease 3CL(pro)

Energy Technology Data Exchange (ETDEWEB)

Bacha,U.; Barilla, J.; Gabelli, S.; Kiso, Y.; Amzel, L.; Freire, E.

2008-01-01

Coronaviruses comprise a large group of RNA viruses with diverse host specificity. The emergence of highly pathogenic strains like the SARS coronavirus (SARS-CoV), and the discovery of two new coronaviruses, NL-63 and HKU1, corroborates the high rate of mutation and recombination that have enabled them to cross species barriers and infect novel hosts. For that reason, the development of broad-spectrum antivirals that are effective against several members of this family is highly desirable. This goal can be accomplished by designing inhibitors against a target, such as the main protease 3CLpro (Mpro), which is highly conserved among all coronaviruses. Here 3CLpro derived from the SARS-CoV was used as the primary target to identify a new class of inhibitors containing a halomethyl ketone warhead. The compounds are highly potent against SARS 3CLpro with Ki's as low as 300 nm. The crystal structure of the complex of one of the compounds with 3CLpro indicates that this inhibitor forms a thioether linkage between the halomethyl carbon of the warhead and the catalytic Cys 145. Furthermore, Structure Activity Relationship (SAR) studies of these compounds have led to the identification of a pharmacophore that accurately defines the essential molecular features required for the high affinity.

The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

International Nuclear Information System (INIS)

Kumar, Purnima; Gunalan, Vithiagaran; Liu Boping; Chow, Vincent T.K.; Druce, Julian; Birch, Chris; Catton, Mike; Fielding, Burtram C.; Tan, Yee-Joo; Lal, Sunil K.

2007-01-01

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication

Feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene.

Science.gov (United States)

Chang, Hui-Wen; de Groot, Raoul J; Egberink, Herman F; Rottier, Peter J M

2010-02-01

Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP virus (FIPV), a virulent mutant of apathogenic feline enteric coronavirus (FECV). We analysed the 3c gene--a proposed virulence marker--in 27 FECV- and 28 FIPV-infected cats. Our findings suggest that functional 3c protein expression is crucial for FECV replication in the gut, but dispensable for systemic FIPV replication. Whilst intact in all FECVs, the 3c gene was mutated in the majority (71.4 %) of FIPVs, but not in all, implying that mutation in 3c is not the (single) cause of FIP. Most cats with FIP had no detectable intestinal feline coronaviruses (FCoVs) and had seemingly cleared the primary FECV infection. In those with detectable intestinal FCoV, the virus always had an intact 3c and seemed to have been acquired by FECV superinfection. Apparently, 3c-inactivated viruses replicate not at all--or only poorly--in the gut, explaining the rare incidence of FIP outbreaks.

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.

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...... and three PCR assays with primers specific for the nucleocapsid protein of human coronavirus strain OC43 were performed. Sporadic positive PCR assays were observed in both patients and controls in some of the PCR assays. However, these results were not reproducible and there was no difference...... 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....

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

Directory of Open Access Journals (Sweden)

Albrecht von Brunn

2007-05-01

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.

Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein

NARCIS (Netherlands)

Godeke, G J; de Haan, Cornelis A M; Rossen, J W; Vennema, H; Rottier, P J

The type I glycoprotein S of coronavirus, trimers of which constitute the typical viral spikes, is assembled into virions through noncovalent interactions with the M protein. Here we demonstrate that incorporation is mediated by the short carboxy-terminal segment comprising the transmembrane and

Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses.

Directory of Open Access Journals (Sweden)

Yutaka Terada

Full Text Available Type II feline coronavirus (FCoV emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV. In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3'-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5'-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently.

Middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies

NARCIS (Netherlands)

F. Song (Fei); R. Fux (Robert); L.B.V. Provacia (Lisette); A. Volz (Asisa); M. Eickmann; S. Becker (Stephan); A.D.M.E. Osterhaus (Albert); B.L. Haagmans (Bart); G. Suttera (Gerd)

2013-01-01

textabstractMiddle East respiratory syndrome coronavirus (MERS-CoV) has recently emerged as a causative agent of severe respiratory disease in humans. Here, we constructed recombinant modified vaccinia virus Ankara (MVA) expressing full-length MERS-CoV spike (S) protein (MVA-MERS-S). The genetic

Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor

Energy Technology Data Exchange (ETDEWEB)

Wu, Kailang; Li, Weikai; Peng, Guiqing; Li, Fang; (Harvard-Med); (UMM-MED)

2010-03-04

NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel {beta}-sandwich core structure consisting of 2 layers of {beta}-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homology in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a 'virus-binding hotspot' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.

Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

International Nuclear Information System (INIS)

Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong

2012-01-01

A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

Energy Technology Data Exchange (ETDEWEB)

Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong, E-mail: yerong24@fudan.edu.cn

2012-06-05

A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

Science.gov (United States)

Licitra, Beth N.; Millet, Jean K.; Regan, Andrew D.; Hamilton, Brian S.; Rinaldi, Vera D.; Duhamel, Gerald E.

2013-01-01

Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses. PMID:23763835

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.

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.

Immune responses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine

International Nuclear Information System (INIS)

Zhao Ping; Cao Jie; Zhao Lanjuan; Qin Zhaolin; Ke Jinshan; Pan Wei; Ren Hao; Yu Jianguo; Qi Zhongtian

2005-01-01

The nucleocapsid (N) protein of SARS-coronavirus (SARS-CoV) is the key protein for the formation of the helical nucleocapsid during virion assembly. This protein is believed to be more conserved than other proteins of the virus, such as spike and membrane glycoprotein. In this study, the N protein of SARS-CoV was expressed in Escherichia coli DH5α and identified with pooled sera from patients in the convalescence phase of SARS. A plasmid pCI-N, encoding the full-length N gene of SARS-CoV, was constructed. Expression of the N protein was observed in COS1 cells following transfection with pCI-N. The immune responses induced by intramuscular immunization with pCI-N were evaluated in a murine model. Serum anti-N immunoglobulins and splenocytes proliferative responses against N protein were observed in immunized BALB/c mice. The major immunoglobulin G subclass recognizing N protein was immunoglobulin G2a, and stimulated splenocytes secreted high levels of gamma interferon and IL-2 in response to N protein. More importantly, the immunized mice produced strong delayed-type hypersensitivity (DTH) and CD8 + CTL responses to N protein. The study shows that N protein of SARS-CoV not only is an important B cell immunogen, but also can elicit broad-based cellular immune responses. The results indicate that the N protein may be of potential value in vaccine development for specific prophylaxis and treatment against SARS

Mosaic Evolution of the Severe Acute Respiratory Syndrome Coronavirus

Science.gov (United States)

Stavrinides, John; Guttman, David S.

2004-01-01

Severe acute respiratory syndrome (SARS) is a deadly form of pneumonia caused by a novel coronavirus, a viral family responsible for mild respiratory tract infections in a wide variety of animals including humans, pigs, cows, mice, cats, and birds. Analyses to date have been unable to identify the precise origin of the SARS coronavirus. We used Bayesian, neighbor-joining, and split decomposition phylogenetic techniques on the SARS virus replicase, surface spike, matrix, and nucleocapsid proteins to reveal the evolutionary origin of this recently emerging infectious agent. The analyses support a mammalian-like origin for the replicase protein, an avian-like origin for the matrix and nucleocapsid proteins, and a mammalian-avian mosaic origin for the host-determining spike protein. A bootscan recombination analysis of the spike gene revealed high nucleotide identity between the SARS virus and a feline infectious peritonitis virus throughout the gene, except for a 200- base-pair region of high identity to an avian sequence. These data support the phylogenetic analyses and suggest a possible past recombination event between mammalian-like and avian-like parent viruses. This event occurred near a region that has been implicated to be the human receptor binding site and may have been directly responsible for the switch of host of the SARS coronavirus from animals to humans. PMID:14671089

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.

Evidence for an ancestral association of human coronavirus 229E with bats

Czech Academy of Sciences Publication Activity Database

Corman, V. M.; Baldwin, H. J.; Tateno, A. F.; Zerbinati, R. M.; Annan, A.; Owusu, M.; Nkrumah, E. E.; Maganga, G. D.; Oppong, S.; Adu-Sarkodie, Y.; Vallo, Peter; da Silva Filho, L. V. R. F.; Leroy, E. M.; Thiel, V.; van der Hoek, L.; Poon, L. L. M.; Tschapka, M.; Drosten, C.; Drexler, J. F.

2015-01-01

Roč. 89, č. 23 (2015), s. 11858-11870 ISSN 0022-538X Institutional support: RVO:68081766 Keywords : respiratory syndrome coronavirus * SARS-coronavirus * genomic characterization * dromedary camels * clinical impact * virus * children * protein * spike * classification Subject RIV: FN - Epidemiology, Contagious Diseases ; Clinical Immunology Impact factor: 4.606, year: 2015

In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins

Directory of Open Access Journals (Sweden)

Jeremiah Athmer

2017-01-01

Full Text Available Coronavirus (CoV replication and transcription are carried out in close proximity to restructured endoplasmic reticulum (ER membranes in replication/transcription complexes (RTC. Many of the CoV nonstructural proteins (nsps are required for RTC function; however, not all of their functions are known. nsp15 contains an endoribonuclease domain that is conserved in the CoV family. While the enzymatic activity and crystal structure of nsp15 are well defined, its role in replication remains elusive. nsp15 localizes to sites of RNA replication, but whether it acts independently or requires additional interactions for its function remains unknown. To begin to address these questions, we created an in situ tagged form of nsp15 using the prototypic CoV, mouse hepatitis virus (MHV. In MHV, nsp15 contains the genomic RNA packaging signal (P/S, a 95-bp RNA stem-loop structure that is not required for viral replication or nsp15 function. Utilizing this knowledge, we constructed an internal hemagglutinin (HA tag that replaced the P/S. We found that nsp15-HA was localized to discrete perinuclear puncta and strongly colocalized with nsp8 and nsp12, both well-defined members of the RTC, but not the membrane (M protein, involved in virus assembly. Finally, we found that nsp15 interacted with RTC-associated proteins nsp8 and nsp12 during infection, and this interaction was RNA independent. From this, we conclude that nsp15 localizes and interacts with CoV proteins in the RTC, suggesting it plays a direct or indirect role in virus replication. Furthermore, the use of in situ epitope tags could be used to determine novel nsp-nsp interactions in coronaviruses.

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

Broad-Spectrum Inhibitors against 3C-Like Proteases of Feline Coronaviruses and Feline Caliciviruses

Science.gov (United States)

Shivanna, Vinay; Narayanan, Sanjeev; Prior, Allan M.; Weerasekara, Sahani; Hua, Duy H.; Kankanamalage, Anushka C. Galasiti; Groutas, William C.; Chang, Kyeong-Ok

2015-01-01

ABSTRACT Feline infectious peritonitis and virulent, systemic calicivirus infection are caused by certain types of feline coronaviruses (FCoVs) and feline caliciviruses (FCVs), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families, the Coronaviridae and the Caliciviridae, respectively, they share a dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally and structurally conserved among these viruses and essential for viral replication, 3CLpro is considered a potential target for the design of antiviral drugs with broad-spectrum activities against these distinct and highly important viral infections. However, small-molecule inhibitors against the 3CLpro enzymes of FCoV and FCV have not been previously identified. In this study, derivatives of peptidyl compounds targeting 3CLpro were synthesized and evaluated for their activities against FCoV and FCV. The structures of compounds that showed potent dual antiviral activities with a wide margin of safety were identified and are discussed. Furthermore, the in vivo efficacy of 3CLpro inhibitors was evaluated using a mouse model of coronavirus infection. Intraperitoneal administration of two 3CLpro inhibitors in mice infected with murine hepatitis virus A59, a hepatotropic coronavirus, resulted in significant reductions in virus titers and pathological lesions in the liver compared to the findings for the controls. These results suggest that the series of 3CLpro inhibitors described here may have the potential to be further developed as therapeutic agents against these important viruses in domestic and wild cats. This study provides important insights into the structure and function relationships of 3CLpro for the design of antiviral drugs with broader antiviral activities. IMPORTANCE Feline infectious peritonitis virus (FIPV) is the leading cause of death in young cats

Isolation of avian infectious bronchitis coronavirus from domestic peafowl (Pavo cristatus) and teal (Anas).

Science.gov (United States)

Liu, Shengwang; Chen, Jianfei; Chen, Jinding; Kong, Xiangang; Shao, Yuhao; Han, Zongxi; Feng, Li; Cai, Xuehui; Gu, Shoulin; Liu, Ming

2005-03-01

Coronavirus-like viruses, designated peafowl/China/LKQ3/2003 (pf/CH/LKQ3/03) and teal/China/LDT3/2003 (tl/CH/LDT3/03), were isolated from a peafowl and a teal during virological surveillance in Guangdong province, China. Partial genomic sequence analysis showed that these isolates had the S-3-M-5-N gene order that is typical of avian coronaviruses. The spike, membrane and nucleocapsid protein genes of pf/CH/LKQ3/03 had >99 % identity to those of the avian infectious bronchitis coronavirus H120 vaccine strain (Massachusetts serotype) and other Massachusetts serotype isolates. Furthermore, when pf/CH/LKQ3/03 was inoculated experimentally into chickens (specific-pathogen-free), no disease signs were apparent. tl/CH/LDT3/03 had a spike protein gene with 95 % identity to that of a Chinese infectious bronchitis virus (IBV) isolate, although more extensive sequencing revealed the possibility that this strain may have undergone recombination. When inoculated into chickens, tl/CH/LDT3/03 resulted in the death of birds from nephritis. Taken together, this information suggests that pf/CH/LKQ3/03 might be a revertant, attenuated vaccine IBV strain, whereas tl/CH/LDT3/03 is a nephropathogenic field IBV strain, generated through recombination. The replication and non-pathogenic nature of IBV in domestic peafowl and teal under field conditions raises questions as to the role of these hosts as carriers of IBV and the potential that they may have to transmit virus to susceptible chicken populations.

Discovery of novel bat coronaviruses in south China that use the same receptor as MERS coronavirus.

Science.gov (United States)

Luo, Chu-Ming; Wang, Ning; Yang, Xing-Lou; Liu, Hai-Zhou; Zhang, Wei; Li, Bei; Hu, Ben; Peng, Cheng; Geng, Qi-Bin; Zhu, Guang-Jian; Li, Fang; Shi, Zheng-Li

2018-04-18

Middle East respiratory syndrome coronavirus (MERS-CoV) has represented a human health threat since 2012. Although several MERS-related CoVs, which belong to the same species as MERS-CoV, have been identified from bats, they do not use the MERS-CoV receptor, dipeptidyl peptidase 4 (DPP4). Here, we screened 1059 bat samples from at least 30 bat species collected in different regions in south China and identified 89 strains of lineage C betacoronaviruses, including Tylonycteris pachypus HKU4 , Pipistrellus pipistrellus HKU5, and MERS-related CoVs. We sequenced the full-length genomes of two positive samples collected from the great evening bat, Ia io , from Guangdong Province. The two genomes were highly similar and exhibited genomic structures identical to those of other lineage C betacoronaviruses. While they exhibited genome-wide nucleotide identities of only 75.3 to 81.2% with other MERS-related CoVs, their gene-coding regions were highly similar to their counterparts, except in the case of the spike proteins. Further protein--protein interaction assays demonstrated that the spike proteins of these MERS-related CoVs bind to the receptor DPP4. Recombination analysis suggested that the newly discovered MERS-related CoVs might have acquired their spike genes from a DPP4-recognizing bat HKU4. Our study provides further evidence that bats represent the evolutionary origins of MERS-CoV. IMPORTANCE Previous studies suggested that the Middle East respiratory syndrome coronavirus (MERS-CoV) may have originated in bats. However, its evolutionary path from bats to humans remains unclear. In this study, we discovered 89 novel lineage C betacoronaviruses (BetaCoVs) in eight bat species. We provide the evidence of a MERS-related CoV derived from the great evening bat that uses the same host receptor as human MERS-CoV. This virus also provides evidence for a natural recombination event between the bat MERS-related CoV and another bat coronavirus HKU4. Our study expands the host

BIOLOGICAL CLONING OF A BOVINE CORONAVIRUS ISOLATE

OpenAIRE

Betancourt, A; Rodríguez, Edisleidy; Relova, Damarys; Barrera, Maritza

2008-01-01

Con el objetivo de obtener un aislado de Coronavirus bovino clonado biológicamente se adaptó el aislado VB73/04 a la multiplicación en la línea celular MDBK. Este aislado indujo la formación de placas, las cuales resultaron homogéneas después del clonaje biológico. La población viral obtenida fue identificada como Coronavirus bovino por RT-PCR y Seroneutralización. In order to obtain a biologically cloned bovine coronavirus isolate, the isolate VB73/04 was adapted to multiplication in MDBK...

Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B.

Science.gov (United States)

Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

2017-02-01

Feline coronavirus (FCoV) causes the fatal disease feline infectious peritonitis, which is currently incurable by drug treatment, and no effective vaccines are available. Cyclosporin A (CsA), a cyclophilin (Cyp) inhibitor, inhibits the replication of FCoV in vitro and in vivo as well as the replication of human and animal coronaviruses. However, the mechanism underlying the regulation of coronavirus replication by CsA is unknown. In this study, we analysed the role of Cyps in FCoV replication using knockdown and knockout cells specific to Cyps. Inhibition of CypA and CypB reduced FCoV replication, with replication in knockout cells being much less than that in knockdown cells. Furthermore, the proteins expressed by CypA and CypB harbouring mutations in their respective predicted peptidyl-prolyl cis-transisomerase active sites, which also alter the affinities between Cyps and CsA, inhibited FCoV replication. These findings indicate that the peptidyl-prolyl cis-transisomerase active sites of Cyps might be required for FCoV replication.

Absence of E protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway

International Nuclear Information System (INIS)

Ortego, Javier; Ceriani, Juan E.; Patino, Cristina; Plana, Juan; Enjuanes, Luis

2007-01-01

A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-ΔE) has been engineered. This deletion mutant only grows in cells expressing E protein (E + cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-ΔE infected BHK-pAPN-E - cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E - cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-ΔE virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless, full virion maturation was blocked. Studies of the rTGEV-ΔE subcellular localization by confocal and immunoelectron microscopy in infected E - cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation

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

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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.

Peptides corresponding to the predicted heptad repeat 2 domain of the feline coronavirus spike protein are potent inhibitors of viral infection.

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I-Jung Liu

Full Text Available BACKGROUND: Feline infectious peritonitis (FIP is a lethal immune-mediated disease caused by feline coronavirus (FCoV. Currently, no therapy with proven efficacy is available. In searching for agents that may prove clinically effective against FCoV infection, five analogous overlapping peptides were designed and synthesized based on the putative heptad repeat 2 (HR2 sequence of the spike protein of FCoV, and the antiviral efficacy was evaluated. METHODS: Plaque reduction assay and MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide cytotoxicity assay were performed in this study. Peptides were selected using a plaque reduction assay to inhibit Feline coronavirus infection. RESULTS: The results demonstrated that peptide (FP5 at concentrations below 20 μM inhibited viral replication by up to 97%. The peptide (FP5 exhibiting the most effective antiviral effect was further combined with a known anti-viral agent, human interferon-α (IFN-α, and a significant synergistic antiviral effect was observed. CONCLUSION: Our data suggest that the synthetic peptide FP5 could serve as a valuable addition to the current FIP prevention methods.

Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core

International Nuclear Information System (INIS)

Ma Guangpeng; Feng Youjun; Gao Feng; Wang Jinzi; Liu Cheng; Li Yijing

2005-01-01

Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle), which has been confirmed by a combined series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host

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

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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.

Mutation of the S and 3c genes in genomes of feline coronaviruses.

Science.gov (United States)

Oguma, Keisuke; Ohno, Megumi; Yoshida, Mayuko; Sentsui, Hiroshi

2018-05-17

Feline coronavirus (FCoV) is classified into two biotypes based on its pathogenicity in cats: a feline enteric coronavirus of low pathogenicity and a highly virulent feline infectious peritonitis virus. It has been suspected that FCoV alters its biotype via mutations in the viral genome. The S and 3c genes of FCoV have been considered the candidates for viral pathogenicity conversion. In the present study, FCoVs were analyzed for the frequency and location of mutations in the S and 3c genes from faecal samples of cats in an animal shelter and the faeces, effusions, and tissues of cats that were referred to veterinary hospitals. Our results indicated that approximately 95% FCoVs in faeces did not carry mutations in the two genes. However, 80% FCoVs in effusion samples exhibited mutations in the S and 3c genes with remainder displaying a mutation in the S or 3c gene. It was also suggested that mutational analysis of the 3c gene could be useful for studying the horizontal transmission of FCoVs in multi-cat environments.

Characterization of HCoV-229E fusion core: Implications for structure basis of coronavirus membrane fusion

International Nuclear Information System (INIS)

Liu Cheng; Feng Youjun; Gao Feng; Zhang Qiangmin; Wang Ming

2006-01-01

Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans for nearly 40 years. However, the detailed molecular mechanism of the membrane fusion mediated by the spike (S) protein of HCoV-229E remains elusive. Here, we report, for the first time, a rationally designed fusion core of HCoV-229E (HR1-SGGRGG-HR2), which was in vitro produced in GST prokaryotic expression system. Multiple lines of experimental data including gel-filtration, chemical cross-linking, and circular diagram (CD) demonstrated that the HCoV-229E fusion core possesses the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle). 3D structure modeling presents its most-likely structure, similar to those of coronaviruses that have been well-documented. Collectively, HCoV-229E S protein belongs to the type I fusion protein, which is characterized by the existence of two heptad-repeat regions (HR1 and HR2), furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting the membrane fusion, a crucial step of HCoV-229E infection

Computational modeling of the bat HKU4 coronavirus 3CLpro inhibitors as a tool for the development of antivirals against the emerging Middle East respiratory syndrome (MERS) coronavirus.

Science.gov (United States)

Abuhammad, Areej; Al-Aqtash, Rua'a A; Anson, Brandon J; Mesecar, Andrew D; Taha, Mutasem O

2017-11-01

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that poses a major challenge to clinical management. The 3C-like protease (3CL pro ) is essential for viral replication and thus represents a potential target for antiviral drug development. Presently, very few data are available on MERS-CoV 3CL pro inhibition by small molecules. We conducted extensive exploration of the pharmacophoric space of a recently identified set of peptidomimetic inhibitors of the bat HKU4-CoV 3CL pro . HKU4-CoV 3CL pro shares high sequence identity (81%) with the MERS-CoV enzyme and thus represents a potential surrogate model for anti-MERS drug discovery. We used 2 well-established methods: Quantitative structure-activity relationship (QSAR)-guided modeling and docking-based comparative intermolecular contacts analysis. The established pharmacophore models highlight structural features needed for ligand recognition and revealed important binding-pocket regions involved in 3CL pro -ligand interactions. The best models were used as 3D queries to screen the National Cancer Institute database for novel nonpeptidomimetic 3CL pro inhibitors. The identified hits were tested for HKU4-CoV and MERS-CoV 3CL pro inhibition. Two hits, which share the phenylsulfonamide fragment, showed moderate inhibitory activity against the MERS-CoV 3CL pro and represent a potential starting point for the development of novel anti-MERS agents. To the best of our knowledge, this is the first pharmacophore modeling study supported by in vitro validation on the MERS-CoV 3CL pro . MERS-CoV is an emerging virus that is closely related to the bat HKU4-CoV. 3CL pro is a potential drug target for coronavirus infection. HKU4-CoV 3CL pro is a useful surrogate model for the identification of MERS-CoV 3CL pro enzyme inhibitors. dbCICA is a very robust modeling method for hit identification. The phenylsulfonamide scaffold represents a potential starting point for MERS coronavirus 3CL pro inhibitors

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; 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

2016-01-01

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 (PLpro), 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 PLpros 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–PLpro fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PLpro 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. PMID:27519799

A molecular arms race between host innate antiviral response and emerging human coronaviruses.

Science.gov (United States)

Wong, Lok-Yin Roy; Lui, Pak-Yin; Jin, Dong-Yan

2016-02-01

Coronaviruses have been closely related with mankind for thousands of years. Community-acquired human coronaviruses have long been recognized to cause common cold. However, zoonotic coronaviruses are now becoming more a global concern with the discovery of highly pathogenic severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses causing severe respiratory diseases. Infections by these emerging human coronaviruses are characterized by less robust interferon production. Treatment of patients with recombinant interferon regimen promises beneficial outcomes, suggesting that compromised interferon expression might contribute at least partially to the severity of disease. The mechanisms by which coronaviruses evade host innate antiviral response are under intense investigations. This review focuses on the fierce arms race between host innate antiviral immunity and emerging human coronaviruses. Particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against SARS and MERS coronavirus infection are discussed. On the other hand, the counter-measures evolved by SARS and MERS coronaviruses to circumvent host defense are also dissected. With a better understanding of the dynamic interaction between host and coronaviruses, it is hoped that insights on the pathogenesis of newly-identified highly pathogenic human coronaviruses and new strategies in antiviral development can be derived.

Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

International Nuclear Information System (INIS)

Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian

2007-01-01

The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection

Coronavirus Attachment and Replication

Science.gov (United States)

1988-03-28

has been shown by serologic and virological methods to infect coyotes. Dual infection with canine coronavirus and canine parvovirus causes fatal... attenuation and characteristics of a coronavirus-like agent. Am. J. Vet. Res. 34:145-150. Mebus, C.A., Stair, E.L., Rhodes, M.B., and Twiehaus, M.J. 1973b

Design, synthesis and crystallographic analysis of nitrile-based broad-spectrum peptidomimetic inhibitors for coronavirus 3C-like proteases.

Science.gov (United States)

Chuck, Chi-Pang; Chen, Chao; Ke, Zhihai; Wan, David Chi-Cheong; Chow, Hak-Fun; Wong, Kam-Bo

2013-01-01

Coronaviral infection is associated with up to 5% of respiratory tract diseases. The 3C-like protease (3CL(pro)) of coronaviruses is required for proteolytic processing of polyproteins and viral replication, and is a promising target for the development of drugs against coronaviral infection. We designed and synthesized four nitrile-based peptidomimetic inhibitors with different N-terminal protective groups and different peptide length, and examined their inhibitory effect on the in-vitro enzymatic activity of 3CL(pro) of severe-acute-respiratory-syndrome-coronavirus. The IC(50) values of the inhibitors were in the range of 4.6-49 μM, demonstrating that the nitrile warhead can effectively inactivate the 3CL(pro) autocleavage process. The best inhibitor, Cbz-AVLQ-CN with an N-terminal carbobenzyloxy group, was ~10x more potent than the other inhibitors tested. Crystal structures of the enzyme-inhibitor complexes showed that the nitrile warhead inhibits 3CL(pro) by forming a covalent bond with the catalytic Cys145 residue, while the AVLQ peptide forms a number of favourable interactions with the S1-S4 substrate-binding pockets. We have further showed that the peptidomimetic inhibitor, Cbz-AVLQ-CN, has broad-spectrum inhibition against 3CL(pro) from human coronavirus strains 229E, NL63, OC43, HKU1, and infectious bronchitis virus, with IC(50) values ranging from 1.3 to 3.7 μM, but no detectable inhibition against caspase-3. In summary, we have shown that the nitrile-based peptidomimetic inhibitors are effective against 3CL(pro), and they inhibit 3CL(pro) from a broad range of coronaviruses. Our results provide further insights into the future design of drugs that could serve as a first line defence against coronaviral infection. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Conserved antigenic sites between MERS-CoV and Bat-coronavirus are revealed through sequence analysis.

Science.gov (United States)

Sharmin, Refat; Islam, Abul B M M K

2016-01-01

MERS-CoV is a newly emerged human coronavirus reported closely related with HKU4 and HKU5 Bat coronaviruses. Bat and MERS corona-viruses are structurally related. Therefore, it is of interest to estimate the degree of conserved antigenic sites among them. It is of importance to elucidate the shared antigenic-sites and extent of conservation between them to understand the evolutionary dynamics of MERS-CoV. Multiple sequence alignment of the spike (S), membrane (M), enveloped (E) and nucleocapsid (N) proteins was employed to identify the sequence conservation among MERS and Bat (HKU4, HKU5) coronaviruses. We used various in silico tools to predict the conserved antigenic sites. We found that MERS-CoV shared 30 % of its S protein antigenic sites with HKU4 and 70 % with HKU5 bat-CoV. Whereas 100 % of its E, M and N protein's antigenic sites are found to be conserved with those in HKU4 and HKU5. This sharing suggests that in case of pathogenicity MERS-CoV is more closely related to HKU5 bat-CoV than HKU4 bat-CoV. The conserved epitopes indicates their evolutionary relationship and ancestry of pathogenicity.

Identification of Aminopeptidase N as a Cellular Receptor for Human Coronavirus-229E

Science.gov (United States)

1992-05-12

hemagglutinating encephalomyelitis virus (HEV), canine coronavirus (CCV), cat FIPV and feline enteric corona virus (FECV), human CVLPs, mouse...While the cat , dog and pig serve as natural hosts for the other coronavirus group 1 viruses , feline infectious peritonitis virus (FIPV), canine...3 2 . Virus Receptors ••••••••.••••••.....•................ 20 3. Viruses Which Cause Common Colds

The glycosylation status of the murine hepatitis coronavirus M protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain

International Nuclear Information System (INIS)

Haan, Cornelis A.M. de; Wit, Marel de; Kuo, Lili; Montalto-Morrison, Cynthia; Haagmans, Bart L.; Weiss, Susan R.; Masters, Paul S.; Rottier, Peter J.M.

2003-01-01

The coronavirus M protein, the most abundant coronaviral envelope component, is invariably glycosylated, which provides the virion with a diffuse, hydrophilic cover on its outer surface. Remarkably, while the group 1 and group 3 coronaviruses all have M proteins with N-linked sugars, the M proteins of the group 2 coronaviruses [e.g., mouse hepatitis virus (MHV)] are O-glycosylated. The conservation of the N- and O-glycosylation motifs suggests that each of these types of carbohydrate modifications is beneficial to their respective virus. Since glycosylation of the M protein is not required for virus assembly, the oligosaccharides are likely to be involved in the virus-host interaction. In order to investigate the role of the M protein glycosylation in the host, two genetically modified MHVs were generated by using targeted RNA recombination. The recombinant viruses carried M proteins that were either N-glycosylated or not glycosylated at all, and these were compared with the parental, O-glycosylated, virus. The M protein glycosylation state did not influence the tissue culture growth characteristics of the recombinant viruses. However, it affected their interferogenic capacity as measured using fixed, virus-infected cells. Viruses containing M proteins with N-linked sugars induced type I interferons to higher levels than viruses carrying M proteins with O-linked sugars. MHV with unglycosylated M proteins appeared to be a poor interferon inducer. In mice, the recombinant viruses differed in their ability to replicate in the liver, but not in the brain, whereas their in vivo interferogenic capacity did not appear to be affected by their glycosylation status. Strikingly, their abilities to replicate in the liver correlated with their in vitro interferogenic capacity. This apparent correlation might be explained by the functioning of lectins, such as the mannose receptor, which are abundantly expressed in the liver but also play a role in the induction of interferon

Molecular and pathological identification of feline coronavirus type I

African Journals Online (AJOL)

DR. NJ TONUKARI

2012-06-05

Jun 5, 2012 ... In this study, we described the isolation and molecular characterization of .... fecv2b) designed in the regions of S-protein gene were used to differentiate ..... The molecular dynamics of feline coronaviruses. Vet. Microbiol.

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors.

Directory of Open Access Journals (Sweden)

Susanne Pfefferle

2011-10-01

Full Text Available Coronaviruses (CoVs are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS in 2002/2003 has demonstrated human vulnerability to (Coronavirus CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B as interaction partners of the CoV non-structural protein 1 (Nsp1. These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

Genomic Analysis and Surveillance of the Coronavirus Dominant in Ducks in China.

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Qing-Ye Zhuang

Full Text Available The genetic diversity, evolution, distribution, and taxonomy of some coronaviruses dominant in birds other than chickens remain enigmatic. In this study we sequenced the genome of a newly identified coronavirus dominant in ducks (DdCoV, and performed a large-scale surveillance of coronaviruses in chickens and ducks using a conserved RT-PCR assay. The viral genome harbors a tandem repeat which is rare in vertebrate RNA viruses. The repeat is homologous to some proteins of various cellular organisms, but its origin remains unknown. Many substitutions, insertions, deletions, and some frameshifts and recombination events have occurred in the genome of the DdCoV, as compared with the coronavirus dominant in chickens (CdCoV. The distances between DdCoV and CdCoV are large enough to separate them into different species within the genus Gammacoronavirus. Our surveillance demonstrated that DdCoVs and CdCoVs belong to different lineages and occupy different ecological niches, further supporting that they should be classified into different species. Our surveillance also demonstrated that DdCoVs and CdCoVs are prevalent in live poultry markets in some regions of China. In conclusion, this study shed novel insight into the genetic diversity, evolution, distribution, and taxonomy of the coronaviruses circulating in chickens and ducks.

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.

Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

NARCIS (Netherlands)

Rabouw, Huib H; Langereis, Martijn A; Knaap, Robert C M; Dalebout, Tim J; Canton, Javier; Sola, Isabel; Enjuanes, Luis; Bredenbeek, Peter J; Kikkert, Marjolein; de Groot, Raoul J; van Kuppeveld, Frank J M

2016-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infections that can be life-threatening. To establish an infection and spread, MERS-CoV, like most other viruses, must navigate through an intricate network of antiviral host responses. Besides the well-known type I

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.

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.

Antibody response to equine coronavirus in horses inoculated with a bovine coronavirus vaccine.

Science.gov (United States)

Nemoto, Manabu; Kanno, Toru; Bannai, Hiroshi; Tsujimura, Koji; Yamanaka, Takashi; Kokado, Hiroshi

2017-11-17

A vaccine for equine coronavirus (ECoV) is so far unavailable. Bovine coronavirus (BCoV) is antigenically related to ECoV; it is therefore possible that BCoV vaccine will induce antibodies against ECoV in horses. This study investigated antibody response to ECoV in horses inoculated with BCoV vaccine. Virus neutralization tests showed that antibody titers against ECoV increased in all six horses tested at 14 days post inoculation, although the antibody titers were lower against ECoV than against BCoV. This study showed that BCoV vaccine provides horses with antibodies against ECoV to some extent. It is unclear whether antibodies provided by BCoV vaccine are effective against ECoV, and therefore ECoV challenge studies are needed to evaluate efficacy of the vaccine in the future.

Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein

Science.gov (United States)

Millet, Jean Kaoru; Whittaker, Gary R.

2014-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified betacoronavirus causing high morbidity and mortality in humans. The coronavirus spike (S) protein is the main determinant of viral entry, and although it was previously shown that MERS-CoV S can be activated by various proteases, the details of the mechanisms of proteolytic activation of fusion are still incompletely characterized. Here, we have uncovered distinctive characteristics of MERS-CoV S. We identify, by bioinformatics and peptide cleavage assays, two cleavage sites for furin, a ubiquitously expressed protease, which are located at the S1/S2 interface and at the S2′ position of the S protein. We show that although the S1/S2 site is proteolytically processed by furin during protein biosynthesis, the S2′ site is cleaved upon viral entry. MERS-CoV pseudovirion infection was shown to be enhanced by elevated levels of furin expression, and entry could be decreased by furin siRNA silencing. Enhanced furin activity appeared to partially override the low pH-dependent nature of MERS-CoV entry. Inhibition of furin activity was shown to decrease MERS-CoV S-mediated entry, as well as infection by the virus. Overall, we show that MERS-CoV has evolved an unusual two-step furin activation for fusion, suggestive of a role during the process of emergence into the human population. The ability of MERS-CoV to use furin in this manner, along with other proteases, may explain the polytropic nature of the virus. PMID:25288733

Feline infectious peritonitis: role of the feline coronavirus 3c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats.

Science.gov (United States)

Pedersen, Niels C; Liu, Hongwei; Scarlett, Jennifer; Leutenegger, Christian M; Golovko, Lyudmila; Kennedy, Heather; Kamal, Farina Mustaffa

2012-04-01

Feline infectious peritonitis virus (FIPV) was presumed to arise from mutations in the 3c of a ubiquitous and largely nonpathogenic feline enteric coronavirus (FECV). However, a recent study found that one-third of FIPV isolates have an intact 3c and suggested that it is not solely involved in FIP but is essential for intestinal replication. In order to confirm these assumptions, 27 fecal and 32 FIP coronavirus isolates were obtained from resident or adopted cats from a large metropolitan shelter during 2008-2009 and their 3a-c, E, and M genes sequenced. Forty percent of coronavirus isolates from FIP tissues had an intact 3c gene, while 60% had mutations that truncated the gene product. The 3c genes of fecal isolates from healthy cats were always intact. Coronavirus from FIP diseased tissues consistently induced FIP when given either oronasally or intraperitoneally (i.p.), regardless of the functional status of their 3c genes, thus confirming them to be FIPVs. In contrast, fecal isolates from healthy cats were infectious following oronasal infection and shed at high levels in feces without causing disease, as expected for FECVs. Only one in three cats shed FECV in the feces following i.p. infection, indicating that FECVs can replicate systemically, but with difficulty. FIPVs having a mutated 3c were not shed in the feces following either oronasal or i.p. inoculation, while FIPVs with intact 3c genes were shed in the feces following oronasal but not i.p. inoculation. Therefore, an intact 3c appears to be essential for intestinal replication. Although FIPVs with an intact 3c were shed in the feces following oronasal inoculation, fecal virus from these cats was not infectious for other cats. Attempts to identify potential FIP mutations in the 3a, 3b, E, and M were negative. However, the 3c gene of FIPVs, even though appearing intact, contained many more non-synonymous amino acid changes in the 3' one-third of the 3c protein than FECVs. An attempt to trace FIPV

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...

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

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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.

Genetic Characteristics of Coronaviruses from Korean Bats in 2016.

Science.gov (United States)

Lee, Saemi; Jo, Seong-Deok; Son, Kidong; An, Injung; Jeong, Jipseol; Wang, Seung-Jun; Kim, Yongkwan; Jheong, Weonhwa; Oem, Jae-Ku

2018-01-01

Bats have increasingly been recognized as the natural reservoir of severe acute respiratory syndrome (SARS), coronavirus, and other coronaviruses found in mammals. However, little research has been conducted on bat coronaviruses in South Korea. In this study, bat samples (332 oral swabs, 245 fecal samples, 38 urine samples, and 57 bat carcasses) were collected at 33 natural bat habitat sites in South Korea. RT-PCR and sequencing were performed for specific coronavirus genes to identify the bat coronaviruses in different bat samples. Coronaviruses were detected in 2.7% (18/672) of the samples: 13 oral swabs from one species of the family Rhinolophidae, and four fecal samples and one carcass (intestine) from three species of the family Vespertiliodae. To determine the genetic relationships of the 18 sequences obtained in this study and previously known coronaviruses, the nucleotide sequences of a 392-nt region of the RNA-dependent RNA polymerase (RdRp) gene were analyzed phylogenetically. Thirteen sequences belonging to SARS-like betacoronaviruses showed the highest nucleotide identity (97.1-99.7%) with Bat-CoV-JTMC15 reported in China. The other five sequences were most similar to MERS-like betacoronaviruses. Four nucleotide sequences displayed the highest identity (94.1-95.1%) with Bat-CoV-HKU5 from Hong Kong. The one sequence from a carcass showed the highest nucleotide identity (99%) with Bat-CoV-SC2013 from China. These results suggest that careful surveillance of coronaviruses from bats should be continued, because animal and human infections may result from the genetic variants present in bat coronavirus reservoirs.

Coronavirus infections in horses in Saudi Arabia and Oman.

Science.gov (United States)

Hemida, M G; Chu, D K W; Perera, R A P M; Ko, R L W; So, R T Y; Ng, B C Y; Chan, S M S; Chu, S; Alnaeem, A A; Alhammadi, M A; Webby, R J; Poon, L L M; Balasuriya, U B R; Peiris, M

2017-12-01

Equine coronaviruses (ECoV) are the only coronavirus known to infect horses. So far, data on ECoV infection in horses remain limited to the USA, France and Japan and its geographic distribution is not well understood. We carried out RT-PCR on 306 nasal and 315 rectal swabs and tested 243 sera for antibodies to detect coronavirus infections in apparently healthy horses in Saudi Arabia and Oman. We document evidence of infection with ECoV and HKU23 coronavirus by RT-PCR. There was no conclusive evidence of Middle East respiratory syndrome coronavirus infection in horses. Serological data suggest that lineage A betacoronavirus infections are commonly infecting horses in Saudi Arabia and Oman but antibody cross-reactivities between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections. © 2017 Blackwell Verlag GmbH.

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

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

Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease

International Nuclear Information System (INIS)

Erles, Kerstin; Toomey, Crista; Brooks, Harriet W.; Brownlie, Joe

2003-01-01

An investigation into the causes of canine infectious respiratory disease was carried out in a large rehoming kennel. Tissue samples taken from the respiratory tract of diseased dogs were tested for the presence of coronaviruses using RT-PCR with conserved primers for the polymerase gene. Sequence analysis of four positive samples showed the presence of a coronavirus with high similarity to both bovine and human coronavirus (strain OC43) in their polymerase and spike genes, whereas there was a low similarity to comparable genes in the enteric canine coronavirus. This canine respiratory coronavirus (CRCV) was detected by RT-PCR in 32/119 tracheal and 20/119 lung samples, with the highest prevalence being detected in dogs with mild clinical symptoms. Serological analysis showed that the presence of antibodies against CRCV on the day of entry into the kennel decreased the risk of developing respiratory disease

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. © 2016 Elsevier Inc. All rights reserved.

Roadmap to developing a recombinant coronavirus S protein receptor-binding domain vaccine for severe acute respiratory syndrome

Science.gov (United States)

Jiang, Shibo; Bottazzi, Maria Elena; Du, Lanying; Lustigman, Sara; Tseng, Chien-Te Kent; Curti, Elena; Jones, Kathryn; Zhan, Bin; Hotez, Peter J

2013-01-01

A subunit vaccine, RBD-S, is under development to prevent severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV), which is classified by the US NIH as a category C pathogen. This vaccine is comprised of a recombinant receptor-binding domain (RBD) of the SARS-CoV spike (S) protein and formulated on alum, together with a synthetic glucopyranosyl lipid A. The vaccine would induce neutralizing antibodies without causing Th2-type immunopathology. Vaccine development is being led by the nonprofit product development partnership; Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development in collaboration with two academic partners (the New York Blood Center and University of Texas Medical Branch); an industrial partner (Immune Design Corporation); and Walter Reed Army Institute of Research. A roadmap for the product development of the RBD-S SARS vaccine is outlined with a goal to manufacture the vaccine for clinical testing within the next 5 years. PMID:23252385

Proteomic analysis of purified coronavirus infectious bronchitis virus particles

Directory of Open Access Journals (Sweden)

Shu Dingming

2010-06-01

Full Text Available Abstract Background Infectious bronchitis virus (IBV is the coronavirus of domestic chickens causing major economic losses to the poultry industry. Because of the complexity of the IBV life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. Toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified IBV particles. Results Apart from the virus-encoded structural proteins, we detected 60 host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins (20%, molecular chaperone (18%, macromolcular biosynthesis proteins (17%, cytoskeletal proteins (15%, signal transport proteins (15%, protein degradation (8%, chromosome associated proteins (2%, ribosomal proteins (2%, and other function proteins (3%. Interestingly, 21 of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, TENP protein, ovalbumin, and scavenger receptor protein. Following identification of the host proteins by proteomic methods, the presence of 4 proteins in the purified IBV preparation was verified by western blotting and immunogold labeling detection. Conclusions The results present the first standard proteomic profile of IBV and may facilitate the understanding of the pathogenic mechanisms.

Inactivation of surrogate coronaviruses on hard surfaces by health care germicides.

Science.gov (United States)

Hulkower, Rachel L; Casanova, Lisa M; Rutala, William A; Weber, David J; Sobsey, Mark D

2011-06-01

In the 2003 severe acute respiratory syndrome outbreak, finding viral nucleic acids on hospital surfaces suggested surfaces could play a role in spread in health care environments. Surface disinfection may interrupt transmission, but few data exist on the effectiveness of health care germicides against coronaviruses on surfaces. The efficacy of health care germicides against 2 surrogate coronaviruses, mouse hepatitis virus (MHV) and transmissible gastroenteritis virus (TGEV), was tested using the quantitative carrier method on stainless steel surfaces. Germicides were o-phenylphenol/p-tertiary amylphenol) (a phenolic), 70% ethanol, 1:100 sodium hypochlorite, ortho-phthalaldehyde (OPA), instant hand sanitizer (62% ethanol), and hand sanitizing spray (71% ethanol). After 1-minute contact time, for TGEV, there was a log(10) reduction factor of 3.2 for 70% ethanol, 2.0 for phenolic, 2.3 for OPA, 0.35 for 1:100 hypochlorite, 4.0 for 62% ethanol, and 3.5 for 71% ethanol. For MHV, log(10) reduction factors were 3.9 for 70% ethanol, 1.3 for phenolic, 1.7 for OPA, 0.62 for 1:100 hypochlorite, 2.7 for 62% ethanol, and 2.0 for 71% ethanol. Only ethanol reduced infectivity of the 2 coronaviruses by >3-log(10) after 1 minute. Germicides must be chosen carefully to ensure they are effective against viruses such as severe acute respiratory syndrome coronavirus. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Coronavirus 229E-related pneumonia in immunocompromised patients.

Science.gov (United States)

Pene, Frédéric; Merlat, Annabelle; Vabret, Astrid; Rozenberg, Flore; Buzyn, Agnès; Dreyfus, François; Cariou, Alain; Freymuth, François; Lebon, Pierre

2003-10-01

Coronaviruses strains 229E and OC43 have been associated with various respiratory illnesses ranging from the self-resolving common cold to severe pneumonia. Although chronic underlying conditions are major determinants of severe respiratory virus infections, few data about coronavirus-related pneumonia in immunocompromised patients are available. Here we report 2 well-documented cases of pneumonia related to coronavirus 229E, each with a different clinical presentation. Diagnosis was made on the basis of viral culture and electron microscopy findings that exhibited typical crown-like particles and through amplification of the viral genome by reverse transcriptase-polymerase chain reaction. On the basis of this report, coronaviruses should be considered as potential causative microorganisms of pneumonia in immunocompromised patients.

Coronavirus Infection and Diversity in Bats in the Australasian Region.

Science.gov (United States)

Smith, C S; de Jong, C E; Meers, J; Henning, J; Wang, L- F; Field, H E

2016-03-01

Following the SARS outbreak, extensive surveillance was undertaken globally to detect and identify coronavirus diversity in bats. This study sought to identify the diversity and prevalence of coronaviruses in bats in the Australasian region. We identified four different genotypes of coronavirus, three of which (an alphacoronavirus and two betacoronaviruses) are potentially new species, having less than 90% nucleotide sequence identity with the most closely related described viruses. We did not detect any SARS-like betacoronaviruses, despite targeting rhinolophid bats, the putative natural host taxa. Our findings support the virus-host co-evolution hypothesis, with the detection of Miniopterus bat coronavirus HKU8 (previously reported in Miniopterus species in China, Hong Kong and Bulgaria) in Australian Miniopterus species. Similarly, we detected a novel betacoronavirus genotype from Pteropus alecto which is most closely related to Bat coronavirus HKU9 identified in other pteropodid bats in China, Kenya and the Philippines. We also detected possible cross-species transmission of bat coronaviruses, and the apparent enteric tropism of these viruses. Thus, our findings are consistent with a scenario wherein the current diversity and host specificity of coronaviruses reflects co-evolution with the occasional host shift.

Crystal structure of Middle East respiratory syndrome coronavirus helicase.

Directory of Open Access Journals (Sweden)

Wei Hao

2017-06-01

Full Text Available Middle East respiratory syndrome coronavirus (MERS-CoV remains a threat to public health worldwide; however, effective vaccine or drug against CoVs remains unavailable. CoV helicase is one of the three evolutionary most conserved proteins in nidoviruses, thus making it an important target for drug development. We report here the first structure of full-length coronavirus helicase, MERS-CoV nsp13. MERS-CoV helicase has multiple domains, including an N-terminal Cys/His rich domain (CH with three zinc atoms, a beta-barrel domain and a C-terminal SF1 helicase core with two RecA-like subdomains. Our structural analyses show that while the domain organization of nsp13 is conserved throughout nidoviruses, the individual domains of nsp13 are closely related to the equivalent eukaryotic domains of Upf1 helicases. The most distinctive feature differentiating CoV helicases from eukaryotic Upf1 helicases is the interaction between CH domain and helicase core.

Identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy.

Science.gov (United States)

Qu, Xiu-Xia; Hao, Pei; Song, Xi-Jun; Jiang, Si-Ming; Liu, Yan-Xia; Wang, Pei-Gang; Rao, Xi; Song, Huai-Dong; Wang, Sheng-Yue; Zuo, Yu; Zheng, Ai-Hua; Luo, Min; Wang, Hua-Lin; Deng, Fei; Wang, Han-Zhong; Hu, Zhi-Hong; Ding, Ming-Xiao; Zhao, Guo-Ping; Deng, Hong-Kui

2005-08-19

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a recently identified human coronavirus. The extremely high homology of the viral genomic sequences between the viruses isolated from human (huSARS-CoV) and those of palm civet origin (pcSARS-CoV) suggested possible palm civet-to-human transmission. Genetic analysis revealed that the spike (S) protein of pcSARS-CoV and huSARS-CoV was subjected to the strongest positive selection pressure during transmission, and there were six amino acid residues within the receptor-binding domain of the S protein being potentially important for SARS progression and tropism. Using the single-round infection assay, we found that a two-amino acid substitution (N479K/T487S) of a huSARS-CoV for those of pcSARS-CoV almost abolished its infection of human cells expressing the SARS-CoV receptor ACE2 but no effect upon the infection of mouse ACE2 cells. Although single substitution of these two residues had no effects on the infectivity of huSARS-CoV, these recombinant S proteins bound to human ACE2 with different levels of reduced affinity, and the two-amino acid-substituted S protein showed extremely low affinity. On the contrary, substitution of these two amino acid residues of pcSARS-CoV for those of huSRAS-CoV made pcSARS-CoV capable of infecting human ACE2-expressing cells. These results suggest that amino acid residues at position 479 and 487 of the S protein are important determinants for SARS-CoV tropism and animal-to-human transmission.

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

International Nuclear Information System (INIS)

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.

Transmission of Middle East respiratory syndrome coronavirus ...

African Journals Online (AJOL)

... hand hygiene, and cough etiquette, would minimize the infection rate among HCPs. The required consumables for maintaining hand hygiene should be readily available to all HCPs. Keywords: Middle East respiratory syndrome coronavirus (MERS-CoV), Systematic review, healthcareassociated infections, Coronaviruses ...

Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus

NARCIS (Netherlands)

Ulasli, M.; Verheije, M.H.; de Haan, C.A.M.; Reggiori, F.M.

2011-01-01

Coronaviruses (CoV) are enveloped positive-strand RNA viruses that induce different membrane rearrangements in infected cells in order to efficiently replicate and assemble. The origin, the protein composition and the function of these structures are not well established. To shed further light on

Mutations of 3c and spike protein genes correlate with the occurrence of feline infectious peritonitis.

Science.gov (United States)

Bank-Wolf, Barbara Regina; Stallkamp, Iris; Wiese, Svenja; Moritz, Andreas; Tekes, Gergely; Thiel, Heinz-Jürgen

2014-10-10

The genes encoding accessory proteins 3a, 3b, 3c, 7a and 7b, the S2 domain of the spike (S) protein gene and the membrane (M) protein gene of feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) samples were amplified, cloned and sequenced. For this faeces and/or ascites samples from 19 cats suffering from feline infectious peritonitis (FIP) as well as from 20 FECV-infected healthy cats were used. Sequence comparisons revealed that 3c genes of animals with FIP were heavily affected by nucleotide deletions and point mutations compared to animals infected with FECV; these alterations resulted either in early termination or destruction of the translation initiation codon. Two ascites-derived samples of cats with FIP which displayed no alterations of ORF3c harboured mutations in the S2 domain of the S protein gene which resulted in amino acid exchanges or deletions. Moreover, changes in 3c were often accompanied by mutations in S2. In contrast, in samples obtained from faeces of healthy cats, the ORF3c was never affected by such mutations. Similarly ORF3c from faecal samples of the cats with FIP was mostly intact and showed only in a few cases the same mutations found in the respective ascites samples. The genes encoding 3a, 3b, 7a and 7b displayed no mutations linked to the feline coronavirus (FCoV) biotype. The M protein gene was found to be conserved between FECV and FIPV samples. Our findings suggest that mutations of 3c and spike protein genes correlate with the occurrence of FIP. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of antibodies against feline coronavirus 7b protein for diagnosis of feline infectious peritonitis in cats.

Science.gov (United States)

Kennedy, Melissa A; Abd-Eldaim, Mohamed; Zika, Sarah E; Mankin, Joseph M; Kania, Stephen A

2008-09-01

To determine whether expression of feline coronavirus (FCoV) 7b protein, as indicated by the presence of specific serum antibodies, consistently correlated with occurrence of feline infectious peritonitis (FIP) in cats. 95 serum samples submitted for various diagnostic assays and 20 samples from specific-pathogen-free cats tested as negative control samples. The 7b gene from a virulent strain of FCoV was cloned into a protein expression vector. The resultant recombinant protein was produced and used in antibody detection assays via western blot analysis of serum samples. Results were compared with those of an immunofluorescence assay (IFA) for FCoV-specific antibody and correlated with health status. Healthy IFA-seronegative cats were seronegative for antibodies against the 7b protein. Some healthy cats with detectable FCoV-specific antibodies as determined via IFA were seronegative for antibodies against the 7b protein. Serum from cats with FIP had antibodies against the 7b protein, including cats with negative results via conventional IFA. However, some healthy cats, as well as cats with conditions other than FIP that were seropositive to FCoV via IFA, were also seropositive for the 7b protein. Expression of the 7b protein, as indicated by detection of antibodies against the protein, was found in most FCoV-infected cats. Seropositivity for this protein was not specific for the FCoV virulent biotype or a diagnosis of FIP.

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavius molecules

International Nuclear Information System (INIS)

Wu, H.-Y.; Guy, James S.; Yoo, Dongwan; Vlasak, Reinhard; Urbach, Ena; Brian, David A.

2003-01-01

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members

ORF7-encoded accessory protein 7a of feline infectious peritonitis virus as a counteragent against IFN-α-induced antiviral response.

Science.gov (United States)

Dedeurwaerder, Annelike; Olyslaegers, Dominique A J; Desmarets, Lowiese M B; Roukaerts, Inge D M; Theuns, Sebastiaan; Nauwynck, Hans J

2014-02-01

The type I IFN-mediated immune response is the first line of antiviral defence. Coronaviruses, like many other viruses, have evolved mechanisms to evade this innate response, ensuring their survival. Several coronavirus accessory genes play a central role in these pathways, but for feline coronaviruses this has never to our knowledge been studied. As it has been demonstrated previously that ORF7 is essential for efficient replication in vitro and virulence in vivo of feline infectious peritonitis virus (FIPV), the role of this ORF in the evasion of the IFN-α antiviral response was investigated. Deletion of ORF7 from FIPV strain 79-1146 (FIPV-Δ7) rendered the virus more susceptible to IFN-α treatment. Given that ORF7 encodes two proteins, 7a and 7b, it was further explored which of these proteins is active in this mechanism. Providing 7a protein in trans rescued the mutant FIPV-Δ7 from IFN sensitivity, which was not achieved by addition of 7b protein. Nevertheless, addition of protein 7a to FIPV-Δ3Δ7, a FIPV mutant deleted in both ORF3 and ORF7, could no longer increase the replication capacity of this mutant in the presence of IFN. These results indicate that FIPV 7a protein is a type I IFN antagonist and protects the virus from the antiviral state induced by IFN, but it needs the presence of ORF3-encoded proteins to exert its antagonistic function.

Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets

NARCIS (Netherlands)

ter Meulen, Jan; Bakker, Alexander B. H.; van den Brink, Edward N.; Weverling, Gerrit J.; Martina, Byron E. E.; Haagmans, Bart L.; Kuiken, Thijs; de Kruif, John; Preiser, Wolfgang; Spaan, Willy; Gelderblom, Hans R.; Goudsmit, Jaap; Osterhaus, Albert D. M. E.

2004-01-01

SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal

HLA-A*0201 T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus nucleocapsid and spike proteins

International Nuclear Information System (INIS)

Tsao, Y.-P.; Lin, J.-Y.; Jan, J.-T.; Leng, C.-H.; Chu, C.-C.; Yang, Y.-C.; Chen, S.-L.

2006-01-01

The immunogenicity of HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins' ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (http://thr.cit.nih.gov/molbio/hla_bind/), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated by T2-cell stabilization assays, with immunogenicity assays revealing peptides N223-231, N227-235, and N317-325 to be First identified HLA-A*0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A*0201-restricted CTL epitopes of S protein (S978-986, S1203-1211, and S1167-1175), here we found two novel peptides S787-795 and S1042-1050 as S-specific CTL epitopes. Moreover, our identified N317-325 and S1042-1050 CTL epitopes could induce recall responses when IFN-γ stimulation of blood CD8 + T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS

Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

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Stobart, Christopher C; Sexton, Nicole R; Munjal, Havisha; Lu, Xiaotao; Molland, Katrina L; Tomar, Sakshi; Mesecar, Andrew D; Denison, Mark R

2013-12-01

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

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

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I-Chueh Huang

2011-01-01

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.

Prevalence of Korean cats with natural feline coronavirus infections

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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.

Suppression of Coronavirus Replication by Cyclophilin Inhibitors

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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.

The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis.

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Wei-Yu Liao

Full Text Available The synthesis of the negative-strand [(--strand] complement of the ∼30 kilobase, positive-strand [(+-strand] coronaviral genome is a necessary early step for genome replication. The identification of cis-acting elements required for (--strand RNA synthesis in coronaviruses, however, has been hampered due to insufficiencies in the techniques used to detect the (--strand RNA species. Here, we employed a method of head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR to detect and quantitate the synthesis of bovine coronavirus (BCoV defective interfering (DI RNA (- strands. Furthermore, using the aforementioned techniques along with Northern blot assay, we specifically defined the cis-acting RNA elements within the 3'-terminal 55 nucleotides (nts which function in the synthesis of (-- or (+-strand BCoV DI RNA. The major findings are as follows: (i nts from -5 to -39 within the 3'-terminal 55 nts are the cis-acting elements responsible for (--strand BCoV DI RNA synthesis, (ii nts from -3 to -34 within the 3'-terminal 55 nts are cis-acting elements required for (+-strand BCoV DI RNA synthesis, and (iii the nucleotide species at the 3'-most position (-1 is important, but not critical, for both (-- and (+-strand BCoV DI RNA synthesis. These results demonstrate that the 3'-terminal 55 nts in BCoV DI RNA harbor cis-acting RNA elements required for both (-- and (+-strand DI RNA synthesis and extend our knowledge on the mechanisms of coronavirus replication. The method of head-to-tail ligation and qRT-PCR employed in the study may also be applied to identify other cis-acting elements required for (--strand RNA synthesis in coronaviruses.

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...

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

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Anna Lundin

2014-05-01

Full Text Available Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs, a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6, a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV, and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Genetic diversity of coronaviruses in bats in Lao PDR and Cambodia.

Science.gov (United States)

Lacroix, Audrey; Duong, Veasna; Hul, Vibol; San, Sorn; Davun, Hull; Omaliss, Keo; Chea, Sokha; Hassanin, Alexandre; Theppangna, Watthana; Silithammavong, Soubanh; Khammavong, Kongsy; Singhalath, Sinpakone; Greatorex, Zoe; Fine, Amanda E; Goldstein, Tracey; Olson, Sarah; Joly, Damien O; Keatts, Lucy; Dussart, Philippe; Afelt, Aneta; Frutos, Roger; Buchy, Philippe

2017-03-01

South-East Asia is a hot spot for emerging zoonotic diseases, and bats have been recognized as hosts for a large number of zoonotic viruses such as Severe Acute Respiratory Syndrome (SARS), responsible for acute respiratory syndrome outbreaks. Thus, it is important to expand our knowledge of the presence of viruses in bats which could represent a risk to humans. Coronaviruses (CoVs) have been reported in bat species from Thailand, China, Indonesia, Taiwan and the Philippines. However no such work was conducted in Cambodia or Lao PDR. Between 2010 and 2013, 1965 bats were therefore sampled at interfaces with human populations in these two countries. They were tested for the presence of coronavirus by consensus reverse transcription-PCR assay. A total of 93 samples (4.7%) from 17 genera of bats tested positive. Sequence analysis revealed the presence of potentially 37 and 56 coronavirus belonging to alpha-coronavirus (αCoV) and beta-CoV (βCoV), respectively. The βCoVs group is known to include some coronaviruses highly pathogenic to human, such as SARS-CoV and MERS-CoV. All coronavirus sequences generated from frugivorous bats (family Pteropodidae) (n=55) clustered with other bat βCoVs of lineage D, whereas one coronavirus from Pipistrellus coromandra fell in the lineage C of βCoVs which also includes the MERS-CoV. αCoVs were all detected in various genera of insectivorous bats and clustered with diverse bat αCoV sequences previously published. A closely related strain of PEDV, responsible for severe diarrhea in pigs (PEDV-CoV), was detected in 2 Myotis bats. We highlighted the presence and the high diversity of coronaviruses circulating in bats from Cambodia and Lao PDR. Three new bat genera and species were newly identified as host of coronaviruses, namely Macroglossus sp., Megaerops niphanae and Myotis horsfieldii. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparative in vivo analysis of recombinant type II feline coronaviruses with truncated and completed ORF3 region.

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Ádám Bálint

Full Text Available Our previous in vitro comparative study on a feline coronavirus (FCoV pair, differing only in the intactness of their ORF3abc regions, showed that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II feline infectious peritonitis virus (FIPV. In the present study, we describe a challenge experiment with the same recombinant FCoVs in order to gain data on the in vivo characteristics on these viruses. While parent virus FIPV DF-2 developed feline infectious peritonitis in all the infected cats, its recombinant virus PBFIPV-DF-2, differing only in seven nucleotides, proved to be surprisingly low virulent, although caused an acute febrile episode similarly to the original FIPV DF-2. PBFIPV-DF-2 infection induced significantly lower virus neutralization titers than its parent virus, and lacked the second phase of viremia and development of fatal course of the disease. The recombinant PBFIPV-DF-2-R3i with completed ORF3abc gained biological properties that differentiate between the feline enteric coronavirus (FECV and FIPV biotypes such as intensive replication in the gut, absence of viremia and weak or no serological response. Using reverse genetic approaches our study is the first experimental proof that ORF3abc is indeed responsible for the restriction of FECV replication to the intestine in vivo.

Comparative In Vivo Analysis of Recombinant Type II Feline Coronaviruses with Truncated and Completed ORF3 Region

Science.gov (United States)

Bálint, Ádám; Farsang, Attila; Zádori, Zoltán; Belák, Sándor

2014-01-01

Our previous in vitro comparative study on a feline coronavirus (FCoV) pair, differing only in the intactness of their ORF3abc regions, showed that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II feline infectious peritonitis virus (FIPV). In the present study, we describe a challenge experiment with the same recombinant FCoVs in order to gain data on the in vivo characteristics on these viruses. While parent virus FIPV DF-2 developed feline infectious peritonitis in all the infected cats, its recombinant virus PBFIPV-DF-2, differing only in seven nucleotides, proved to be surprisingly low virulent, although caused an acute febrile episode similarly to the original FIPV DF-2. PBFIPV-DF-2 infection induced significantly lower virus neutralization titers than its parent virus, and lacked the second phase of viremia and development of fatal course of the disease. The recombinant PBFIPV-DF-2-R3i with completed ORF3abc gained biological properties that differentiate between the feline enteric coronavirus (FECV) and FIPV biotypes such as intensive replication in the gut, absence of viremia and weak or no serological response. Using reverse genetic approaches our study is the first experimental proof that ORF3abc is indeed responsible for the restriction of FECV replication to the intestine in vivo. PMID:24586385

Group 2 coronaviruses prevent immediate early interferon induction by protection of viral RNA from host cell recognition

International Nuclear Information System (INIS)

Versteeg, Gijs A.; Bredenbeek, Peter J.; Worm, Sjoerd H.E. van den; Spaan, Willy J.M.

2007-01-01

Many viruses encode antagonists to prevent interferon (IFN) induction. Infection of fibroblasts with the murine hepatitis coronavirus (MHV) and SARS-coronavirus (SARS-CoV) did not result in nuclear translocation of interferon-regulatory factor 3 (IRF3), a key transcription factor involved in IFN induction, and induction of IFN mRNA transcription. Furthermore, MHV and SARS-CoV infection could not prevent IFN induction by poly (I:C) or Sendai virus, suggesting that these CoVs do not inactivate IRF3-mediated transcription regulation, but apparently prevent detection of replicative RNA by cellular sensory molecules. Our data indicate that shielding of viral RNA to host cell sensors might be the main general mechanism for coronaviruses to prevent IFN induction

Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme.

Science.gov (United States)

Huang, Xingchuan; Dong, Wenjuan; Milewska, Aleksandra; Golda, Anna; Qi, Yonghe; Zhu, Quan K; Marasco, Wayne A; Baric, Ralph S; Sims, Amy C; Pyrc, Krzysztof; Li, Wenhui; Sui, Jianhua

2015-07-01

Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity. Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic

Middle East Respiratory Syndrome Coronavirus Nonstructural Protein 16 Is Necessary for Interferon Resistance and Viral Pathogenesis

Energy Technology Data Exchange (ETDEWEB)

Menachery, Vineet D.; Gralinski, Lisa E.; Mitchell, Hugh D.; Dinnon, Kenneth H.; Leist, Sarah R.; Yount, Boyd L.; Graham, Rachel L.; McAnarney, Eileen T.; Stratton, Kelly G.; Cockrell, Adam S.; Debbink, Kari; Sims, Amy C.; Waters, Katrina M.; Baric, Ralph S.; Fernandez-Sesma, Ana

2017-11-15

Coronaviruses (CoVs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. In this study, we focused on highly conserved nonstructural protein 16 (NSP16), a viral 2'O-methyltransferase (2'O-MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of Middle East respiratory syndrome CoV (MERS-CoV) NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2'O-MTase activity had only a marginal impact on propagation and replication in Vero cells, dNSP16 mutant MERS-CoV demonstrated significant attenuation relative to the control both in primary human airway cell cultures andin vivo. Further examination indicated that dNSP16 mutant MERS-CoV had a type I interferon (IFN)-based attenuation and was partially restored in the absence of molecules of IFN-induced proteins with tetratricopeptide repeats. Importantly, the robust attenuation permitted the use of dNSP16 mutant MERS-CoV as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2'O-MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting.

IMPORTANCECoronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that

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

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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.

Transmissible gastroenteritis coronavirus genome packaging signal is located at the 5' end of the genome and promotes viral RNA incorporation into virions in a replication-independent process.

Science.gov (United States)

Morales, Lucia; Mateos-Gomez, Pedro A; Capiscol, Carmen; del Palacio, Lorena; Enjuanes, Luis; Sola, Isabel

2013-11-01

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5' end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3' end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies.

A mouse model for MERS coronavirus-induced acute respiratory distress syndrome.

Science.gov (United States)

Cockrell, Adam S; Yount, Boyd L; Scobey, Trevor; Jensen, Kara; Douglas, Madeline; Beall, Anne; Tang, Xian-Chun; Marasco, Wayne A; Heise, Mark T; Baric, Ralph S

2016-11-28

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that emerged in 2012, causing acute respiratory distress syndrome (ARDS), severe pneumonia-like symptoms and multi-organ failure, with a case fatality rate of ∼36%. Limited clinical studies indicate that humans infected with MERS-CoV exhibit pathology consistent with the late stages of ARDS, which is reminiscent of the disease observed in patients infected with severe acute respiratory syndrome coronavirus. Models of MERS-CoV-induced severe respiratory disease have been difficult to achieve, and small-animal models traditionally used to investigate viral pathogenesis (mouse, hamster, guinea-pig and ferret) are naturally resistant to MERS-CoV. Therefore, we used CRISPR-Cas9 gene editing to modify the mouse genome to encode two amino acids (positions 288 and 330) that match the human sequence in the dipeptidyl peptidase 4 receptor, making mice susceptible to MERS-CoV infection and replication. Serial MERS-CoV passage in these engineered mice was then used to generate a mouse-adapted virus that replicated efficiently within the lungs and evoked symptoms indicative of severe ARDS, including decreased survival, extreme weight loss, decreased pulmonary function, pulmonary haemorrhage and pathological signs indicative of end-stage lung disease. Importantly, therapeutic countermeasures comprising MERS-CoV neutralizing antibody treatment or a MERS-CoV spike protein vaccine protected the engineered mice against MERS-CoV-induced ARDS.

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. © 2015 The Authors.

Central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the SARS coronavirus spike glycoprotein

International Nuclear Information System (INIS)

Duquerroy, Stephane; Vigouroux, Armelle; Rottier, Peter J.M.; Rey, Felix A.; Jan Bosch, Berend

2005-01-01

The coronavirus spike glycoprotein is a class I membrane fusion protein with two characteristic heptad repeat regions (HR1 and HR2) in its ectodomain. Here, we report the X-ray structure of a previously characterized HR1/HR2 complex of the severe acute respiratory syndrome coronavirus spike protein. As expected, the HR1 and HR2 segments are organized in antiparallel orientations within a rod-like molecule. The HR1 helices form an exceptionally long (120 A) internal coiled coil stabilized by hydrophobic and polar interactions. A striking arrangement of conserved asparagine and glutamine residues of HR1 propagates from two central chloride ions, providing hydrogen-bonding 'zippers' that strongly constrain the path of the HR2 main chain, forcing it to adopt an extended conformation at either end of a short HR2 α-helix

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

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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.

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

[Prevalence and clinical characteristics of coronavirus NL63 infection in children hospitalized for acute lower respiratory tract infections in Changsha].

Science.gov (United States)

Zhang, Fei; Zhang, Bing; Xie, Zhi-Ping; Gao, Han-Chun; Zhao, Xin; Zhong, Li-Li; Zhou, Qiong-Hua; Hou, Yun-De; Duan, Zhao-Jun

2012-04-01

The main objective of this study was to explore the prevalence and clinical characteristics of human coronavirus NL63 infection in hospitalized children with acute lower respiratory tract infection (ALRTI) in Changsha. Nasopharyngeal aspirates (NPA) samples were collected from 1185 hospitalized children with ALRTI at the People's Hospital of Hunan province, between September 2008 and October 2010. Reverse transcriptase polymerase chain reaction (RT-PCR) was employed to screen for coronavirus NL63, which is a 255 bp fragment of a part of N gene. All positive amplification products were confirmed by sequencing and compared with those in GenBank. The overall frequency of coronavirus NL63 infection was 0.8%, 6 (60%) out of the coronavirus NL63 positive patients were detected in summer, 2 in autumn, 1 in spring and winter, respectively. The patients were from 2 months to two and a half years old. The clinical diagnosis was bronchopneumonia (60%), bronchiolitis (30%), and acute laryngotracheal bronchitis (10%). Four of the 10 cases had critical illness, 4 cases had underlying diseases, and 7 cases had mixed infection with other viruses. The homogeneity of coronavirus NL63 with those published in the GenBank at nucleotide levels was 97%-100%. Coronavirus NL63 infection exists in hospitalized children with acute lower respiratory tract infection in Changsha. Coronavirus NL63 infections are common in children under 3 years of age. There is significant difference in the infection rate between the boys and the girls: the boys had higher rate than the girls. The peak of prevalence of the coronavirus NL63 was in summer. A single genetic lineage of coronavirus NL63 was revealed in human subjects in Changsha. Coronavirus NL63 may also be one of the lower respiratory pathogen in China.

Construction of recombinant Newcastle disease virus expressing the S1 protein of Turkey enteric coronavirus for use as a bivalent vaccine

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Turkey enteric coronavirus (TCoV) causes a contagious form of enteritis in turkeys, generally recognized in the field by outward signs including diarrhea and decreased weight gain, resulting in severe economic losses for the poultry industry in the US. To date there is no commercial vaccine availab...

A study on antigenicity and receptor-binding ability of fragment 450-650 of the spike protein of SARS coronavirus

International Nuclear Information System (INIS)

Zhao Jincun; Wang Wei; Yuan Zhihong; Jia Rujing; Zhao Zhendong; Xu Xiaojun; Lv Ping; Zhang Yan; Jiang Chengyu; Gao Xiaoming

2007-01-01

The spike (S) protein of SARS coronavirus (SARS-CoV) is responsible for viral binding with ACE2 molecules. Its receptor-binding motif (S-RBM) is located between residues 424 and 494, which folds into 2 anti-parallel β-sheets, β5 and β6. We have previously demonstrated that fragment 450-650 of the S protein (S450-650) is predominantly recognized by convalescent sera of SARS patients. The N-terminal 60 residues (450-510) of the S450-650 fragment covers the entire β6 strand of S-RBM. In the present study, we demonstrate that patient sera predominantly recognized 2 linear epitopes outside the β6 fragment, while the mouse antisera, induced by immunization of BALB/c mice with recombinant S450-650, mainly recognized the β6 strand-containing region. Unlike patient sera, however, the mouse antisera were unable to inhibit the infectivity of S protein-expressing (SARS-CoV-S) pseudovirus. Fusion protein between green fluorescence protein (GFP) and S450-650 (S450-650-GFP) was able to stain Vero E6 cells and deletion of the β6 fragment rendered the fusion product (S511-650-GFP) unable to do so. Similarly, recombinant S450-650, but not S511-650, was able to block the infection of Vero E6 cells by the SARS-CoV-S pseudovirus. Co-precipitation experiments confirmed that S450-650 was able to specifically bind with ACE2 molecules in lysate of Vero E6 cells. However, the ability of S450-510, either alone or in fusion with GFP, to bind with ACE2 was significantly poorer compared with S450-650. Our data suggest a possibility that, although the β6 strand alone is able to bind with ACE2 with relatively high affinity, residues outside the S-RBM could also assist the receptor binding of SARS-CoV-S protein

Diagnostic Methods for Feline Coronavirus: A Review

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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.

Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

Science.gov (United States)

Morales, Lucia; Mateos-Gomez, Pedro A.; Capiscol, Carmen; del Palacio, Lorena; Sola, Isabel

2013-01-01

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3′ end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies. PMID:23966403

Discovery of a novel canine respiratory coronavirus support genetic recombination among betacoronavirus1.

Science.gov (United States)

Lu, Shuai; Wang, Yanqun; Chen, Yingzhu; Wu, Bingjie; Qin, Kun; Zhao, Jincun; Lou, Yongliang; Tan, Wenjie

2017-06-02

Although canine respiratory coronavirus (CRCoV) is an important respiratory pathogen that is prevalent in many countries, only one complete genome sequence of CRCoV (South Korea strain K37) has been obtained to date. Genome-wide analyses and recombination have rarely been conducted, as small numbers of samples and limited genomic characterization have previously prevented further analyses. Herein, we report a unique CRCoV strain, denoted strain BJ232, derived from a CRCoV-positive dog with a mild respiratory infection. Phylogenetic analysis based on complete genome of all available coronaviruses consistently show that CRCoV BJ232 is most closely related to human coronavirus OC43 (HCoV-OC43) and BCoV, forming a separate clade that split off early from other Betacoronavirus 1. Based on the phylogenetic and SimPlot analysis we propose that CRCoV-K37 was derived from genetic recombination between CRCoV-BJ232 and BCoV. In detail, spike (S) gene of CRCoV-K37 clustered with CRCoV-BJ232. However orf1ab, membrane (M) and nucleocapsid (N) genes were more related to Bovine coronavirus (BCoV) than CRCoV-B232. Molecular epidemic analysis confirmed the prevalence of CRCoV-BJ232 lineage around the world for a long time. Recombinant events among Betacoronavirus 1 may have implications for CRCoV transmissibility. All these findings provide further information regarding the origin of CRCoV. Copyright © 2017. Published by Elsevier B.V.

Evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses.

Science.gov (United States)

Agnihothram, Sudhakar; Gopal, Robin; Yount, Boyd L; Donaldson, Eric F; Menachery, Vineet D; Graham, Rachel L; Scobey, Trevor D; Gralinski, Lisa E; Denison, Mark R; Zambon, Maria; Baric, Ralph S

2014-04-01

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012, causing severe acute respiratory disease and pneumonia, with 44% mortality among 136 cases to date. Design of vaccines to limit the virus spread or diagnostic tests to track newly emerging strains requires knowledge of antigenic and serologic relationships between MERS-CoV and other CoVs.  Using synthetic genomics and Venezuelan equine encephalitis virus replicons (VRPs) expressing spike and nucleocapsid proteins from MERS-CoV and other human and bat CoVs, we characterize the antigenic responses (using Western blot and enzyme-linked immunosorbent assay) and serologic responses (using neutralization assays) against 2 MERS-CoV isolates in comparison with those of other human and bat CoVs.  Serologic and neutralization responses against the spike glycoprotein were primarily strain specific, with a very low level of cross-reactivity within or across subgroups. CoV N proteins within but not across subgroups share cross-reactive epitopes with MERS-CoV isolates. Our findings were validated using a convalescent-phase serum specimen from a patient infected with MERS-CoV (NA 01) and human antiserum against SARS-CoV, human CoV NL63, and human CoV OC43.  Vaccine design for emerging CoVs should involve chimeric spike protein containing neutralizing epitopes from multiple virus strains across subgroups to reduce immune pathology, and a diagnostic platform should include a panel of nucleocapsid and spike proteins from phylogenetically distinct CoVs.

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis

International Nuclear Information System (INIS)

Sawicki, S.G.; Sawicki, D.L.

1986-01-01

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [ 3 H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minis-strand RNA synthesis was three- to fourfold more sensitive to inhibition of cycloheximide than was plus-strand synthesis

Characterisation of different forms of the accessory gp3 canine coronavirus type I protein identified in cats.

Science.gov (United States)

d'Orengiani, Anne-Laure Pham-Hung d'Alexandry; Duarte, Lidia; Pavio, Nicole; Le Poder, Sophie

2015-04-16

ORF3 is a supplemental open reading frame coding for an accessory glycoprotein gp3 of unknown function, only present in genotype I canine strain (CCoV-I) and some atypical feline FCoV strains. In these latter hosts, the ORF3 gene systematically displays one or two identical deletions leading to the synthesis of truncated proteins gp3-Δ1 and gp3-Δ2. As deletions in CoV accessory proteins have already been involved in tissue or host switch, studies of these different gp3 proteins were conducted in canine and feline cell. All proteins oligomerise through covalent bonds, are N-glycosylated and are maintained in the ER in non-infected but also in CCoV-II infected cells, without any specific retention signal. However, deletions influence their level of expression. In canine cells, all proteins are expressed with similar level whereas in feline cells, the expression of gp3-Δ1 is higher than the two other forms of gp3. None of the gp3 proteins modulate the viral replication cycle of heterologous genotype II CCoV in canine cell line, leading to the conclusion that the gp3 proteins are probably advantageous only for CCoV-I and atypical FCoV strains. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Eveline Kindler

2017-02-01

Full Text Available 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.

Suppression of feline coronavirus replication in vitro by cyclosporin A

Directory of Open Access Journals (Sweden)

Tanaka Yoshikazu

2012-04-01

Full Text Available Abstract The feline infectious peritonitis virus (FIPV is a member of the feline coronavirus family that causes FIP, which is incurable and fatal in cats. Cyclosporin A (CsA, an immunosuppressive agent that targets the nuclear factor pathway of activated T-cells (NF-AT to bind cellular cyclophilins (CyP, dose-dependently inhibited FIPV replication in vitro. FK506 (an immunosuppressor of the pathway that binds cellular FK506-binding protein (FKBP but not CyP did not affect FIPV replication. Neither cell growth nor viability changed in the presence of either CsA or FK506, and these factors did not affect the NF-AT pathway in fcwf-4 cells. Therefore, CsA does not seem to exert inhibitory effects via the NF-AT pathway. In conclusion, CsA inhibited FIPV replication in vitro and further studies are needed to verify the practical value of CsA as an anti-FIPV treatment in vivo.

Fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency

OpenAIRE

Szczawinska‐Poplonyk, Aleksandra; Jonczyk‐Potoczna, Katarzyna; Breborowicz, Anna; Bartkowska‐Sniatkowska, Alicja; Figlerowicz, Magdalena

2012-01-01

Please cite this paper as: Szczawinska‐Poplonyk et al. (2012) Fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency. Influenza and Other Respiratory Viruses DOI: 10.1111/irv.12059. 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, particularl...

On the biased nucleotide composition of the human coronavirus RNA genome

NARCIS (Netherlands)

Berkhout, Ben; van Hemert, Formijn

2015-01-01

We investigated the nucleotide composition of the RNA genome of the six human coronaviruses. Some general coronavirus characteristics were apparent (e.g. high U, low C count), but we also detected species-specific signatures. Most strikingly, the high U and low C proportions are quite variable and

Identification of Alpha and Beta Coronavirus in Wildlife Species in France: Bats, Rodents, Rabbits, and Hedgehogs

Directory of Open Access Journals (Sweden)

Elodie Monchatre-Leroy

2017-11-01

Full Text Available Coronaviruses are closely monitored in the context of emerging diseases and, as illustrated with Severe Acute Respiratory Syndrome coronavirus (SARS-CoV and Middle East Respiratory Syndrome-coronavirus (MERS-CoV, are known to cross the species barrier and eventually to move from wildlife to humans. Knowledge of the diversity of coronaviruses in wildlife is therefore essential to better understand and prevent emergence events. This study explored the presence of coronaviruses in four wild mammal orders in France: Bats, rodents, lagomorphs, and hedgehogs. Betacoronavirus and Alphacoronavirus genera were identified. The results obtained suggest the circulation of potentially evolving virus strains, with the potential to cross the species barrier.

Structural Insights into Immune Recognition of the Severe Acute Respiratory Syndrome Coronavirus S Protein Receptor Binding Domain

Energy Technology Data Exchange (ETDEWEB)

Pak, J.; Sharon, C; Satkunarajah, M; Thierry, C; Cameron, C; Kelvin, D; Seetharaman, J; Cochrane, A; Plummer, F; et. al.

2009-01-01

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for host cell attachment and fusion of the viral and host cell membranes. Within S the receptor binding domain (RBD) mediates the interaction with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV host cell receptor. Both S and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Reported here is the X-ray crystal structure of the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The RBD-F26G19 Fab complex represents the first example of the structural characterization of an antibody elicited by an immune response to SARS-CoV or any fragment of it. The structure reveals that the RBD surface recognized by F26G19 overlaps significantly with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction.

Deficient incorporation of spike protein into virions contributes to the lack of infectivity following establishment of a persistent, non-productive infection in oligodendroglial cell culture by murine coronavirus

International Nuclear Information System (INIS)

Liu Yin; Herbst, Werner; Cao Jianzhong; Zhang Xuming

2011-01-01

Infection of mouse oligodendrocytes with a recombinant mouse hepatitis virus (MHV) expressing a green fluorescence protein facilitated specific selection of virus-infected cells and subsequent establishment of persistence. Interestingly, while viral genomic RNAs persisted in infected cells over 14 subsequent passages with concomitant synthesis of viral subgenomic mRNAs and structural proteins, no infectious virus was isolated beyond passage 2. Further biochemical and electron microscopic analyses revealed that virions, while assembled, contained little spike in the envelope, indicating that lack of infectivity during persistence was likely due to deficiency in spike incorporation. This type of non-lytic, non-productive persistence in oligodendrocytes is unique among animal viruses and resembles MHV persistence previously observed in the mouse central nervous system. Thus, establishment of such a culture system that can recapitulate the in vivo phenomenon will provide a powerful approach for elucidating the mechanisms of coronavirus persistence in glial cells at the cellular and molecular levels.

Human coronavirus NL63, France

NARCIS (Netherlands)

Vabret, Astrid; Mourez, Thomas; Dina, Julia; van der Hoek, Lia; Gouarin, Stéphanie; Petitjean, Joëlle; Brouard, Jacques; Freymuth, François

2005-01-01

The human coronavirus NL63 (HCoV-NL63) was first identified in The Netherlands, and its circulation in France has not been investigated. We studied HCoV-NL63 infection in hospitalized children diagnosed with respiratory tract infections. From November 2002 to April 2003, we evaluated 300 respiratory

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

Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus

International Nuclear Information System (INIS)

Ricagno, Stéfano; Coutard, Bruno; Grisel, Sacha; Brémond, Nicolas; Dalle, Karen; Tocque, Fabienne; Campanacci, Valérie; Lichière, Julie; Lantez, Violaine; Debarnot, Claire; Cambillau, Christian; Canard, Bruno; Egloff, Marie-Pierre

2006-01-01

Crystals of Nsp15 from the aetiological agent of SARS have been grown at room temperature. Crystals have cubic symmetry and diffract to a maximum resolution of 2.7 Å. The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4 1 32 or P4 3 32, with unit-cell parameters a = b = c = 166.8 Å. Diffraction data were collected to a maximum resolution of 2.7 Å

Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus

Energy Technology Data Exchange (ETDEWEB)

Ricagno, Stéfano; Coutard, Bruno; Grisel, Sacha; Brémond, Nicolas; Dalle, Karen; Tocque, Fabienne; Campanacci, Valérie; Lichière, Julie; Lantez, Violaine; Debarnot, Claire; Cambillau, Christian; Canard, Bruno; Egloff, Marie-Pierre, E-mail: marie-pierre.egloff@afmb.univ-mrs.fr [Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d’Ingénieurs de Luminy-Case 925, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France)

2006-04-01

Crystals of Nsp15 from the aetiological agent of SARS have been grown at room temperature. Crystals have cubic symmetry and diffract to a maximum resolution of 2.7 Å. The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4{sub 1}32 or P4{sub 3}32, with unit-cell parameters a = b = c = 166.8 Å. Diffraction data were collected to a maximum resolution of 2.7 Å.

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

Coronaviruses in guano from Pteropus medius bats in Peradeniya, Sri Lanka.

Science.gov (United States)

Kudagammana, H D W S; Thevanesam, V; Chu, D K W; Eriyagama, N B; Peiris, J S M; Noordeen, F

2018-03-02

Bats are a unique group of mammals well suited to be hosts for emerging viruses. With current rates of deforestation and urbanization, redistribution of bat habitats to urban and suburban areas may bring bats into closer contact with livestock and humans. Common flying fox, Pteropus medius (previously known as Pteropus giganteus), forms large communal roosts on treetops, often in close proximity to human habitation in Sri Lanka. This report describes the detection of coronavirus RNA in P. medius bat guano collected in Peradeniya, Sri Lanka. These viruses had >97% nucleotide identity with coronaviruses detected in Cynopterus sphinx, Scotophilus heathii and S. kuhlii bats in Thailand. Pteropus medius is widespread in Asia and appears to excrete group D coronaviruses, which are hitherto confined to bats; however, these findings may have public health implications in the future. © 2018 Blackwell Verlag GmbH.

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

Human Coronaviruses 229E and NL63: Close Yet Still So Far

NARCIS (Netherlands)

Dijkman, Ronald; van der Hoek, Lia

2009-01-01

HCoV-NL63 and HCoV-229E are two of the four human coronaviruses that circulate worldwide. These two viruses are unique in their relationship towards each other. Phylogenetically, the viruses are more closely related to each other than to any other human coronavirus, yet they only share 65% sequence

MERS-coronavirus: From discovery to intervention

NARCIS (Netherlands)

W. Widagdo; N.M.A. 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

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

A phylogenetically distinct Middle East respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in Dubai with rising seroprevalence with age

OpenAIRE

Wernery, Ulrich; Rasoul, IHassab El; Wong, Emily YM; Joseph, Marina; Chen, Yixin; Jose, Shanty; Tsang, Alan KL; Patteril, Nissy Annie Georgy; Chen, Honglin; Elizabeth, Shyna K; Yuen, Kwok-Yung; Joseph, Sunitha; Xia, Ningshao; Wernery, Renate; Lau, Susanna KP

2015-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) was detected by monoclonal antibody-based nucleocapsid protein-capture enzyme-linked immunosorbent assay (ELISA), RNA detection, and viral culture from the nasal sample of a 1-month-old dromedary calf in Dubai with sudden death. Whole genome phylogeny showed that this MERS-CoV strain did not cluster with the other MERS-CoV strains from Dubai that we reported recently. Instead, it formed a unique branch more closely related to other MERS-...

Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method.

Science.gov (United States)

Takeda, Mitsuhiro; Chang, Chung-ke; Ikeya, Teppei; Güntert, Peter; Chang, Yuan-hsiang; Hsu, Yen-lan; Huang, Tai-huang; Kainosho, Masatsune

2008-07-18

The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform (13)C and (15)N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the beta-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.

Detection of Coronaviruses in Bats of Various Species in Italy

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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.

A rare cause of acute flaccid paralysis: Human coronaviruses

OpenAIRE

Turgay, Cokyaman; Emine, Tekin; Ozlem, Koken; Muhammet, S. Paksu; Haydar, A. Tasdemir

2015-01-01

Acute flaccid paralysis (AFP) is a life-threatening clinical entity characterized by weakness in the whole body muscles often accompanied by respiratory and bulbar paralysis. The most common cause is Gullian-Barre syndrome, but infections, spinal cord diseases, neuromuscular diseases such as myasthenia gravis, drugs and toxins, periodic hypokalemic paralysis, electrolyte disturbances, and botulism should be considered as in the differential diagnosis. Human coronaviruses (HCoVs) cause common ...

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|info:eu-repo/dai/nl/273306049; 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

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

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

DEFF Research Database (Denmark)

Knudsen, T B; Kledal, T N; Andersen, O

2003-01-01

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...

False-Positive Results in a Recombinant Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV) Nucleocapsid Enzyme-Linked Immunosorbent Assay Due to HCoV-OC43 and HCoV-229E Rectified by Western Blotting with Recombinant SARS-CoV Spike Polypeptide

OpenAIRE

Woo, Patrick C. Y.; Lau, Susanna K. P.; Wong, Beatrice H. L.; Chan, Kwok-Hung; Hui, Wai-Ting; Kwan, Grace S. W.; Peiris, J. S. Malik; Couch, Robert B.; Yuen, Kwok-Yung

2004-01-01

Using paired serum samples obtained from patients with illness associated with increases in anti-human coronavirus OC43 (HCoV-OC43) or anti-HCoV-229E antibodies, we examined the possibility of false-positive results detected in a recombinant severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein immunoglobulin G enzyme-linked immunosorbent assay (ELISA). Three of the 21 and 1 of the 7 convalescent-phase serum samples from persons with increases in anti...

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

International Nuclear Information System (INIS)

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

2009-01-01

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.

Clinico-epidemiological characteristics of acute respiratory infections caused by coronavirus OC43, NL63 and 229E.

Science.gov (United States)

Reina, J; López-Causapé, C; Rojo-Molinero, E; Rubio, R

2014-12-01

Acute respiratory infection is a very common condition in the general population. The majority of these infections are due to viruses. This study attempted to determine the clinical and epidemiological characteristics of adult patients with respiratory infection by the coronavirus OC43, NL63 and 229E. Between January 2013 and February 2014, we prospectively studied all patients with suspected clinical respiratory infection by taking throat swabs and performing a reverse transcription polymerase chain reaction in search of coronavirus. In 48 cases (7.0% of the 686 enrolled patients; 12.6% of the 381 in whom a virus was detected) the presence of a coronavirus demonstrated. In 24 cases, the virus was OC43 (50%); in 14 cases, the virus was NL63 (29%); and in 10 cases, the virus was 229E (21%). The mean age was 54.5 years, with a slight predominance of men. The most common clinical presentations were nonspecific influenza symptoms (43.7%), pneumonia (29.2%) and chronic obstructive pulmonary disease exacerbation (8.3%). Fifty-two percent of the patients required hospitalization, and 2 patients required intensive care. There were no deaths. Acute respiratory infections caused by coronavirus mainly affect middle-aged male smokers, who are often affected by previous diseases. The most common clinical picture has been nonspecific influenza symptoms. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

Lack of association between infection with a novel human coronavirus (HCoV), HCoV-NH, and Kawasaki disease in Taiwan

NARCIS (Netherlands)

Chang, Luan-Yin; Chiang, Bor-Luen; Kao, Chuan-Liang; Wu, Mei-Hwan; Chen, Pei-Jer; Berkhout, Ben; Yang, Hui-Ching; Huang, Li-Min

2006-01-01

We investigated whether infection with a novel human coronavirus (HCoV), called "New Haven coronavirus" (HCoV-NH)--which is similar to and likely represents the same species as another novel HCoV, HCoV-NL63--is associated with Kawasaki disease (KD) in Taiwan. Fifty-three patients with KD were

Human Coronaviruses 229E and NL63: Close Yet Still So Far

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Ronald Dijkman

2009-04-01

Full Text Available HCoV-NL63 and HCoV-229E are two of the four human coronaviruses that circulate worldwide. These two viruses are unique in their relationship towards each other. Phylogenetically, the viruses are more closely related to each other than to any other human coronavirus, yet they only share 65% sequence identity. Moreover, the viruses use different receptors to enter their target cell. HCoV-NL63 is associated with croup in children, whereas all signs suggest that the virus probably causes the common cold in healthy adults. HCoV-229E is a proven common cold virus in healthy adults, so it is probable that both viruses induce comparable symptoms in adults, even though their mode of infection differs. Here, we present an overview of the current knowledge on both human coronaviruses, focusing on similarities and differences.

A virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses.

Science.gov (United States)

Wu, Kailang; Chen, Lang; Peng, Guiqing; Zhou, Wenbo; Pennell, Christopher A; Mansky, Louis M; Geraghty, Robert J; Li, Fang

2011-06-01

How viruses evolve to select their receptor proteins for host cell entry is puzzling. We recently determined the crystal structures of NL63 coronavirus (NL63-CoV) and SARS coronavirus (SARS-CoV) receptor-binding domains (RBDs), each complexed with their common receptor, human angiotensin-converting enzyme 2 (hACE2), and proposed the existence of a virus-binding hot spot on hACE2. Here we investigated the function of this hypothetical hot spot using structure-guided biochemical and functional assays. The hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. Mutations that disturb the hot spot structure have significant effects on virus/receptor interactions, revealing critical energy contributions from the hot spot structure. The tunnel structure at the NL63-CoV/hACE2 interface is more compact than that at the SARS-CoV/hACE2 interface, and hence RBD/hACE2 binding affinities are decreased either by NL63-CoV mutations decreasing the tunnel space or by SARS-CoV mutations increasing the tunnel space. Furthermore, NL63-CoV RBD inhibits hACE2-dependent transduction by SARS-CoV spike protein, a successful application of the hot spot theory that has the potential to become a new antiviral strategy against SARS-CoV infections. These results suggest that the structural features of the hot spot on hACE2 were among the driving forces for the convergent evolution of NL63-CoV and SARS-CoV.

Molecular epidemiology of bovine coronavirus on the basis of comparative analyses of the S gene

DEFF Research Database (Denmark)

Liu, Lihong; Hägglund, Sara; Hakhverdyan, Mikhayil

2006-01-01

Bovine coronavirus (BCoV), a group 2 member of the genus Coronavirus in the family Coronaviridae, is an important pathogen in cattle worldwide. It causes diarrhea in adult animals (winter dysentery), as well as enteric and respiratory diseases in calves. The annual occurrence of BCoV epidemics...

Renin-angiotensin system in human coronavirus pathogenesis

NARCIS (Netherlands)

Wevers, Brigitte A.; van der Hoek, Lia

2010-01-01

Although initially considered relatively harmless pathogens, human coronaviruses (HCoVs) are nowadays known to be associated with more severe clinical complications. Still, their precise pathogenic potential is largely unknown, particularly regarding the most recently identified species HCoV-NL63

Coronavirus gene 7 counteracts host defenses and modulates virus virulence.

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Jazmina L G Cruz

2011-06-01

Full Text Available Transmissible gastroenteritis virus (TGEV genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7. Both the mutant and the parental (rTGEV-wt viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c, a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the

The Paradox of Feline Coronavirus Pathogenesis: A Review

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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.

Two deletion variants of Middle East respiratory syndrome coronavirus found in a patient with characteristic symptoms.

Science.gov (United States)

Xie, Qian; Cao, Yujuan; Su, Juan; Wu, Jie; Wu, Xianbo; Wan, Chengsong; He, Mingliang; Ke, Changwen; Zhang, Bao; Zhao, Wei

2017-08-01

Significant sequence variation of Middle East respiratory syndrome coronavirus (MERS CoV) has never been detected since it was first reported in 2012. A MERS patient came from Korea to China in late May 2015. The patient was 44 years old and had symptoms including high fever, dry cough with a little phlegm, and shortness of breath, which are roughly consistent with those associated with MERS, and had had close contact with individuals with confirmed cases of MERS.After one month of therapy with antiviral, anti-infection, and immune-enhancing agents, the patient recovered in the hospital and was discharged. A nasopharyngeal swab sample was collected for direct sequencing, which revealed two deletion variants of MERS CoV. Deletions of 414 and 419 nt occurred between ORF5 and the E protein, resulting in a partial protein fusion or truncation of ORF5 and the E protein. Functional analysis by bioinformatics and comparison to previous studies implied that the two variants might be defective in their ability to package MERS CoV. However, the mechanism of how these deletions occurred and what effects they have need to be further investigated.

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.

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

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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.

Chimeric Feline Coronaviruses That Encode Type II Spike Protein on Type I Genetic Background Display Accelerated Viral Growth and Altered Receptor Usage▿

Science.gov (United States)

Tekes, Gergely; Hofmann-Lehmann, Regina; Bank-Wolf, Barbara; Maier, Reinhard; Thiel, Heinz-Jürgen; Thiel, Volker

2010-01-01

Persistent infection of domestic cats with feline coronaviruses (FCoVs) can lead to a highly lethal, immunopathological disease termed feline infectious peritonitis (FIP). Interestingly, there are two serotypes, type I and type II FCoVs, that can cause both persistent infection and FIP, even though their main determinant of host cell tropism, the spike (S) protein, is of different phylogeny and displays limited sequence identity. In cell culture, however, there are apparent differences. Type II FCoVs can be propagated to high titers by employing feline aminopeptidase N (fAPN) as a cellular receptor, whereas the propagation of type I FCoVs is usually difficult, and the involvement of fAPN as a receptor is controversial. In this study we have analyzed the phenotypes of recombinant FCoVs that are based on the genetic background of type I FCoV strain Black but encode the type II FCoV strain 79-1146 S protein. Our data demonstrate that recombinant FCoVs expressing a type II FCoV S protein acquire the ability to efficiently use fAPN for host cell entry and corroborate the notion that type I FCoVs use another main host cell receptor. We also observed that recombinant FCoVs display a large-plaque phenotype and, unexpectedly, accelerated growth kinetics indistinguishable from that of type II FCoV strain 79-1146. Thus, the main phenotypic differences for type I and type II FCoVs in cell culture, namely, the growth kinetics and the efficient usage of fAPN as a cellular receptor, can be attributed solely to the FCoV S protein. PMID:19906918

The persistent prevalence and evolution of cross-family recombinant coronavirus GCCDC1 among a bat population: a two-year follow-up.

Science.gov (United States)

Obameso, Joseph O; Li, Hong; Jia, Hao; Han, Min; Zhu, Shiyan; Huang, Canping; Zhao, Yuhui; Zhao, Min; Bai, Yu; Yuan, Fei; Zhao, Honglan; Peng, Xia; Xu, Wen; Tan, Wenjie; Zhao, Yingze; Yuen, Kwok-Yung; Liu, William J; Lu, Lin; Gao, George F

2017-12-01

Bats are connected with the increasing numbers of emerging and re-emerging viruses that may break the species barrier and spread into the human population. Coronaviruses are one of the most common viruses discovered in bats, which were considered as the natural source of recent human-susceptible coronaviruses, i.e. SARS-COV and MERS-CoV. Our previous study reported the discovery of a bat-derived putative cross-family recombinant coronavirus with a reovirus gene p10, named as Ro-BatCoV GCCDC1. In this report, through a two-year follow-up of a special bat population in one specific cave of south China, we illustrate that Ro-BatCoV GCCDC1 persistently circulates among bats. Notably, through the longitudinal observation, we identified the dynamic evolution of Ro-BatCoV GCCDC1 in bats represented by continuously recombination events. Our study provides the first glimpse of the virus evolution in one longitudinally observed bat population cohort and underlines the surveillance and pre-warning of potential interspecies transmittable viruses in bats.

Detection by radioimmunoassay and enzyme-linked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions.

Science.gov (United States)

Rodak, L; Babiuk, L A; Acres, S D

1982-07-01

The sensitivity of a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), and a serum neutralization assay (SN) for detecting antibodies to bovine coronavirus in serum and colostrum were compared. Although there proved to be a good correlation among all three assays (r = 0.915 and 0.964 for RIA with SN and ELISA, respectively), RIA and ELISA proved to be at least 10 times more sensitive than neutralization tests. By using these techniques, it was possible to detect a time-dependent decrease in antibody levels in bovine colostrum after parturition. Using ELISA, we demonstrated that 12 of 12 herds in Saskatchewan, and 109 of 110 animals tested, and antibody to bovine coronavirus. There was no elevated antibody response in serum or lacteal secretions of cows vaccinated once or twice with a commercially available modified live rota-coronavirus vaccine. In addition to being more sensitive than SN, ELISA and RIA proved to have other advantages for measuring antibody levels to bovine coronavirus and therefore warrant wider use as tools in diagnostic virology.

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.

Crystallization and diffraction analysis of the SARS coronavirus nsp10–nsp16 complex

International Nuclear Information System (INIS)

Debarnot, Claire; Imbert, Isabelle; Ferron, François; Gluais, Laure; Varlet, Isabelle; Papageorgiou, Nicolas; Bouvet, Mickaël; Lescar, Julien; Decroly, Etienne; Canard, Bruno

2011-01-01

The expression, purification and crystallization of the SARS coronavirus nsp16 RNA-cap AdoMet-dependent (nucleoside-2′O)-methyltransferase in complex with its activating factor nsp10 are reported. To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1–16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2′-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress

A Rare Case of Human Coronavirus 229E Associated with Acute Respiratory Distress Syndrome in a Healthy Adult

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Foula Vassilara

2018-01-01

Full Text Available Human coronavirus 229E (HCoV-229E is one of the first coronavirus strains being described. It is linked to common cold symptoms in healthy adults. Younger children and the elderly are considered vulnerable to developing lower respiratory tract infections (LRTIs. In particular, immunocompromised patients have been reported with severe and life-threatening LRTIs attributed to HCoV-229E. We report for the first time a case of LRTI and acute respiratory distress syndrome developed in a healthy adult with no comorbidities and HCoV-229E strain identified as the only causative agent. A 45-year-old female with a clear medical history presented with fever, cough, and headache. Respiratory tract infection was diagnosed, and empirical antibiotics were started. Within two days, she developed bilateral pleural effusions, diffuse consolidations, and ground glass opacities involving all lung fields. She needed immediate oxygen supply, while ABGs deteriorated and chest imaging and PaO2/FiO2 indicated ARDS. Early administration of systemic corticosteroids led to gradual clinical improvement. Multiplex PCR from nasal secretions was positive only for HCoV-229E and negative for multiple other pathogens. It remains to be elucidated how an immunocompetent adult developed a life-threatening LRTI caused by a “benign considered” coronavirus strain, the HCoV-229E.

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.

2012-01-01

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

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.

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

Rapid detection of MERS coronavirus-like viruses in bats: pote1ntial for tracking MERS coronavirus transmission and animal origin.

Science.gov (United States)

Woo, Patrick C Y; Lau, Susanna K P; Chen, Yixin; Wong, Emily Y M; Chan, Kwok-Hung; Chen, Honglin; Zhang, Libiao; Xia, Ningshao; Yuen, Kwok-Yung

2018-03-07

Recently, we developed a monoclonal antibody-based rapid nucleocapsid protein detection assay for diagnosis of MERS coronavirus (MERS-CoV) in humans and dromedary camels. In this study, we examined the usefulness of this assay to detect other lineage C betacoronaviruses closely related to MERS-CoV in bats. The rapid MERS-CoV nucleocapsid protein detection assay was tested positive in 24 (88.9%) of 27 Tylonycteris bat CoV HKU4 (Ty-BatCoV-HKU4) RNA-positive alimentary samples of Tylonycteris pachypus and 4 (19.0%) of 21 Pipistrellus bat CoV HKU5 (Pi-BatCoV-HKU5) RNA-positive alimentary samples of Pipistrellus abramus. There was significantly more Ty-BatCoV-HKU4 RNA-positive alimentary samples than Pi-BatCoV-HKU5 RNA-positive alimentary samples that were tested positive by the rapid MERS-CoV nucleocapsid protein detection assay (P < 0.001 by Chi-square test). The rapid assay was tested negative in all 51 alimentary samples RNA-positive for alphacoronaviruses (Rhinolophus bat CoV HKU2, Myotis bat CoV HKU6, Miniopterus bat CoV HKU8 and Hipposideros batCoV HKU10) and 32 alimentary samples positive for lineage B (SARS-related Rhinolophus bat CoV HKU3) and lineage D (Rousettus bat CoV HKU9) betacoronaviruses. No significant difference was observed between the viral loads of Ty-BatCoV-HKU4/Pi-BatCoV-HKU5 RNA-positive alimentary samples that were tested positive and negative by the rapid test (Mann-Witney U test). The rapid MERS-CoV nucleocapsid protein detection assay is able to rapidly detect lineage C betacoronaviruses in bats. It detected significantly more Ty-BatCoV-HKU4 than Pi-BatCoV-HKU5 because MERS-CoV is more closely related to Ty-BatCoV-HKU4 than Pi-BatCoV-HKU5. This assay will facilitate rapid on-site mass screening of animal samples for ancestors of MERS-CoV and tracking transmission in the related bat species.

Isolation and molecular characterization of type I and type II feline coronavirus in Malaysia

OpenAIRE

Amer, Alazawy; Siti Suri, Arshad; Abdul Rahman, Omar; Mohd, Hair Bejo; Faruku, Bande; Saeed, Sharif; Tengku Azmi, Tengku Ibrahim

2012-01-01

Abstract Background Feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first...

Feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis.

Science.gov (United States)

Longstaff, Louise; Porter, Emily; Crossley, Victoria J; Hayhow, Sophie E; Helps, Christopher R; Tasker, Séverine

2017-02-01

Objectives The aim of the study was to determine whether feline coronavirus (FCoV) RNA in effusion samples can be used as a diagnostic marker of feline infectious peritonitis (FIP); and in FCoV RNA-positive samples to examine amino acid codons in the FCoV spike protein at positions 1058 and 1060 where leucine and alanine, respectively, have been associated with systemic or virulent (FIP) FCoV infection. Methods Total RNA was extracted from effusion samples from 20 cats with confirmed FIP and 23 cats with other diseases. Feline coronavirus RNA was detected using a reverse transcriptase quantitative polymerase chain reaction assay (qRT-PCR), and positive samples underwent pyrosequencing of position 1058 with or without Sanger sequencing of position 1060 in the FCoV spike protein. Results Seventeen (85%) of the effusion samples from 20 cats with FIP were positive for FCoV RNA, whereas none of the 23 cats with other diseases were positive. Pyrosequencing of the 17 FCoV-positive samples showed that 11 (65%) of the cats had leucine and two (12%) had methionine at position 1058. Of the latter two samples with methionine, one had alanine at position 1060. Conclusions and relevance A positive FCoV qRT-PCR result on effusions appears specific for FIP and may be a useful diagnostic marker for FIP in cats with effusions. The majority of FCoVs contained amino acid changes previously associated with systemic spread or virulence (FIP) of the virus.

A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination

NARCIS (Netherlands)

van Beurden, Steven J; Berends, Alinda J; Krämer-Kühl, Annika; Spekreijse, Dieuwertje; Chénard, Gilles; Philipp, Hans-Christian; Mundt, Egbert; Rottier, Peter J M; Verheije, M Hélène

2017-01-01

BACKGROUND: Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for

Cross host transmission in the emergence of MERS coronavirus

NARCIS (Netherlands)

C.B.E.M. Reusken (Chantal); V.S. Raj (Stalin); M.P.G. Koopmans D.V.M. (Marion); B.L. Haagmans (Bart)

2016-01-01

textabstractCoronaviruses (CoVs) able to infect humans emerge through cross-host transmission from animals. There is substantial evidence that the recent Middle East respiratory syndrome (MERS)-CoV outbreak is fueled by zoonotic transmission from dromedary camels. This is largely based on the fact

Structural and Functional Analyses of the Severe Acute Respiratory Syndrome Coronavirus Endoribonuclease Nsp15

Energy Technology Data Exchange (ETDEWEB)

Bhardwaj, Kanchan; Palaninathan, Satheesh; Alcantara, Joanna Maria Ortiz; Yi, Lillian Li; Guarino, Linda; Sacchettini, James C.; Kao, C. Cheng (TAM)

2008-03-31

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3' of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.

Vacuolating encephalitis in mice infected by human coronavirus OC43

International Nuclear Information System (INIS)

Jacomy, Helene; Talbot, Pierre J.

2003-01-01

Involvement of viruses in human neurodegenerative diseases and the underlying pathologic mechanisms remain generally unclear. Human respiratory coronaviruses (HCoV) can infect neural cells, persist in human brain, and activate myelin-reactive T cells. As a means of understanding the human infection, we characterized in vivo the neurotropic and neuroinvasive properties of HCoV-OC43 through the development of an experimental animal model. Virus inoculation of 21-day postnatal C57BL/6 and BALB/c mice led to a generalized infection of the whole CNS, demonstrating HCoV-OC43 neuroinvasiveness and neurovirulence. This acute infection targeted neurons, which underwent vacuolation and degeneration while infected regions presented strong microglial reactivity and inflammatory reactions. Damage to the CNS was not immunologically mediated and microglial reactivity was instead a consequence of direct virus-mediated neuronal injury. Although this acute encephalitis appears generally similar to that induced by murine coronaviruses, an important difference rests in the prominent spongiform-like degeneration that could trigger neuropathology in surviving animals

The roles of transportation and transportation hubs in the propagation of influenza and coronaviruses: a systematic review.

Science.gov (United States)

Browne, Annie; Ahmad, Sacha St-Onge; Beck, Charles R; Nguyen-Van-Tam, Jonathan S

2016-01-01

Respiratory viruses spread in humans across wide geographical areas in short periods of time, resulting in high levels of morbidity and mortality. We undertook a systematic review to assess the evidence that air, ground and sea mass transportation systems or hubs are associated with propagating influenza and coronaviruses. Healthcare databases and sources of grey literature were searched using pre-defined criteria between April and June 2014. Two reviewers screened all identified records against the protocol, undertook risk of bias assessments and extracted data using a piloted form. Results were analysed using a narrative synthesis. Forty-one studies met the eligibility criteria. Risk of bias was high in the observational studies, moderate to high in the reviews and moderate to low in the modelling studies. In-flight influenza transmission was identified substantively on five flights with up to four confirmed and six suspected secondary cases per affected flight. Five studies highlighted the role of air travel in accelerating influenza spread to new areas. Influenza outbreaks aboard cruise ships affect 2-7% of passengers. Influenza transmission events have been observed aboard ground transport vehicles. High heterogeneity between studies and the inability to exclude other sources of infection means that the risk of influenza transmission from an index case to other passengers cannot be accurately quantified. A paucity of evidence was identified describing severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus transmission events associated with transportation systems or hubs. Air transportation appears important in accelerating and amplifying influenza propagation. Transmission occurs aboard aeroplanes, at the destination and possibly at airports. Control measures to prevent influenza transmission on cruise ships are needed to reduce morbidity and mortality. There is no recent evidence of sea transport accelerating influenza

Tissue Distribution of the MERS-Coronavirus Receptor in Bats

NARCIS (Netherlands)

W. Widagdo; L. Begeman (Lineke); D. Schipper (Debby); P.R.W.A. van Run (Peter); Cunningham, A.A. (Andrew A); Kley, N. (Nils); C.B.E.M. Reusken (Chantal); B.L. Haagmans (Bart); J.M.A. van den Brand (Judith)

2017-01-01

textabstractMiddle East respiratory syndrome coronavirus (MERS-CoV) has been shown to infect both humans and dromedary camels using dipeptidyl peptidase-4 (DPP4) as its receptor.The distribution of DPP4 in the respiratory tract tissues of humans and camels reflects MERS-CoV tropism.Apart from

Tissue Distribution of the MERS-Coronavirus Receptor in Bats

NARCIS (Netherlands)

Widagdo, W; Begeman, Lineke; Schipper, Debby; van Run, Peter R; Cunningham, Andrew A; Kley, Nils; Reusken, Chantal B E M; Haagmans, Bart L; van den Brand, Judith M A

2017-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) has been shown to infect both humans and dromedary camels using dipeptidyl peptidase-4 (DPP4) as its receptor. The distribution of DPP4 in the respiratory tract tissues of humans and camels reflects MERS-CoV tropism. Apart from dromedary

First full length sequences of the S gene of European isolates reveal further diversity among turkey coronaviruses.

OpenAIRE

2011-01-01

Abstract An increasing incidence of enteric disorders clinically evocative of the poult enteritis complex has been observed in turkeys in France since 2003. Using a newly designed real-time RT-PCR assay specific for the nucleocapsid (N) gene of infectious bronchitis virus (IBV) and turkey coronaviruses (TCoV), coronaviruses were identified in 37 % of the intestinal samples collected from diseased turkey flocks. The full length Spike (S) gene of these viruses was amplified, cloned a...

The cytoplasmic tails of infectious bronchitis virus E and M proteins mediate their interaction

International Nuclear Information System (INIS)

Corse, Emily; Machamer, Carolyn E.

2003-01-01

Virus-like particle (VLP) formation by the coronavirus E and M proteins suggests that interactions between these proteins play a critical role in coronavirus assembly. We studied interactions between the infectious bronchitis virus (IBV) E and M proteins using in vivo crosslinking and VLP assembly assays. We show that IBV E and M can be crosslinked to each other in IBV-infected and transfected cells, indicating that they interact. The cytoplasmic tails of both proteins are important for this interaction. We also examined the ability of the mutant and chimeric E and M proteins to form VLPs. IBV M proteins that are missing portions of their cytoplasmic tails or transmembrane regions were not able to support VLP formation, regardless of their ability to be crosslinked to IBV E. Interactions between the E and M proteins and the membrane bilayer are likely to play an important role in VLP formation and virus budding

Sero-prevalence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) specific antibodies in Dromedary Camels in Tabuk, Saudi Arabia.

Science.gov (United States)

Harrath, Rafik; Duhier, Faisel M Abu

2018-04-16

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a novel Coronavirus which was responsible of the first case of human acute respiratory syndrome in the Kingdom of Saudi Arabia (KSA), 2012. Dromedary camels are considered as potential reservoirs for the virus and seem to be the only animal host which may transmit the infection to human. Further studies are required to better understand the animal sources of zoonotic transmission route and the risks of this infection. A primary sero-prevalence study of MERS-CoV preexisting neutralizing antibodies in Dromedary camel serum was conducted in Tabuk, western north region of KSA, in order to assess the seopositivity of these animals and to explain their possible role in the transmission of the infection to Human. One hundred seventy one (171) serum samples were collected from healthy dromedary camels with different ages and genders in Tabuk city and tested for specific serum IgG by ELISA using the receptor-binding S1 subunits of spike proteins of MERS-CoV. 144 (84,21%) of the total camel sera shown the presence of protein-specific antibodies against MERS-CoV. These results may provide evidence that MERS-CoV has previously infected dromedary camels in Tabuk and may support the possible role of camels in the human infection. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

NARCIS (Netherlands)

Vergara-Alert, Júlia; van den Brand, Judith M A; Widagdo, W; Muñoz, Marta; Raj, V Stalin; Schipper, Debby; Solanes, David; Cordón, Ivan; Bensaid, Albert; Haagmans, Bart L; Segalés, Joaquim

Middle East respiratory syndrome (MERS) cases continue to be reported, predominantly in Saudi Arabia and occasionally other countries. Although dromedaries are the main reservoir, other animal species might be susceptible to MERS coronavirus (MERS-CoV) infection and potentially serve as reservoirs.

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).

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

Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA.

Science.gov (United States)

Fukushi, Shuetsu; Fukuma, Aiko; Kurosu, Takeshi; Watanabe, Shumpei; Shimojima, Masayuki; Shirato, Kazuya; Iwata-Yoshikawa, Naoko; Nagata, Noriyo; Ohnishi, Kazuo; Ato, Manabu; Melaku, Simenew Keskes; Sentsui, Hiroshi; Saijo, Masayuki

2018-01-01

Since discovering the Middle East respiratory syndrome coronavirus (MERS-CoV) as a causative agent of severe respiratory illness in the Middle East in 2012, serological testing has been conducted to assess antibody responses in patients and to investigate the zoonotic reservoir of the virus. Although the virus neutralization test is the gold standard assay for MERS diagnosis and for investigating the zoonotic reservoir, it uses live virus and so must be performed in high containment laboratories. Competitive ELISA (cELISA), in which a labeled monoclonal antibody (MAb) competes with test serum antibodies for target epitopes, may be a suitable alternative because it detects antibodies in a species-independent manner. In this study, novel MAbs against the spike protein of MERS-CoV were produced and characterized. One of these MAbs was used to develop a cELISA. The cELISA detected MERS-CoV-specific antibodies in sera from MERS-CoV-infected rats and rabbits immunized with the spike protein of MERS-CoV. The MAb-based cELISA was validated using sera from Ethiopian dromedary camels. Relative to the neutralization test, the cELISA detected MERS-CoV-specific antibodies in 66 Ethiopian dromedary camels with a sensitivity and specificity of 98% and 100%, respectively. The cELISA and neutralization test results correlated well (Pearson's correlation coefficients=0.71-0.76, depending on the cELISA serum dilution). This cELISA may be useful for MERS epidemiological investigations on MERS-CoV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Middle East respiratory syndrome coronavirus in dromedary camels: An outbreak investigation

NARCIS (Netherlands)

B.L. Haagmans (Bart); S.H.S. Al Dhahiry (Said); C.B.E.M. Reusken (Chantal); V.S. Raj (Stalin); M. Galiano (Monica); R.H. Myers (Richard); G-J. Godeke (Gert-Jan); M. Jonges (Marcel); E. Farag (Elmoubasher); A. Diab (Ayman); H. Ghobashy (Hazem); F. Alhajri (Farhoud); M. Al-Thani (Mohamed); S.A. Al-Marri (Salih); H.E. Al Romaihi (Hamad); A. Al Khal (Abdullatif); A. Bermingham (Alison); A.D.M.E. Osterhaus (Albert); M.M. AlHajri (Mohd); M.P.G. Koopmans D.V.M. (Marion)

2014-01-01

textabstractBackground: Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe lower respiratory tract infection in people. Previous studies suggested dromedary camels were a reservoir for this virus. We tested for the presence of MERS-CoV in dromedary camels from a farm in Qatar

Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences

Czech Academy of Sciences Publication Activity Database

Drexler, J. F.; Gloza-Rausch, F.; Glende, J.; Corman, V. M.; Muth, D.; Goettsche, M.; Seebens, A.; Niedrig, M.; Pfefferle, S.; Yordanov, S.; Zhelyazkov, L.; Hermanns, U.; Vallo, Peter; Lukashev, A.; Müller, M. A.; Deng, H.; Herrler, G.; Drosten, C.

2010-01-01

Roč. 84, č. 21 (2010), s. 11336-11349 ISSN 0022-538X Institutional research plan: CEZ:AV0Z60930519 Keywords : cross-species transmission * SARS-like coronavirus es * reservoir hosts * horseshoe bats Subject RIV: EE - Microbiology, Virology Impact factor: 5.189, year: 2010

MERS Coronavirus Neutralizing Antibodies in Camels, Eastern Africa, 1983-1997

NARCIS (Netherlands)

Müller, Marcel A; Corman, Victor Max; Jores, Joerg; Meyer, Benjamin; Younan, Mario; Liljander, Anne; Bosch, Berend-Jan; Lattwein, Erik; Hilali, Mosaad; Musa, Bakri E; Bornstein, Set; Drosten, Christian

2014-01-01

To analyze the distribution of Middle East respiratory syndrome coronavirus (MERS-CoV)-seropositive dromedary camels in eastern Africa, we tested 189 archived serum samples accumulated during the past 30 years. We identified MERS-CoV neutralizing antibodies in 81.0% of samples from the main

Amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis.

Science.gov (United States)

Porter, Emily; Tasker, Séverine; Day, Michael J; Harley, Ross; Kipar, Anja; Siddell, Stuart G; Helps, Christopher R

2014-04-25

Recent evidence suggests that a mutation in the spike protein gene of feline coronavirus (FCoV), which results in an amino acid change from methionine to leucine at position 1058, may be associated with feline infectious peritonitis (FIP). Tissue and faecal samples collected post mortem from cats diagnosed with or without FIP were subjected to RNA extraction and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to detect FCoV RNA. In cats with FIP, 95% of tissue, and 81% of faecal samples were PCR-positive, as opposed to 22% of tissue, and 60% of faecal samples in cats without FIP. Relative FCoV copy numbers were significantly higher in the cats with FIP, both in tissues (P < 0.001) and faeces (P = 0.02). PCR-positive samples underwent pyrosequencing encompassing position 1058 of the FCoV spike protein. This identified a methionine codon at position 1058, consistent with the shedding of an enteric form of FCoV, in 77% of the faecal samples from cats with FIP, and in 100% of the samples from cats without FIP. In contrast, 91% of the tissue samples from cats with FIP and 89% from cats without FIP had a leucine codon at position 1058, consistent with a systemic form of FCoV. These results suggest that the methionine to leucine substitution at position 1058 in the FCoV spike protein is indicative of systemic spread of FCoV from the intestine, rather than a virus with the potential to cause FIP.

Coronavirus in Pigs: Significance and Presentation of Swine Epidemic Diarrhea Virus (PEDV in Colombia

Directory of Open Access Journals (Sweden)

Ricardo Piñeros

2015-05-01

Full Text Available The article seeks to study general aspects of the main coronaviruses affecting pigs, their presentation in Colombia, and particular aspects of porcine epidemic diarrhea virus (PEDV, emerging in different countries and generating a great impact on the health and economy of the swine industry. The main coronaviruses affecting swine are porcine transmissible gastroenteritis virus (TGEV, porcine respiratory coronavirus (PRCV, porcine hemagglutinating encephalomyelitis virus (PHEV, PEDV, and porcine deltacoronavirus (PDCoV. Long ago in Colombia there had been reports of TGEV and PRCV associated with the importation of animals from the United States, which was controlled in the infected farms and in quarantine units. PEDV was first detected in Colombia in mid-March 2014; the Colombian Agricultural Institute issued a health alert in Neiva (Huila, Fusagasugá and Silvania (Cundinamarca, and Puerto López (Meta due to the unusual presentation of epidemic vomiting and diarrhea in young and adult animals, abortion in pregnant sows, with high mortality rates (up to 100% in animals during the first week of age. At present the disease has been reported in other municipalities of the country as well as in different countries with similar clinical conditions and mortality rates in pigs with high economic losses for the swine sector.

Immunogenicity of recombinant feline infectious peritonitis virus spike protein in mice and kittens

NARCIS (Netherlands)

Horzinek, M.C.; Vennema, H.; Groot, R. de; Harbour, D.A.; Dalderup, M.; Gruffydd-Jones, T.; Spaan, W.J.M.

1990-01-01

The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis (FIVP) was recombined into the genome of vaccinia virus, strain WR. The recombinant induced spike protein specific, in vitro neutralizing antibodies in mkice. When kittens were immunized with the

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.

Middle East Respiratory Syndrome (MERS) coronavirus seroprevalence in domestic livestock in Saudi Arabia, 2010 to 2013.

Science.gov (United States)

Hemida, M G; Perera, R A; Wang, P; Alhammadi, M A; Siu, L Y; Li, M; Poon, L L; Saif, L; Alnaeem, A; Peiris, M

2013-12-12

In Saudi Arabia, including regions of Riyadh and Al Ahsa, pseudoparticle neutralisation (ppNT) and microneutralisation (MNT) tests detected no antibodies to Middle East Respiratory Syndrome coronavirus (MERS-CoV) in sheep (n= 100), goats (n= 45), cattle (n= 50) and chickens (n= 240). Dromedary camels however, had a high prevalence of MERS-CoV antibodies. Bovine coronavirus (BCoV) infected sera from cattle had no cross-reactivity in MERS-CoV ppNT or MNT, while many dromedary camels’ sera reacted to both BCoV and MERS-CoV. Some nevertheless displayed specific serologic reaction profiles to MERS-CoV.

An eight-year epidemiologic study based on baculovirus-expressed type-specific spike proteins for the differentiation of type I and II feline coronavirus infections

Science.gov (United States)

2014-01-01

Background Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV). FCoVs are divided into two serotypes with markedly different infection rates among cat populations around the world. A baculovirus-expressed type-specific domain of the spike proteins of FCoV was used to survey the infection of the two viruses over the past eight years in Taiwan. Results An immunofluorescence assay based on cells infected with the recombinant viruses that was capable of distinguishing between the two types of viral infection was established. A total of 833 cases from a teaching hospital was surveyed for prevalence of different FCoV infections. Infection of the type I FCoV was dominant, with a seropositive rate of 70.4%, whereas 3.5% of cats were infected with the type II FCoV. In most cases, results derived from serotyping and genotyping were highly agreeable. However, 16.7% (4/24) FIP cats and 9.8% (6/61) clinically healthy cats were found to possess antibodies against both viruses. Moreover, most of the cats (84.6%, 22/26) infected with a genotypic untypable virus bearing a type I FCoV antibody. Conclusion A relatively simple serotyping method to distinguish between two types of FCoV infection was developed. Based on this method, two types of FCoV infection in Taiwan was first carried out. Type I FCoV was found to be predominant compared with type II virus. Results derived from serotyping and genotyping support our current understanding of evolution of disease-related FCoV and transmission of FIP. PMID:25123112

Structures of Two Coronavirus Main Proteases: Implications for Substrate Binding and Antiviral Drug Design

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Xue, Xiaoyu; Yu, Hongwei; Yang, Haitao; Xue, Fei; Wu, Zhixin; Shen, Wei; Li, Jun; Zhou, Zhe; Ding, Yi; Zhao, Qi; Zhang, Xuejun C.; Liao, Ming; Bartlam, Mark; Rao, Zihe (SCAU); (Tsinghua); (Chinese Aca. Sci.)

2008-07-21

Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) MP{sup pro} and a severe acute respiratory syndrome CoV (SARS-CoV) M{sup pro} mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of M{sup pro}. A monomeric form of IBV M{sup pro} was identified for the first time in CoV M{sup pro} structures. A comparison of these two structures to other available M{sup pro} structures provides new insights for the design of substrate-based inhibitors targeting CoV M{sup pro}s. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV M{sup pro} and was found to demonstrate in vitro inactivation of IBV M{sup pro} and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV M{sup pro}.

Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

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Ruili Ma

Full Text Available The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV-infected swine testicular (ST cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1, caspase-8, and heat shock protein 90 alpha (HSP90α were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

The role of accessory proteins in the replication of feline infectious peritonitis virus in peripheral blood monocytes.

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Dedeurwaerder, Annelike; Desmarets, Lowiese M; Olyslaegers, Dominique A J; Vermeulen, Ben L; Dewerchin, Hannah L; Nauwynck, Hans J

2013-03-23

The ability to productively infect monocytes/macrophages is the most important difference between the low virulent feline enteric coronavirus (FECV) and the lethal feline infectious peritonitis virus (FIPV). In vitro, the replication of FECV in peripheral blood monocytes always drops after 12h post inoculation, while FIPV sustains its replication in the monocytes from 45% of the cats. The accessory proteins of feline coronaviruses have been speculated to play a prominent role in virulence as deletions were found to be associated with attenuated viruses. Still, no functions have been ascribed to them. In order to investigate if the accessory proteins of FIPV are important for sustaining its replication in monocytes, replication kinetics were determined for FIPV 79-1146 and its deletion mutants, lacking either accessory protein open reading frame 3abc (FIPV-Δ3), 7ab (FIPV-Δ7) or both (FIPV-Δ3Δ7). Results showed that the deletion mutants FIPV-Δ7 and FIPV-Δ3Δ7 could not maintain their replication, which was in sharp contrast to wt-FIPV. FIPV-Δ3 could still sustain its replication, but the percentage of infected monocytes was always lower compared to wt-FIPV. In conclusion, this study showed that ORF7 is crucial for FIPV replication in monocytes/macrophages, giving an explanation for its importance in vivo, its role in the development of FIP and its conservation in field strains. The effect of an ORF3 deletion was less pronounced, indicating only a supportive role of ORF3 encoded proteins during the infection of the in vivo target cell by FIPVs. Copyright © 2012 Elsevier B.V. All rights reserved.

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

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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.

SARS-Coronavirus ancestor's foot-prints in South-East Asian bat colonies and the refuge theory.

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Gouilh, Meriadeg Ar; Puechmaille, Sébastien J; Gonzalez, Jean-Paul; Teeling, Emma; Kittayapong, Pattamaporn; Manuguerra, Jean-Claude

2011-10-01

One of the great challenges in the ecology of infectious diseases is to understand what drives the emergence of new pathogens including the relationship between viruses and their hosts. In the case of the emergence of SevereAcute Respiratory Syndrome Coronavirus (SARS-CoV), several studies have shown coronavirus diversity in bats as well as the existence of SARS-CoV infection in apparently healthy bats, suggesting that bats may be a crucial host in the genesis of this disease. To elucidate the biogeographic origin of SARS-CoV and investigate the role that bats played in its emergence, we amplified coronavirus sequences from bat species captured throughout Thailand and assessed the phylogenetic relationships to each other and to other published coronavirus sequences. To this end, RdRp sequence of Coronavirinae was targeted by RT-PCR in non-invasive samples from bats collected in Thailand. Two new coronaviruses were detected in two bat species: one Betacoronavirus in Hipposideros larvatus and one Alphacoronavirus in Hipposiderosarmiger. Interestingly, these viruses from South-East Asia are related to those previously detected in Africa (Betacoronavirus-b) or in Europe (Alphacoronavirus & Betacoronavirus-b). These findings illuminate the origin and the evolutionary history of the SARS-CoV group found in bats by pushing forward the hypothesis of a Betacoronavirus spill-over from Hipposideridae to Rhinolophidae and then from Rhinolophidae to civets and Human. All reported Betacoronaviruses-b (SARS-CoV group) of Hipposideridae and Rhinolophidae respectively cluster in two groups despite their broad geographic distribution and the sympatry of their hosts, which is in favor of an ancient and genetically independent evolution of Betacoronavirus-b clusters in these families. Moreover, despite its probable pathogenicity, we found that a Betacoronavirus-b can persistently infect a medium-sized hipposiderid bat colony. These findings illustrate the importance of the host

Competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions.

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Al-Mulla, Hawaa M N; Turrell, Lauren; Smith, Nicola M; Payne, Luke; Baliji, Surendranath; Züst, Roland; Thiel, Volker; Baker, Susan C; Siddell, Stuart G; Neuman, Benjamin W

2014-04-01

Positive-stranded viruses synthesize their RNA in membrane-bound organelles, but it is not clear how this benefits the virus or the host. For coronaviruses, these organelles take the form of double-membrane vesicles (DMVs) interconnected by a convoluted membrane network. We used electron microscopy to identify murine coronaviruses with mutations in nsp3 and nsp14 that replicated normally while producing only half the normal amount of DMVs under low-temperature growth conditions. Viruses with mutations in nsp5 and nsp16 produced small DMVs but also replicated normally. Quantitative reverse transcriptase PCR (RT-PCR) confirmed that the most strongly affected of these, the nsp3 mutant, produced more viral RNA than wild-type virus. Competitive growth assays were carried out in both continuous and primary cells to better understand the contribution of DMVs to viral fitness. Surprisingly, several viruses that produced fewer or smaller DMVs showed a higher fitness than wild-type virus at the reduced temperature, suggesting that larger and more numerous DMVs do not necessarily confer a competitive advantage in primary or continuous cell culture. For the first time, this directly demonstrates that replication and organelle formation may be, at least in part, studied separately during infection with positive-stranded RNA virus. IMPORTANCE The viruses that cause severe acute respiratory syndrome (SARS), poliomyelitis, and hepatitis C all replicate in double-membrane vesicles (DMVs). The big question about DMVs is why they exist in the first place. In this study, we looked at thousands of infected cells and identified two coronavirus mutants that made half as many organelles as normal and two others that made typical numbers but smaller organelles. Despite differences in DMV size and number, all four mutants replicated as efficiently as wild-type virus. To better understand the relative importance of replicative organelles, we carried out competitive fitness experiments. None

Proteomic analysis of chicken embryonic trachea and kidney tissues after infection in ovo by avian infectious bronchitis coronavirus

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Kong Xiangang

2011-03-01

Full Text Available Abstract Background Avian infectious bronchitis (IB is one of the most serious diseases of economic importance in chickens; it is caused by the avian infectious coronavirus (IBV. Information remains limited about the comparative protein expression profiles of chicken embryonic tissues in response to IBV infection in ovo. In this study, we analyzed the changes of protein expression in trachea and kidney tissues from chicken embryos, following IBV infection in ovo, using two-dimensional gel electrophoresis (2-DE coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS. Results 17 differentially expressed proteins from tracheal tissues and 19 differentially expressed proteins from kidney tissues were identified. These proteins mostly related to the cytoskeleton, binding of calcium ions, the stress response, anti-oxidative, and macromolecular metabolism. Some of these altered proteins were confirmed further at the mRNA level using real-time RT-PCR. Moreover, western blotting analysis further confirmed the changes of annexin A5 and HSPB1 during IBV infection. Conclusions To the best of our knowledge, we have performed the first analysis of the proteomic changes in chicken embryonic trachea and kidney tissues during IBV infection in ovo. The data obtained should facilitate a better understanding of the pathogenesis of IBV infection.

Presence of Middle East respiratory syndrome coronavirus antibodies in Saudi Arabia : a nationwide, cross-sectional, serological study

NARCIS (Netherlands)

Müller, Marcel A; Meyer, Benjamin; Corman, Victor M; Al-Masri, Malak; Turkestani, Abdulhafeez; Ritz, Daniel; Sieberg, Andrea; Aldabbagh, Souhaib; Bosch, Berend-J; Lattwein, Erik; Alhakeem, Raafat F; Assiri, Abdullah M; Albarrak, Ali M; Al-Shangiti, Ali M; Al-Tawfiq, Jaffar A; Wikramaratna, Paul; Alrabeeah, Abdullah A; Drosten, Christian; Memish, Ziad A

2015-01-01

BACKGROUND: Scientific evidence suggests that dromedary camels are the intermediary host for the Middle East respiratory syndrome coronavirus (MERS-CoV). However, the actual number of infections in people who have had contact with camels is unknown and most index patients cannot recall any such

Mechanism for Controlling the Dimer-Monomer Switch and Coupling Dimerization to Catalysis of the Severe Acute Respiratory Syndrome Coronavirus 3C-Like Protease

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Shi,J.; Sivaraman, J.; Song, J.

2008-01-01

Unlike 3C protease, the severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) is only enzymatically active as a homodimer and its catalysis is under extensive regulation by the unique extra domain. Despite intense studies, two puzzles still remain: (i) how the dimer-monomer switch is controlled and (ii) why dimerization is absolutely required for catalysis. Here we report the monomeric crystal structure of the SARS-CoV 3CLpro mutant R298A at a resolution of 1.75 Angstroms . Detailed analysis reveals that Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimer to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 310-helix from an active-site loop. Remarkably, dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis.

Human Infection with MERS coronavirus after exposure to infected camels, Saudi Arabia, 2013

NARCIS (Netherlands)

Memish, Ziad A.; Cotten, Matthew; Meyer, Benjamin; Watson, Simon J.; Alsahafi, Abdullah J.; Al Rabeeah, Abdullah A.; Corman, Victor Max; Sieberg, Andrea; Makhdoom, Hatem Q.; Assiri, Abdullah; Al Masri, Malaki; Aldabbagh, Souhaib; Bosch, Berend Jan|info:eu-repo/dai/nl/273306049; Beer, Martin; Müller, Marcel A.; Kellam, Paul; Drosten, Christian

2014-01-01

We investigated a case of human infection with Middle East respiratory syndrome coronavirus (MERS-CoV) after exposure to infected camels. Analysis of the whole human-derived virus and 15% of the camel-derived virus sequence yielded nucleotide polymorphism signatures suggestive of cross-species

Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

OpenAIRE

Morales, Lucia; Mateos-Gomez, Pedro A.; Capiscol, Carmen; del Palacio, Lorena; Enjuanes, Luis; Sola, Isabel

2013-01-01

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. Th...

Molecular tweezers modulate 14-3-3 protein-protein interactions

Science.gov (United States)

Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

2013-03-01

Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

Antibodies against MERS coronavirus in dromedaries, United Arab Emirates, 2003 and 2013

NARCIS (Netherlands)

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

2014-01-01

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

The Middle East respiratory syndrome coronavirus (MERS-CoV does not replicate in Syrian hamsters.

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Emmie de Wit

Full Text Available In 2012 a novel coronavirus, MERS-CoV, associated with severe respiratory disease emerged in the Arabian Peninsula. To date, 55 human cases have been reported, including 31 fatal cases. Several of the cases were likely a result of human-to-human transmission. The emergence of this novel coronavirus prompts the need for a small animal model to study the pathogenesis of this virus and to test the efficacy of potential intervention strategies. In this study we explored the use of Syrian hamsters as a small animal disease model, using intratracheal inoculation and inoculation via aerosol. Clinical signs of disease, virus replication, histological lesions, cytokine upregulation nor seroconversion were observed in any of the inoculated animals, indicating that MERS-CoV does not replicate in Syrian hamsters.

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.

Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus

NARCIS (Netherlands)

Kusters, J G; Jager, E J; Niesters, H G; van der Zeijst, B A

1990-01-01

Under laboratory conditions coronaviruses were shown to have a high frequency of recombination. In The Netherlands, vaccination against infectious bronchitis virus (IBV) is performed with vaccines that contain several life-attenuated virus strains. These highly effective vaccines may create ideal

Middle East Respiratory Syndrome Coronavirus Antibodies in Dromedary Camels, Bangladesh, 2015

Science.gov (United States)

Islam, Ariful; Rostal, Melinda K.; Islam, Shariful; Rahman, Mohammed Ziaur; Hossain, Mohammed Enayet; Uzzaman, Mohammed Salim; Munster, Vincent J.; Peiris, Malik; Flora, Meerjady Sabrina; Rahman, Mahmudur; Daszak, Peter

2018-01-01

Dromedary camels are bred domestically and imported into Bangladesh. In 2015, of 55 camels tested for Middle East respiratory syndrome coronavirus in Dhaka, 17 (31%) were seropositive, including 1 bred locally. None were PCR positive. The potential for infected camels in urban markets could have public health implications and warrants further investigation. PMID:29664373

Detection of potentially novel paramyxovirus and coronavirus viral RNA in bats and rats in the Mekong Delta region of southern Viet Nam.

Science.gov (United States)

Berto, A; Anh, P H; Carrique-Mas, J J; Simmonds, P; Van Cuong, N; Tue, N T; Van Dung, N; Woolhouse, M E; Smith, I; Marsh, G A; Bryant, J E; Thwaites, G E; Baker, S; Rabaa, M A

2018-02-01

Bats and rodents are being increasingly recognized as reservoirs of emerging zoonotic viruses. Various studies have investigated bat viruses in tropical regions, but to date there are no data regarding viruses with zoonotic potential that circulate in bat and rat populations in Viet Nam. To address this paucity of data, we sampled three bat farms and three wet markets trading in rat meat in the Mekong Delta region of southern Viet Nam. Faecal and urine samples were screened for the presence of RNA from paramyxoviruses, coronaviruses and filoviruses. Paramyxovirus RNA was detected in 4 of 248 (1%) and 11 of 222 (4.9%) bat faecal and urine samples, respectively. Coronavirus RNA was detected in 55 of 248 (22%) of bat faecal samples; filovirus RNA was not detected in any of the bat samples. Further, coronavirus RNA was detected in 12 of 270 (4.4%) of rat faecal samples; all samples tested negative for paramyxovirus. Phylogenetic analysis revealed that the bat paramyxoviruses and bat and rat coronaviruses were related to viruses circulating in bat and rodent populations globally, but showed no cross-species mixing of viruses between bat and rat populations within Viet Nam. Our study shows that potentially novel variants of paramyxoviruses and coronaviruses commonly circulate in bat and rat populations in Viet Nam. Further characterization of the viruses and additional human and animal surveillance is required to evaluate the likelihood of viral spillover and to assess whether these viruses pose a risk to human health. © 2017 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.

Down-regulation of transcription of the proapoptotic gene BNip3 in cultured astrocytes by murine coronavirus infection

International Nuclear Information System (INIS)

Cai Yingyun; Liu Yin; Yu Dongdong; Zhang Xuming

2003-01-01

Murine coronavirus mouse hepatitis virus (MHV) causes encephalitis and demyelination in the central nervous system of susceptible rodents. Astrocytes are the major target for MHV persistence. However, the mechanisms by which astrocytes survive MHV infection and permit viral persistence are not known. Here we performed DNA microarray analysis on differential gene expression in astrocyte DBT cells by MHV infection and found that the mRNA of the proapoptotic gene BNip3 was significantly decreased following MHV infection. This finding was further confirmed by quantitative reverse transcription-polymerase chain reaction, Western blot analysis, and BNip3-promoter-luciferase reporter system. Interestingly, infection with live and ultraviolet light-inactivated viruses equally repressed BNip3 expression, indicating that the down-regulation of BNip3 expression does not require virus replication and is mediated during cell entry. Furthermore, treatment of cells with chloroquine, which blocks the acidification of endosomes, significantly inhibited the repression of the BNip3 promoter activity induced by the acidic pH-dependent MHV mutant OBLV60, which enters cells via endocytosis, indicating that the down-regulation of BNip3 expression is mediated by fusion between viral envelope and cell membranes during entry. Deletion analysis showed that the sequence between nucleotides 262 and 550 of the 588-base-pair BNip3 promoter is necessary and sufficient for driving the BNip3 expression and that it contains signals that are responsible for MHV-induced down-regulation of BNip3 expression in DBT cells. These results may provide insights into the mechanisms by which MHV evades host antiviral defense and promotes cell survival, thereby allowing its persistence in the host astrocytes

Association of seropositivity for influenza and coronaviruses with history of mood disorders and suicide attempts.

Science.gov (United States)

Okusaga, Olaoluwa; Yolken, Robert H; Langenberg, Patricia; Lapidus, Manana; Arling, Timothy A; Dickerson, Faith B; Scrandis, Debra A; Severance, Emily; Cabassa, Johanna A; Balis, Theodora; Postolache, Teodor T

2011-04-01

Anecdotal reports of mood disorder following infection with common respiratory viruses with neurotropic potential have been in existence since the last century. Nevertheless, systematic studies on the association between these viruses and mood disorders are lacking. Influenza A, B and coronavirus antibody titers were measured in 257 subjects with recurrent unipolar and bipolar disorder and healthy controls, by SCID. Pearson's χ² tests and logistic regression models were used to analyze associations between seropositivity for coronaviruses, influenza A and B viruses and the following: a) history of recurrent mood disorders b) having attempted suicide in the past c) uni- vs. bi-polarity and d) presence of psychotic symptoms during mood episodes. Seropositivity for influenza A (p=0.004), B (pmood disorders but not with the specific diagnosis of unipolar or bipolar depression. Seropositivity for influenza B was significantly associated with a history of suicide attempt (p=0.001) and history of psychotic symptoms (p=0.005). The design was cross-sectional. Socioeconomic factors, inflammatory markers, and axis II psychopathology were not assessed. The association of seropositivity for influenza and coronaviruses with a history of mood disorders, and influenza B with suicidal behavior require replication in larger longitudinal samples. The need for these studies is additionally supported by the high incidence of these viral infections, the high prevalence of mood disorders, and resilience of suicide epidemics. Copyright © 2010 Elsevier B.V. All rights reserved.

Genomic Analysis of 15 Human Coronaviruses OC43 (HCoV-OC43s Circulating in France from 2001 to 2013 Reveals a High Intra-Specific Diversity with New Recombinant Genotypes

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Nathalie Kin

2015-05-01

Full Text Available Human coronavirus OC43 (HCoV-OC43 is one of five currently circulating human coronaviruses responsible for respiratory infections. Like all coronaviruses, it is characterized by its genome’s high plasticity. The objectives of the current study were to detect genetically distinct genotypes and eventually recombinant genotypes in samples collected in Lower Normandy between 2001 and 2013. To this end, we sequenced complete nsp12, S, and N genes of 15 molecular isolates of HCoV-OC43 from clinical samples and compared them to available data from the USA, Belgium, and Hong-Kong. A new cluster E was invariably detected from nsp12, S, and N data while the analysis of nsp12 and N genes revealed the existence of new F and G clusters respectively. The association of these different clusters of genes in our specimens led to the description of thirteen genetically distinct genotypes, among which eight recombinant viruses were discovered. Identification of these recombinant viruses, together with temporal analysis and tMRCA estimation, provides important information for understanding the dynamics of the evolution of these epidemic coronaviruses.

Genome-wide analysis of codon usage bias in Bovine Coronavirus

OpenAIRE

Castells, Mat?as; Victoria, Mat?as; Colina, Rodney; Musto, H?ctor; Cristina, Juan

2017-01-01

Background Bovine coronavirus (BCoV) belong to the genus Betacoronavirus of the family Coronaviridae. BCoV are widespread around the world and cause enteric or respiratory infections among cattle, leading to important economic losses to the beef and dairy industry worldwide. To study the relation of codon usage among viruses and their hosts is essential to understand host-pathogen interaction, evasion from host?s immune system and evolution. Methods We performed a comprehensive analysis of co...

Coronavirus and influenza virus proteolytic priming takes place in tetraspanin-enriched membrane microdomains.

Science.gov (United States)

Earnest, James T; Hantak, Michael P; Park, Jung-Eun; Gallagher, Tom

2015-06-01

Coronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases. Enveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

International Nuclear Information System (INIS)

Collomb, J.; Finance, C.; Alabouch, S.; Laporte, J.

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32 P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors)

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

Energy Technology Data Exchange (ETDEWEB)

Collomb, J; Finance, C; Alabouch, S [Lab. de Microbiologie Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques, Univ. de Nancy I, Nancy (France); Laporte, J [Station de Virologie et d' Immunologie Moleculaires, INRA, Jouy-en-Josas (France)

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).

Evidence for an Ancestral Association of Human Coronavirus 229E with Bats

NARCIS (Netherlands)

Corman, Victor Max; Baldwin, Heather J.; Tateno, Adriana Fumie; Zerbinati, Rodrigo Melim; Annan, Augustina; Owusu, Michael; Nkrumah, Evans Ewald; Maganga, Gael Darren; Oppong, Samuel; Adu-Sarkodie, Yaw; Vallo, Peter; da Silva Filho, Luiz Vicente Ribeiro Ferreira; Leroy, Eric M.; Thiel, Volker; van der Hoek, Lia; Poon, Leo L. M.; Tschapka, Marco; Drosten, Christian; Drexler, Jan Felix

2015-01-01

We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related

Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture.

OpenAIRE

Harrison , Sally; Tarpey , Ian; Rothwell , Lisa; Kasier , Pete; Hiscox , Julian

2007-01-01

Abstract The avian coronavirus infectious bronchitis virus (IBV) is a major economic pathogen of domestic poultry which, despite vaccination, causes mortality and significant losses in production. During replication of the RNA genome there is a high frequency of mutation and recombination which has given rise to many strains of IBV and results in the potential for new and emerging strains. Currently the live-attenuated vaccine gives poor cross strain immunity. Effective antivira...

The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus

Directory of Open Access Journals (Sweden)

Enjuanes Luis

2011-09-01

Full Text Available Abstract Background Transmissible gastroenteritis virus (TGEV has a sialic acid binding activity that is believed to be important for enteropathogenicity, but that has so far appeared to be dispensable for infection of cultured cells. The aims of this study were to determine the effect of sialic acid binding for the infection of cultured cells under unfavorable conditions, and comparison of TGEV strains and mutants, as well as the avian coronavirus IBV concerning their dependence on the sialic acid binding activity. Methods The infectivity of different viruses was analyzed by a plaque assay after adsorption times of 5, 20, and 60 min. Prior to infection, cultured cells were either treated with neuraminidase to deplete sialic acids from the cell surface, or mock-treated. In a second approach, pre-treatment of the virus with porcine intestinal mucin was performed, followed by the plaque assay after a 5 min adsorption time. A student's t-test was used to verify the significance of the results. Results Desialylation of cells only had a minor effect on the infection by TGEV strain Purdue 46 when an adsorption period of 60 min was allowed for initiation of infection. However, when the adsorption time was reduced to 5 min the infectivity on desialylated cells decreased by more than 60%. A TGEV PUR46 mutant (HAD3 deficient in sialic acid binding showed a 77% lower titer than the parental virus after a 5 min adsorption time. After an adsorption time of 60 min the titer of HAD3 was 58% lower than that of TGEV PUR46. Another TGEV strain, TGEV Miller, and IBV Beaudette showed a reduction in infectivity after neuraminidase treatment of the cultured cells irrespective of the virion adsorption time. Conclusions Our results suggest that the sialic acid binding activity facilitates the infection by TGEV under unfavorable environmental conditions. The dependence on the sialic acid binding activity for an efficient infection differs in the analyzed TGEV strains.

Immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling.

Science.gov (United States)

Zhang, Qingzhan; Yoo, Dongwan

2016-12-02

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are emerged and reemerging viruses in pigs, and together with transmissible gastroenteritis virus (TGEV), pose significant economic concerns to the swine industry. These viruses infect epithelial cells of the small intestine and cause watery diarrhea, dehydration, and a high mortality in neonatal piglets. Type I interferons (IFN-α/β) are major antiviral cytokines forming host innate immunity, and in turn, these enteric coronaviruses have evolved to modulate the host innate immune signaling during infection. Accumulating evidence however suggests that IFN induction and signaling in the intestinal epithelial cells differ from other epithelial cells, largely due to distinct features of the gut epithelial mucosal surface and commensal microflora, and it appears that type III interferon (IFN-λ) plays a key role to maintain the antiviral state in the gut. This review describes the recent understanding on the immune evasion strategies of porcine enteric coronaviruses and the role of different types of IFNs for intestinal antiviral innate immunity. Copyright © 2016 Elsevier B.V. All rights reserved.

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. PMID:20663472

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

Directory of Open Access Journals (Sweden)

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.

Cytoplasmic tail of Coronavirus spike protein has intracellular ...

Indian Academy of Sciences (India)

58

1Department of Biological Sciences, Indian Institute of Science Education and ... Introduction .... A major effort is underway to understand the interplay of .... For protein analysis, cells were plated on 100 mm tissue culture dishes and transfected.

Clinical, hematological, and biochemical findings in puppies with coronavirus and parvovirus enteritis

Science.gov (United States)

Castro, Tatiana X.; Cubel Garcia, Rita de Cássia N.; Gonçalves, Luciana P. S.; Costa, Erika M.; Marcello, Gracy C.G.; Labarthe, Norma V.; Mendes-de-Almeida, Flavya

2013-01-01

The clinical and laboratory findings in puppies naturally infected with canine coronavirus (CCoV) and/or canine parvovirus (CPV) were compared with findings in uninfected puppies. Lymphopenia was the only parameter related to CCoV infection that was statistically significant; vomiting, anorexia, lethargy, hemorrhagic fluid diarrhea, leukopenia, lymphopenia, thrombocytopenia, hypoglycemia, and hypoproteinemia were correlated with CPV infection. PMID:24155496

Novel coronavirus and astrovirus in Delaware Bay shorebirds.

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Kirsi S Honkavuori

Full Text Available Wild birds are an important but to some extent under-studied reservoir for emerging pathogens. We used unbiased sequencing methods for virus discovery in shorebird samples from the Delaware Bay, USA; an important feeding ground for thousands of migratory birds.Analysis of shorebird fecal samples indicated the presence of a novel astrovirus and coronavirus. A sanderling sample yielded sequences with distant homology to avian nephritis virus 1, an astrovirus associated with acute nephritis in poultry. A ruddy turnstone sample yielded sequences with homology to deltacoronaviruses.Our findings highlight shorebirds as a virus reservoir and the need to closely monitor wild bird populations for the emergence of novel virus variants.

Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal

OpenAIRE

Narayanan, Krishna; Chen, Chun-Jen; Maeda, Junko; Makino, Shinji

2003-01-01

For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into vi...

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...

Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections.

Science.gov (United States)

Xiong, Xiaoli; Tortorici, M Alejandra; Snijder, Joost; Yoshioka, Craig; Walls, Alexandra C; Li, Wentao; McGuire, Andrew T; Rey, Félix A; Bosch, Berend-Jan; Veesler, David

2017-11-01

Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle-East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5 Å resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified delta genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved when compared with the human respiratory coronavirus HCoV-NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S 2 ' activation loop, containing a reduced number of basic amino acids, which participates to rendering the spike largely protease-resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity. IMPORTANCE Coronaviruses use transmembrane spike (S) glycoprotein trimers to promote host attachment and fusion of the viral and cellular membranes. We determined a near-atomic resolution cryo-electron microscopy structure of the S ectodomain trimer from the pathogenic porcine deltacoronavirus (PDCoV), which is responsible for diarrhea in piglets and has had devastating consequences for the swine industry worldwide. Structural and glycoproteomics data reveal that PDCoV S is

Novel coronaviruses, astroviruses, adenoviruses and circoviruses in insectivorous bats from northern China.

Science.gov (United States)

Han, H-J; Wen, H-L; Zhao, L; Liu, J-W; Luo, L-M; Zhou, C-M; Qin, X-R; Zhu, Y-L; Liu, M-M; Qi, R; Li, W-Q; Yu, H; Yu, X-J

2017-12-01

Bats are considered as the reservoirs of several emerging infectious disease, and novel viruses are continually found in bats all around the world. Studies conducted in southern China found that bats carried a variety of viruses. However, few studies have been conducted on bats in northern China, which harbours a diversity of endemic insectivorous bats. It is important to understand the prevalence and diversity of viruses circulating in bats in northern China. In this study, a total of 145 insectivorous bats representing six species were collected from northern China and screened with degenerate primers for viruses belonging to six families, including coronaviruses, astroviruses, hantaviruses, paramyxoviruses, adenoviruses and circoviruses. Our study found that four of the viruses screened for were positive and the overall detection rates for astroviruses, coronaviruses, adenoviruses and circoviruses in bats were 21.4%, 15.9%, 20% and 37.2%, respectively. In addition, we found that bats in northern China harboured a diversity of novel viruses. Common Serotine (Eptesicus serotinu), Fringed long-footed Myotis (Myotis fimriatus) and Peking Myotis (Myotis pequinius) were investigated in China for the first time. Our study provided new information on the ecology and phylogeny of bat-borne viruses. © 2017 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.

Swine enteric coronavirus disease: A review of 4 years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the United States and Canada.

Science.gov (United States)

Niederwerder, M C; Hesse, R A

2018-06-01

Swine enteric coronaviruses, including porcine epidemic diarrhoea virus (PEDV) and porcine deltacoronavirus (PDCoV), have emerged and spread throughout the North American swine industry over the last four years. These diseases cause significant losses within the pork industry and within the first year after PEDV introduction, approximately 10% of the US herd died due to the disease. Similar to other enteric coronaviruses, such as transmissible gastroenteritis virus (TGEV), these emerging swine enteric coronavirus diseases (SECD) are age-dependent, with high morbidity and mortality in neonatal pigs. Since the introduction of SECD, research has focused on investigating viral pathogenesis through experimental inoculation, increasing maternal antibody for neonatal protection, understanding transmission risks through feed and transportation, and outlining the importance of biosecurity in preventing SECD introduction and spread. A survey of swine professionals conducted for this review revealed that the majority of respondents (75%) believe SECD can be eradicated and that most herds have been successful at long-term elimination of SECD after exposure (80%). However, unique properties of SECD, such as ineffective immunity through parenteral vaccination and a low oral infectious dose, play a major role in management of SECD. This review serves to describe the current knowledge of SECD and the characteristics of these viruses which provide both opportunities and challenges for long-term disease control and potential eradication from the US swine population. © 2018 Blackwell Verlag GmbH.

Molecular dynamics of Middle East Respiratory Syndrome Coronavirus (MERS CoV) fusion heptad repeat trimers

KAUST Repository

Kandeel, Mahmoud; Al-Taher, Abdulla; Li, Huifang; Schwingenschlö gl, Udo; Alnazawi, Mohamed

2018-01-01

Structural studies related to Middle East Respiratory Syndrome Coronavirus (MERS CoV) infection process are so limited. In this study, molecular dynamics (MD) simulation was carried out to unravel changes in the MERS CoV heptad repeat domains (HRs

Knowledge, Attitudes and Behaviours of Healthcare Workers in the Kingdom of Saudi Arabia to MERS Coronavirus and Other Emerging Infectious Diseases

Directory of Open Access Journals (Sweden)

Abdullah J. Alsahafi

2016-12-01

Full Text Available Background: The Kingdom of Saudi Arabia has experienced a prolonged outbreak of Middle East Respiratory Syndrome (MERS coronavirus since 2012. Healthcare workers (HCWs form a significant risk group for infection. Objectives: The aim of this survey was to assess the knowledge, attitudes, infection control practices and educational needs of HCWs in the Kingdom of Saudi Arabia to MERS coronavirus and other emerging infectious diseases. Methods: 1500 of HCWs from Saudi Ministry of Health were invited to fill a questionnaire developed to cover the survey objectives from 9 September 2015 to 8 November 2015. The response rate was about 81%. Descriptive statistics was used to summarise the responses. Results: 1216 HCWs were included in this survey. A total of 56.5% were nurses and 22% were physicians. The most common sources of MERS-coronavirus (MERS-CoV information were the Ministry of Health (MOH memo (74.3%. Only (47.6% of the physicians, (30.4% of the nurses and (29.9% of the other HCWs were aware that asymptomatic MERS-CoV was described. Around half of respondents who having been investigated for MERS-CoV reported that their work performance decreased while they have suspicion of having MERS-CoV and almost two thirds reported having psychological problems during this period. Almost two thirds of the HCWs (61.2% reported anxiety about contracting MERS-CoV from patients. Conclusions: The knowledge about emerging infectious diseases was poor and there is need for further education and training programs particularly in the use of personal protective equipment, isolation and infection control measures. The self-reported infection control practices were sub-optimal and seem to be overestimated.

The first complete genome sequences of clinical isolates of human coronavirus 229E

NARCIS (Netherlands)

Farsani, Seyed Mohammad Jazaeri; Dijkman, Ronald; Jebbink, Maarten F.; Goossens, Herman; Ieven, Margareta; Deijs, Martin; Molenkamp, Richard; van der Hoek, Lia

2012-01-01

Human coronavirus 229E has been identified in the mid-1960s, yet still only one full-genome sequence is available. This full-length sequence has been determined from the cDNA-clone Inf-1 that is based on the lab-adapted strain VR-740. Lab-adaptation might have resulted in genomic changes, due to

Altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus.

Science.gov (United States)

Jung, Kwonil; Alekseev, Konstantin P; Zhang, Xinsheng; Cheon, Doo-Sung; Vlasova, Anastasia N; Saif, Linda J

2007-12-01

The pathogenesis and optimal treatments for severe acute respiratory syndrome (SARS) are unclear, although corticosteroids were used to reduce lung and systemic inflammation. Because the pulmonary pathology of porcine respiratory coronavirus (PRCV) in pigs resembles SARS, we used PRCV as a model to clarify the effects of the corticosteroid dexamethasone (DEX) on coronavirus (CoV)-induced pneumonia. Conventional weaned pigs (n = 130) in one of four groups (PRCV/phosphate-buffered saline [PBS] [n = 41], PRCV/DEX [n = 41], mock/PBS [n = 23], and mock/DEX [n = 25]) were inoculated intranasally and intratracheally with the ISU-1 strain of PRCV (1 x 10(7) PFU) or cell culture medium. DEX was administered (once daily, 2 mg/kg of body weight/day, intramuscularly) from postinoculation day (PID) 1 to 6. In PRCV/DEX pigs, significantly milder pneumonia, fewer PRCV-positive cells, and lower viral RNA titers were present in lungs early at PID 2; however, at PID 4, 10, and 21, severe bronchointerstitial pneumonia, significantly higher numbers of PRCV-positive cells, and higher viral RNA titers were observed compared to results for PRCV/PBS pigs. Significantly lower numbers of CD2(+), CD3(+), CD4(+), and CD8(+) T cells were also observed in lungs of PRCV/DEX pigs than in those of PRCV/PBS pigs at PID 8 and 10, coincident with fewer gamma interferon (IFN-gamma)-secreting cells in the tracheobronchial lymph nodes as determined by enzyme-linked immunospot assay. Our results confirm that DEX treatment alleviates PRCV pneumonia early (PID 2) in the infection but continued use through PID 6 exacerbates later stages of infection (PID 4, 10, and 21), possibly by decreasing cellular immune responses in the lungs (IFN-gamma-secreting T cells), thereby creating an environment for more-extensive viral replication. These data have potential implications for corticosteroid use with SARS-CoV patients and suggest a precaution against prolonged use based on their unproven efficacy in humans

Survey of feline leukemia virus and feline coronaviruses in captive neotropical wild felids from Southern Brazil.

Science.gov (United States)

Guimaraes, Ana M S; Brandão, Paulo E; de Moraes, Wanderlei; Cubas, Zalmir S; Santos, Leonilda C; Villarreal, Laura Y B; Robes, Rogério R; Coelho, Fabiana M; Resende, Mauricio; Santos, Renata C F; Oliveira, Rosangela C; Yamaguti, Mauricio; Marques, Lucas M; Neto, Renata L; Buzinhani, Melissa; Marques, Regina; Messick, Joanne B; Biondo, Alexander W; Timenetsky, Jorge

2009-06-01

A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refúgio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.

Seroprevalence and risk factors for infection with equine coronavirus in healthy horses in the USA

NARCIS (Netherlands)

Kooijman, L.J.; James, K.; Mapes, S.M.; Theelen, M.J.P.; Pusterla, N.

2017-01-01

Equine coronavirus (ECoV) is considered an enteric pathogen of foals and has only recently been associated with infections in adult horses. Seroprevalence data is needed to better understand the epidemiology of ECoV in adult horses, evaluate diagnostic modalities and develop preventive measures. The

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

DEFF Research Database (Denmark)

Knudsen, T B; Kledal, T N; Andersen, O

2003-01-01

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...... 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...

A case of imported Middle East Respiratory Syndrome coronavirus infection and public health response, Greece, April 2014.

Science.gov (United States)

Tsiodras, S; Baka, A; Mentis, A; Iliopoulos, D; Dedoukou, X; Papamavrou, G; Karadima, S; Emmanouil, M; Kossyvakis, A; Spanakis, N; Pavli, A; Maltezou, H; Karageorgou, A; Spala, G; Pitiriga, V; Kosmas, E; Tsiagklis, S; Gkatzias, S; Koulouris, Ng; Koutsoukou, A; Bakakos, P; Markozanhs, E; Dionellis, G; Pontikis, K; Rovina, N; Kyriakopoulou, M; Efstathiou, P; Papadimitriou, T; Kremastinou, J; Tsakris, A; Saroglou, G

2014-04-24

On 18 April 2014, a case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) infection was laboratory confirmed in Athens, Greece in a patient returning from Jeddah, Saudi Arabia. Main symptoms upon initial presentation were protracted fever and diarrhoea, during hospitalisation he developed bilateral pneumonia and his condition worsened. During 14 days prior to onset of illness, he had extensive contact with the healthcare environment in Jeddah. Contact tracing revealed 73 contacts, no secondary cases had occurred by 22 April.

Structural Analysis of Major Species Barriers between Humans and Palm Civets for Severe Acute Respiratory Syndrome Coronavirus Infections

Energy Technology Data Exchange (ETDEWEB)

Li, Fang (UMM)

2008-09-23

It is believed that a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), was passed from palm civets to humans and caused the epidemic of SARS in 2002 to 2003. The major species barriers between humans and civets for SARS-CoV infections are the specific interactions between a defined receptor-binding domain (RBD) on a viral spike protein and its host receptor, angiotensin-converting enzyme 2 (ACE2). In this study a chimeric ACE2 bearing the critical N-terminal helix from civet and the remaining peptidase domain from human was constructed, and it was shown that this construct has the same receptor activity as civet ACE2. In addition, crystal structures of the chimeric ACE2 complexed with RBDs from various human and civet SARS-CoV strains were determined. These structures, combined with a previously determined structure of human ACE2 complexed with the RBD from a human SARS-CoV strain, have revealed a structural basis for understanding the major species barriers between humans and civets for SARS-CoV infections. They show that the major species barriers are determined by interactions between four ACE2 residues (residues 31, 35, 38, and 353) and two RBD residues (residues 479 and 487), that early civet SARS-CoV isolates were prevented from infecting human cells due to imbalanced salt bridges at the hydrophobic virus/receptor interface, and that SARS-CoV has evolved to gain sustained infectivity for human cells by eliminating unfavorable free charges at the interface through stepwise mutations at positions 479 and 487. These results enhance our understanding of host adaptations and cross-species infections of SARS-CoV and other emerging animal viruses.

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.

Altered Pathogenesis of Porcine Respiratory Coronavirus in Pigs due to Immunosuppressive Effects of Dexamethasone: Implications for Corticosteroid Use in Treatment of Severe Acute Respiratory Syndrome Coronavirus▿

OpenAIRE

Jung, Kwonil; Alekseev, Konstantin P.; Zhang, Xinsheng; Cheon, Doo-Sung; Vlasova, Anastasia N.; Saif, Linda J.

2007-01-01

The pathogenesis and optimal treatments for severe acute respiratory syndrome (SARS) are unclear, although corticosteroids were used to reduce lung and systemic inflammation. Because the pulmonary pathology of porcine respiratory coronavirus (PRCV) in pigs resembles SARS, we used PRCV as a model to clarify the effects of the corticosteroid dexamethasone (DEX) on coronavirus (CoV)-induced pneumonia. Conventional weaned pigs (n = 130) in one of four groups (PRCV/phosphate-buffered saline [PBS] ...

Selection of SARS-Coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice

International Nuclear Information System (INIS)

Yu Hua; Jiang Lifang; Fang Danyun; Yan Huijun; Zhou Jingjiao; Zhou Junmei; Liang Yu; Gao Yang; Zhao, Wei; Long Beiguo

2007-01-01

Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed

Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses

Science.gov (United States)

2013-01-01

Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen- and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79–1683 and WSU 79–1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79–1683 still replicated significantly more efficient compared to FCoV WSU 79–1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats. PMID:23964891

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

Directory of Open Access Journals (Sweden)

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.

Limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis.

Science.gov (United States)

Barker, Emily N; Stranieri, Angelica; Helps, Chris R; Porter, Emily L; Davidson, Andrew D; Day, Michael J; Knowles, Toby; Kipar, Anja; Tasker, Séverine

2017-10-05

Feline infectious peritonitis (FIP) is a fatal disease of cats, and a sequela of systemic feline coronavirus (FCoV) infection. Mutations in the viral spike (S) gene have been associated with FCoVs found in tissues from cats with FIP, but not FCoVs found in faeces from healthy cats, and are implicated in monocyte/macrophage tropism and systemic spread. This study was designed to determine whether S gene mutation analysis can reliably diagnose FIP. Cats were categorised as with FIP (n = 57) or without FIP (n = 45) based on gross post-mortem and histopathological examination including immunohistochemistry for FCoV antigen. RNA was purified from available tissue, fluid and faeces. Reverse-transcriptase quantitative-PCR (RT-qPCR) was performed on all samples using FCoV-specific primers, followed by sequencing of a section of the S gene on RT-qPCR positive samples. Samples were available from a total of 102 cats. Tissue, fluid, and faecal samples from cats with FIP were more likely to be FCoV RT-qPCR-positive (90.4, 78.4 and 64.6% respectively) than those from cats without FIP (7.8, 2.1 and 20% respectively). Identification of S gene mutated FCoVs as an additional step to the detection of FCoV alone, only moderately increased specificity for tissue samples (from 92.6 to 94.6%) but specificity was unchanged for fluid samples (97.9%) for FIP diagnosis; however, sensitivity was markedly decreased for tissue (from 89.8 to 80.9%) and fluid samples (from 78.4 to 60%) for FIP diagnosis. These findings demonstrate that S gene mutation analysis in FCoVs does not substantially improve the ability to diagnose FIP as compared to detection of FCoV alone.

A phylogenetically distinct Middle East respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in Dubai with rising seroprevalence with age.

Science.gov (United States)

Wernery, Ulrich; El Rasoul, I Hassab; Wong, Emily Y M; Joseph, Marina; Chen, Yixin; Jose, Shanty; Tsang, Alan K L; Patteril, Nissy Annie Georgy; Chen, Honglin; Elizabeth, Shyna K; Yuen, Kwok-Yung; Joseph, Sunitha; Xia, Ningshao; Wernery, Renate; Lau, Susanna K P; Woo, Patrick C Y

2015-12-02

Middle East respiratory syndrome coronavirus (MERS-CoV) was detected by monoclonal antibody-based nucleocapsid protein-capture enzyme-linked immunosorbent assay (ELISA), RNA detection, and viral culture from the nasal sample of a 1-month-old dromedary calf in Dubai with sudden death. Whole genome phylogeny showed that this MERS-CoV strain did not cluster with the other MERS-CoV strains from Dubai that we reported recently. Instead, it formed a unique branch more closely related to other MERS-CoV strains from patients in Qatar and Hafr-Al-Batin in Saudi Arabia, as well as the MERS-CoV strains from patients in the recent Korean outbreak, in which the index patient acquired the infection during travel in the eastern part of the Arabian Peninsula. Non-synonymous mutations, resulting in 11 unique amino acid differences, were observed between the MERS-CoV genome from the present study and all the other available MERS-CoV genomes. Among these 11 unique amino acid differences, four were found in ORF1ab, three were found in the S1 domain of the spike protein, and one each was found in the proteins encoded by ORF4b, ORF5, envelope gene, and ORF8. MERS-CoV detection for all other 254 dromedaries in this closed dairy herd was negative by nucleocapsid protein-capture ELISA and RNA detection. MERS-CoV IgG sero-positivity gradually increased in dromedary calves with increasing age, with positivity rates of 75% at zero to three months, 79% at four months, 89% at five to six months, and 90% at seven to twelve months. The development of a rapid antigen detection kit for instantaneous diagnosis is warranted.Emerging Microbes & Infections (2015) 4, e74; doi:10.1038/emi.2015.74; published online 2 December 2015.

A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes.

Science.gov (United States)

Mesel-Lemoine, Mariana; Millet, Jean; Vidalain, Pierre-Olivier; Law, Helen; Vabret, Astrid; Lorin, Valérie; Escriou, Nicolas; Albert, Matthew L; Nal, Béatrice; Tangy, Frédéric

2012-07-01

Human coronaviruses are associated with upper respiratory tract infections that occasionally spread to the lungs and other organs. Although airway epithelial cells represent an important target for infection, the respiratory epithelium is also composed of an elaborate network of dendritic cells (DCs) that are essential sentinels of the immune system, sensing pathogens and presenting foreign antigens to T lymphocytes. In this report, we show that in vitro infection by human coronavirus 229E (HCoV-229E) induces massive cytopathic effects in DCs, including the formation of large syncytia and cell death within only few hours. In contrast, monocytes are much more resistant to infection and cytopathic effects despite similar expression levels of CD13, the membrane receptor for HCoV-229E. While the differentiation of monocytes into DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 requires 5 days, only 24 h are sufficient for these cytokines to sensitize monocytes to cell death and cytopathic effects when infected by HCoV-229E. Cell death induced by HCoV-229E is independent of TRAIL, FasL, tumor necrosis factor alpha, and caspase activity, indicating that viral replication is directly responsible for the observed cytopathic effects. The consequence of DC death at the early stage of HCoV-229E infection may have an impact on the early control of viral dissemination and on the establishment of long-lasting immune memory, since people can be reinfected multiple times by HCoV-229E.

Virological and serological analysis of a recent Middle East respiratory syndrome coronavirus infection case on a triple combination antiviral regimen.

Science.gov (United States)

Spanakis, Nikolaos; Tsiodras, Sotirios; Haagmans, Bart L; Raj, V Stalin; Pontikis, Kostantinos; Koutsoukou, Antonia; Koulouris, Nikolaos G; Osterhaus, Albert D M E; Koopmans, Marion P G; Tsakris, Athanassios

2014-12-01

Serological, molecular and phylogenetic analyses of a recently imported case of Middle East respiratory syndrome coronavirus (MERS-CoV) in Greece are reported. Although MERS-CoV remained detectable in the respiratory tract secretions of the patient until the fourth week of illness, viraemia was last detected 2 days after initiation of triple combination therapy with pegylated interferon, ribavirin and lopinavir/ritonavir, administered from Day 13 of illness. Phylogenetic analysis of the virus showed close similarity with other human MERS-CoVs from the recent Jeddah outbreak in Saudi Arabia. Immunoglobulin G (IgG) titres peaked 3 weeks after the onset of illness, whilst IgM levels remained constantly elevated during the follow-up period (second to fifth week of illness). Serological testing confirmed by virus neutralisation assay detected an additional case that was a close contact of the patient. Copyright © 2014. Published by Elsevier B.V.

Evidence for an Ancestral Association of Human Coronavirus 229E with Bats.

Science.gov (United States)

Corman, Victor Max; Baldwin, Heather J; Tateno, Adriana Fumie; Zerbinati, Rodrigo Melim; Annan, Augustina; Owusu, Michael; Nkrumah, Evans Ewald; Maganga, Gael Darren; Oppong, Samuel; Adu-Sarkodie, Yaw; Vallo, Peter; da Silva Filho, Luiz Vicente Ribeiro Ferreira; Leroy, Eric M; Thiel, Volker; van der Hoek, Lia; Poon, Leo L M; Tschapka, Marco; Drosten, Christian; Drexler, Jan Felix

2015-12-01

We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related viruses by reverse transcription-PCR (RT-PCR). Only hipposiderid bats tested positive. To compare the genetic diversity of bat viruses and HCoV-229E, we tested historical isolates and diagnostic specimens sampled globally over 10 years. Bat viruses were 5- and 6-fold more diversified than HCoV-229E in the RNA-dependent RNA polymerase (RdRp) and spike genes. In phylogenetic analyses, HCoV-229E strains were monophyletic and not intermixed with animal viruses. Bat viruses formed three large clades in close and more distant sister relationships. A recently described 229E-related alpaca virus occupied an intermediate phylogenetic position between bat and human viruses. According to taxonomic criteria, human, alpaca, and bat viruses form a single CoV species showing evidence for multiple recombination events. HCoV-229E and the alpaca virus showed a major deletion in the spike S1 region compared to all bat viruses. Analyses of four full genomes from 229E-related bat CoVs revealed an eighth open reading frame (ORF8) located at the genomic 3' end. ORF8 also existed in the 229E-related alpaca virus. Reanalysis of HCoV-229E sequences showed a conserved transcription regulatory sequence preceding remnants of this ORF, suggesting its loss after acquisition of a 229E-related CoV by humans. These data suggested an evolutionary origin of 229E-related CoVs in hipposiderid bats, hypothetically with camelids as intermediate hosts preceding the establishment of HCoV-229E. The ancestral origins of major human coronaviruses (HCoVs) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV-229E in hipposiderid bats by analyzing a

Detection of ascitic feline coronavirus RNA from cats with clinically suspected feline infectious peritonitis.

Science.gov (United States)

Soma, Takehisa; Wada, Makoto; Taharaguchi, Satoshi; Tajima, Tomoko

2013-10-01

Ascitic feline coronavirus (FCoV) RNA was examined in 854 cats with suspected feline infectious peritonitis (FIP) by RT-PCR. The positivity was significantly higher in purebreds (62.2%) than in crossbreds (34.8%) (P<0.0001). Among purebreds, the positivities in the Norwegian forest cat (92.3%) and Scottish fold (77.6%) were significantly higher than the average of purebreds (P=0.0274 and 0.0251, respectively). The positivity was significantly higher in males (51.5%) than in females (35.7%) (P<0.0001), whereas no gender difference has generally been noted in FCoV antibody prevalence, indicating that FIP more frequently develops in males among FCoV-infected cats. Genotyping was performed for 377 gene-positive specimens. Type I (83.3%) was far more predominantly detected than type II (10.6%) (P<0.0001), similar to previous serological and genetic surveys.

Genetic diversity of bats coronaviruses in the Atlantic Forest hotspot biome, Brazil.

Science.gov (United States)

Góes, Luiz Gustavo Bentim; Campos, Angélica Cristine de Almeida; Carvalho, Cristiano de; Ambar, Guilherme; Queiroz, Luzia Helena; Cruz-Neto, Ariovaldo Pereira; Munir, Muhammad; Durigon, Edison Luiz

2016-10-01

Bats are notorious reservoirs of genetically-diverse and high-profile pathogens, and are playing crucial roles in the emergence and re-emergence of viruses, both in human and in animals. In this report, we identified and characterized previously unknown and diverse genetic clusters of bat coronaviruses in the Atlantic Forest Biome, Brazil. These results highlight the virus richness of bats and their possible roles in the public health. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Bovine coronavirus in naturally andexperimentally exposed calves; viralshedding and the potential for transmission

OpenAIRE

Oma, Veslemøy Sunniva; Tråven, Madeleine; Alenius, S.; Myrmel, Mette; Stokstad, Maria

2016-01-01

Background Bovine coronavirus (BCoV) is a widely distributed pathogen, causing disease and economic losses in the cattle industry worldwide. Prevention of virus spread is impeded by a lack of basic knowledge concerning viral shedding and transmission potential in individual animals. The aims of the study were to investigate the duration and quantity of BCoV shedding in feces and nasal secretions related to clinical signs, the presence of virus in blood and tissues and to test the hypothesis t...

Human coronavirus OC43 causes influenza-like illness in residents and staff of aged-care facilities in Melbourne, Australia.

Science.gov (United States)

Birch, C. J.; Clothier, H. J.; Seccull, A.; Tran, T.; Catton, M. C.; Lambert, S. B.; Druce, J. D.

2005-01-01

Three outbreaks of respiratory illness associated with human coronavirus HCoV-OC43 infection occurred in geographically unrelated aged-care facilities in Melbourne, Australia during August and September 2002. On clinical and epidemiological grounds the outbreaks were first thought to be caused by influenza virus. HCoV-OC43 was detected by RT-PCR in 16 out of 27 (59%) specimens and was the only virus detected at the time of sampling. Common clinical manifestations were cough (74%), rhinorrhoea (59%) and sore throat (53%). Attack rates and symptoms were similar in residents and staff across the facilities. HCoV-OC43 was also detected in surveillance and diagnostic respiratory samples in the same months. These outbreaks establish this virus as a cause of morbidity in aged-care facilities and add to increasing evidence of the significance of coronavirus infections. PMID:15816152

The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment

DEFF Research Database (Denmark)

Hansen, Gert Helge; Delmas, B; Besnardeau, L

1998-01-01

Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive...

Myeloablation-associated deletion of ORF4 in a human coronavirus 229E infection.

Science.gov (United States)

Greninger, Alexander L; Pepper, Gregory; Shean, Ryan C; Cent, Anne; Palileo, Isabel; Kuypers, Jane M; Schiffer, Joshua T; Jerome, Keith R

2017-01-01

We describe metagenomic next-generation sequencing (mNGS) of a human coronavirus 229E from a patient with AML and persistent upper respiratory symptoms, who underwent hematopoietic cell transplantation (HCT). mNGS revealed a 548-nucleotide deletion, which comprised the near entirety of the ORF4 gene, and no minor allele variants were detected to suggest a mixed infection. As part of her pre-HCT conditioning regimen, the patient received myeloablative treatment with cyclophosphamide and 12 Gy total body irradiation. Iterative sequencing and RT-PCR confirmation of four respiratory samples over the 4-week peritransplant period revealed that the pre-conditioning strain contained an intact ORF4 gene, while the deletion strain appeared just after conditioning and persisted over a 2.5-week period. This sequence represents one of the largest genomic deletions detected in a human RNA virus and describes large-scale viral mutation associated with myeloablation for HCT.

Expression, crystallization and preliminary crystallographic study of mouse hepatitis virus (MHV) nucleocapsid protein C-terminal domain

International Nuclear Information System (INIS)

Tong, Xiaohang; Ma, Yanlin; Li, Xuemei

2010-01-01

The C-terminal domain of mouse hepatitis virus nucleocapsid protein has been overexpressed in E. coli, purified and crystallized. The crystal belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å, and diffracted to 2.20 Å resolution. Mouse hepatitis virus (MHV) belongs to the group II coronaviruses. The virus produces nine genes encoding 11 proteins that could be recognized as structural proteins and nonstructural proteins and are crucial for viral RNA synthesis. The nucleocapsid (N) protein, one of the structural proteins, interacts with the 30.4 kb virus genomic RNA to form the helical nucleocapsid and associates with the membrane glycoprotein via its C-terminus to stabilize virion assembly. Here, the expression and crystallization of the MHV nucleocapsid protein C-terminal domain are reported. The crystals diffracted to 2.20 Å resolution and belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content is 43.0% (V M = 2.16 Å 3 Da −1 )

Origin and Possible Genetic Recombination of the Middle East Respiratory Syndrome Coronavirus from the First Imported Case in China: Phylogenetics and Coalescence Analysis.

Science.gov (United States)

Wang, Yanqun; Liu, Di; Shi, Weifeng; Lu, Roujian; Wang, Wenling; Zhao, Yanjie; Deng, Yao; Zhou, Weimin; Ren, Hongguang; Wu, Jun; Wang, Yu; Wu, Guizhen; Gao, George F; Tan, Wenjie

2015-09-08

The Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe acute respiratory tract infection with a high fatality rate in humans. Coronaviruses are capable of infecting multiple species and can evolve rapidly through recombination events. Here, we report the complete genomic sequence analysis of a MERS-CoV strain imported to China from South Korea. The imported virus, provisionally named ChinaGD01, belongs to group 3 in clade B in the whole-genome phylogenetic tree and also has a similar tree topology structure in the open reading frame 1a and -b (ORF1ab) gene segment but clusters with group 5 of clade B in the tree constructed using the S gene. Genetic recombination analysis and lineage-specific single-nucleotide polymorphism (SNP) comparison suggest that the imported virus is a recombinant comprising group 3 and group 5 elements. The time-resolved phylogenetic estimation indicates that the recombination event likely occurred in the second half of 2014. Genetic recombination events between group 3 and group 5 of clade B may have implications for the transmissibility of the virus. The recent outbreak of MERS-CoV in South Korea has attracted global media attention due to the speed of spread and onward transmission. Here, we present the complete genome of the first imported MERS-CoV case in China and demonstrate genetic recombination events between group 3 and group 5 of clade B that may have implications for the transmissibility of MERS-CoV. Copyright © 2015 Wang et al.

Mutation of Asn28 Disrupts the Dimerization and Enzymatic Activity of SARS 3CL

Energy Technology Data Exchange (ETDEWEB)

Barrila, J.; Gabelli, S; Bacha, U; Amzel, M; Freire, E

2010-01-01

Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL{sup pro}, is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL{sup pro} and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 {angstrom} from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K{sub d}. The crystallographic structure of the N28A mutant determined at 2.35 {angstrom} resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL{sup pro}.

Differential expression of the MERS-coronavirus receptor in the upper respiratory tract of humans and dromedary camels

NARCIS (Netherlands)

Widagdo, W; Raj, V Stalin; Schipper, Debby; Kolijn, Kimberley; van Leenders, Geert J L H; Bosch, Berend J; Bensaid, Albert; Segalés, Joaquim; Baumgärtner, Wolfgang; Osterhaus, Albert D M E; Koopmans, Marion P; van den Brand, Judith M A; Haagmans, Bart L

Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor - dipeptidyl peptidase 4 (DPP4) - is expressed in the upper respiratory tract epithelium of camels but not

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...

3dRPC: a web server for 3D RNA-protein structure prediction.

Science.gov (United States)

Huang, Yangyu; Li, Haotian; Xiao, Yi

2018-04-01

RNA-protein interactions occur in many biological processes. To understand the mechanism of these interactions one needs to know three-dimensional (3D) structures of RNA-protein complexes. 3dRPC is an algorithm for prediction of 3D RNA-protein complex structures and consists of a docking algorithm RPDOCK and a scoring function 3dRPC-Score. RPDOCK is used to sample possible complex conformations of an RNA and a protein by calculating the geometric and electrostatic complementarities and stacking interactions at the RNA-protein interface according to the features of atom packing of the interface. 3dRPC-Score is a knowledge-based potential that uses the conformations of nucleotide-amino-acid pairs as statistical variables and that is used to choose the near-native complex-conformations obtained from the docking method above. Recently, we built a web server for 3dRPC. The users can easily use 3dRPC without installing it locally. RNA and protein structures in PDB (Protein Data Bank) format are the only needed input files. It can also incorporate the information of interface residues or residue-pairs obtained from experiments or theoretical predictions to improve the prediction. The address of 3dRPC web server is http://biophy.hust.edu.cn/3dRPC. yxiao@hust.edu.cn.

Plaque assay for human coronavirus NL63 using human colon carcinoma cells

Directory of Open Access Journals (Sweden)

Drosten Christian

2008-11-01

Full Text Available Abstract Background Coronaviruses cause a broad range of diseases in animals and humans. Human coronavirus (hCoV NL63 is associated with up to 10% of common colds. Viral plaque assays enable the characterization of virus infectivity and allow for purifying virus stock solutions. They are essential for drug screening. Hitherto used cell cultures for hCoV-NL63 show low levels of virus replication and weak and diffuse cytopathogenic effects. It has not yet been possible to establish practicable plaque assays for this important human pathogen. Results 12 different cell cultures were tested for susceptibility to hCoV-NL63 infection. Human colon carcinoma cells (CaCo-2 replicated virus more than 100 fold more efficiently than commonly used African green monkey kidney cells (LLC-MK2. CaCo-2 cells showed cytopathogenic effects 4 days post infection. Avicel, agarose and carboxymethyl-cellulose overlays proved suitable for plaque assays. Best results were achieved with Avicel, which produced large and clear plaques from the 4th day of infection. The utility of plaque assays with agrose overlay was demonstrated for purifying virus, thereby increasing viral infectivity by 1 log 10 PFU/mL. Conclusion CaCo-2 cells support hCoV-NL63 better than LLC-MK2 cells and enable cytopathogenic plaque assays. Avicel overlay is favourable for plaque quantification, and agarose overlay is preferred for plaque purification. HCoV-NL63 virus stock of increased infectivity will be beneficial in antiviral screening, animal modelling of disease, and other experimental tasks.

Evaluation of bovine coronavirus antibody levels, virus shedding, and respiratory disease incidence throughout the beef cattle production cycle

Science.gov (United States)

Objective- Determine how levels of serum antibody to bovine coronavirus (BCV) are related to virus shedding patterns and respiratory disease incidence in beef calves at various production stages. Animals- 890 crossbred beef calves from four separately managed herds at the U.S. Meat Animal Research C...

Oncofetal protein IMP3, a new cancer biomarker.

Science.gov (United States)

Gong, Yuna; Woda, Bruce A; Jiang, Zhong

2014-05-01

IMP3 is a member of a family of RNA-binding proteins that consists of IMP1, IMP2 and IMP3. These proteins contain 2 RNA recognition motifs and 4 K-homology domains that allow them to bind RNAs strongly and specifically. IMP3 is an oncofetal protein involved in embryogenesis and its expression is associated with a number of malignant neoplasms. IMP3 is associated with aggressive and advanced cancers and is specifically expressed in malignant tumors but is not found in adjacent benign tissues. Moreover, in vitro studies have shown that IMP3 promotes tumor cell proliferation, adhesion, and invasion. This review focuses on the studies of IMP3 expression in different cancers and emphasizes the potential utility of IMP3 in routine surgical pathology practice. We also discuss IMP3 as a prognostic biomarker for cancer patients' outcomes.

14-3-3 proteins in plant physiology.

Science.gov (United States)

Denison, Fiona C; Paul, Anna-Lisa; Zupanska, Agata K; Ferl, Robert J

2011-09-01

Plant 14-3-3 isoforms, like their highly conserved homologues in mammals, function by binding to phosphorylated client proteins to modulate their function. Through the regulation of a diverse range of proteins including kinases, transcription factors, structural proteins, ion channels and pathogen defense-related proteins, they are being implicated in an expanding catalogue of physiological functions in plants. 14-3-3s themselves are affected, both transcriptionally and functionally, by the extracellular and intracellular environment of the plant. They can modulate signaling pathways that transduce inputs from the environment and also the downstream proteins that elicit the physiological response. This review covers some of the key emerging roles for plant 14-3-3s including their role in the response to the plant extracellular environment, particularly environmental stress, pathogens and light conditions. We also address potential key roles in primary metabolism, hormone signaling, growth and cell division. Copyright © 2011 Elsevier Ltd. All rights reserved.

Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway.

Directory of Open Access Journals (Sweden)

Jianhua Sui

2008-11-01

Full Text Available Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S protein of animal and human SARS coronaviruses (SARS-CoVs during and between two zoonotic transfers (2002/03 and 2003/04 are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID "hot spot" in a light chain CDR (complementarity determining region alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural

Dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection.

Directory of Open Access Journals (Sweden)

Tomoki Yoshikawa

2010-01-01

Full Text Available Human lung epithelial cells are likely among the first targets to encounter invading severe acute respiratory syndrome-associated coronavirus (SARS-CoV. Not only can these cells support the growth of SARS-CoV infection, but they are also capable of secreting inflammatory cytokines to initiate and, eventually, aggravate host innate inflammatory responses, causing detrimental immune-mediated pathology within the lungs. Thus, a comprehensive evaluation of the complex epithelial signaling to SARS-CoV is crucial for paving the way to better understand SARS pathogenesis. Based on microarray-based functional genomics, we report here the global gene response of 2B4 cells, a cloned bronchial epithelial cell line derived from Calu-3 cells. Specifically, we found a temporal and spatial activation of nuclear factor (NFkappaB, activator protein (AP-1, and interferon regulatory factor (IRF-3/7 in infected 2B4 cells at 12-, 24-, and 48-hrs post infection (p.i., resulting in the activation of many antiviral genes, including interferon (IFN-beta, -lambdas, inflammatory mediators, and many IFN-stimulated genes (ISGs. We also showed, for the first time, that IFN-beta and IFN-lambdas were capable of exerting previously unrecognized, non-redundant, and complementary abilities to limit SARS-CoV replication, even though their expression could not be detected in infected 2B4 bronchial epithelial cells until 48 hrs p.i. Collectively, our results highlight the mechanics of the sequential events of antiviral signaling pathway/s triggered by SARS-CoV in bronchial epithelial cells and identify novel cellular targets for future studies, aiming at advancing strategies against SARS.

Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis

NARCIS (Netherlands)

Felten, Sandra; Leutenegger, Christian M.; Balzer, Hans Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman|info:eu-repo/dai/nl/089740890; Hartmann, Katrin

2017-01-01

Background: Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse

The sample of choice for detecting Middle East respiratory syndrome coronavirus in asymptomatic dromedary camels using real-time reversetranscription polymerase chain reaction.

NARCIS (Netherlands)

Mohran, K A; Farag, E A B; Reusken, C B E; Raj, V S; Lamers, M M; Pas, S D; Voermans, J; Smits, S L; Alhajri, M M; Alhajri, F; Al-Romaihi, H E; Ghobashy, H; El-Maghraby, M M; Al Dhahiry, S H S; Al-Mawlawi, N; El-Sayed, A M; Al-Thani, M; Al-Marri, S A; Haagmans, B L; Koopmans, M P G

2016-01-01

The newly identified Middle East respiratory syndrome coronavirus (MERS-CoV), which causes severe respiratory disease, particularly in people with comorbidities, requires further investigation. Studies in Qatar and elsewhere have provided evidence that dromedary camels are a reservoir for the virus,

3DProIN: Protein-Protein Interaction Networks and Structure Visualization.

Science.gov (United States)

Li, Hui; Liu, Chunmei

2014-06-14

3DProIN is a computational tool to visualize protein-protein interaction networks in both two dimensional (2D) and three dimensional (3D) view. It models protein-protein interactions in a graph and explores the biologically relevant features of the tertiary structures of each protein in the network. Properties such as color, shape and name of each node (protein) of the network can be edited in either 2D or 3D views. 3DProIN is implemented using 3D Java and C programming languages. The internet crawl technique is also used to parse dynamically grasped protein interactions from protein data bank (PDB). It is a java applet component that is embedded in the web page and it can be used on different platforms including Linux, Mac and Window using web browsers such as Firefox, Internet Explorer, Chrome and Safari. It also was converted into a mac app and submitted to the App store as a free app. Mac users can also download the app from our website. 3DProIN is available for academic research at http://bicompute.appspot.com.

Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6

International Nuclear Information System (INIS)

Fang, Puxian; Fang, Liurong; Liu, Xiaorong; Hong, Yingying; Wang, Yongle; Dong, Nan; Ma, Panpan; Bi, Jing; Wang, Dang; Xiao, Shaobo

2016-01-01

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus. Accessory proteins are genus-specific for coronavirus, and two putative accessory proteins, NS6 and NS7, are predicted to be encoded by PDCoV; however, this remains to be confirmed experimentally. Here, we identified the leader-body junction sites of NS6 subgenomic RNA (sgRNA) and found that the actual transcription regulatory sequence (TRS) utilized by NS6 is non-canonical and is located upstream of the predicted TRS. Using the purified NS6 from an Escherichia coli expression system, we obtained two anti-NS6 monoclonal antibodies that could detect the predicted NS6 in cells infected with PDCoV or transfected with NS6-expressing plasmids. Further studies revealed that NS6 is always localized in the cytoplasm of PDCoV-infected cells, mainly co-localizing with the endoplasmic reticulum (ER) and ER-Golgi intermediate compartments, as well as partially with the Golgi apparatus. Together, our results identify the NS6 sgRNA and demonstrate its expression in PDCoV-infected cells. -- Highlights: •The leader-body fusion site of NS6 sgRNA is identified. •NS6 sgRNA uses a non-canonical transcription regulatory sequence (TRS). •NS6 can be expressed in PDCoV-infected cell. •NS6 predominantly localize to the ER complex and ER-Golgi intermediate compartment.

Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6

Energy Technology Data Exchange (ETDEWEB)

Fang, Puxian; Fang, Liurong; Liu, Xiaorong; Hong, Yingying; Wang, Yongle; Dong, Nan; Ma, Panpan [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Bi, Jing [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); Department of Immunology and Aetology, College of Basic Medicine, Hubei University of Chinese Medicine, Wuhan 430065 (China); Wang, Dang [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Xiao, Shaobo, E-mail: vet@mail.hzau.edu.cn [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China)

2016-12-15

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus. Accessory proteins are genus-specific for coronavirus, and two putative accessory proteins, NS6 and NS7, are predicted to be encoded by PDCoV; however, this remains to be confirmed experimentally. Here, we identified the leader-body junction sites of NS6 subgenomic RNA (sgRNA) and found that the actual transcription regulatory sequence (TRS) utilized by NS6 is non-canonical and is located upstream of the predicted TRS. Using the purified NS6 from an Escherichia coli expression system, we obtained two anti-NS6 monoclonal antibodies that could detect the predicted NS6 in cells infected with PDCoV or transfected with NS6-expressing plasmids. Further studies revealed that NS6 is always localized in the cytoplasm of PDCoV-infected cells, mainly co-localizing with the endoplasmic reticulum (ER) and ER-Golgi intermediate compartments, as well as partially with the Golgi apparatus. Together, our results identify the NS6 sgRNA and demonstrate its expression in PDCoV-infected cells. -- Highlights: •The leader-body fusion site of NS6 sgRNA is identified. •NS6 sgRNA uses a non-canonical transcription regulatory sequence (TRS). •NS6 can be expressed in PDCoV-infected cell. •NS6 predominantly localize to the ER complex and ER-Golgi intermediate compartment.

Vaccines for emerging infectious diseases: Lessons from MERS coronavirus and Zika virus.

Science.gov (United States)

Maslow, Joel N

2017-12-02

The past decade and a half has been characterized by numerous emerging infectious diseases. With each new threat, there has been a call for rapid vaccine development. Pathogens such as the Middle East Respiratory Syndrome coronavirus (MERS-CoV) and the Zika virus represent either new viral entities or viruses emergent in new geographic locales and characterized by novel complications. Both serve as paradigms for the global spread that can accompany new pathogens. In this paper, we review the epidemiology and pathogenesis of MERS-CoV and Zika virus with respect to vaccine development. The challenges in vaccine development and the approach to clinical trial design to test vaccine candidates for disease entities with a changing epidemiology are discussed.

Severe respiratory illness associated with a novel coronavirus--Saudi Arabia and Qatar, 2012.

Science.gov (United States)

2012-10-12

CDC is working closely with the World Health Organization (WHO) and other partners to better understand the public health risk presented by a recently detected, novel coronavirus. This virus has been identified in two patients, both previously healthy adults who suffered severe respiratory illness. The first patient, a man aged 60 years from Saudi Arabia, was hospitalized in June 2012 and died; the second patient, a man aged 49 years from Qatar with onset of symptoms in September 2012 was transported to the United Kingdom for intensive care. He remains hospitalized on life support with both pulmonary and renal failure. Person-to-person or health-care-associated transmission has not been identified to date. Interim case definitions based on acute respiratory illness and travel history were issued by WHO on September 29 and include criteria for "patient under investigation," "probable case," and "confirmed case". This information is current as of October 4. Updates on the investigation and the WHO case definition are available at http://www.who.int/csr/don/en/index.html.

14-3-3 Proteins in Guard Cell Signaling.

Science.gov (United States)

Cotelle, Valérie; Leonhardt, Nathalie

2015-01-01

Guard cells are specialized cells located at the leaf surface delimiting pores which control gas exchanges between the plant and the atmosphere. To optimize the CO2 uptake necessary for photosynthesis while minimizing water loss, guard cells integrate environmental signals to adjust stomatal aperture. The size of the stomatal pore is regulated by movements of the guard cells driven by variations in their volume and turgor. As guard cells perceive and transduce a wide array of environmental cues, they provide an ideal system to elucidate early events of plant signaling. Reversible protein phosphorylation events are known to play a crucial role in the regulation of stomatal movements. However, in some cases, phosphorylation alone is not sufficient to achieve complete protein regulation, but is necessary to mediate the binding of interactors that modulate protein function. Among the phosphopeptide-binding proteins, the 14-3-3 proteins are the best characterized in plants. The 14-3-3s are found as multiple isoforms in eukaryotes and have been shown to be involved in the regulation of stomatal movements. In this review, we describe the current knowledge about 14-3-3 roles in the regulation of their binding partners in guard cells: receptors, ion pumps, channels, protein kinases, and some of their substrates. Regulation of these targets by 14-3-3 proteins is discussed and related to their function in guard cells during stomatal movements in response to abiotic or biotic stresses.

Dendritic Cell Targeted Chitosan Nanoparticles for Nasal DNA Immunization against SARS CoV Nucleocapsid Protein

OpenAIRE

Raghuwanshi, Dharmendra; Mishra, Vivek; Das, Dipankar; Kaur, Kamaljit; Suresh, Mavanur R.

2012-01-01

This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for non-invasive receptor mediated gene delivery to na...

Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein, Visinin-Like Protein 3.

Science.gov (United States)

Li, Congmin; Lim, Sunghyuk; Braunewell, Karl H; Ames, James B

2016-01-01

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.

The SGS3 protein involved in PTGS finds a family

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Bateman Alex

2002-08-01

Full Text Available Abstract Background Post transcriptional gene silencing (PTGS is a recently discovered phenomenon that is an area of intense research interest. Components of the PTGS machinery are being discovered by genetic and bioinformatics approaches, but the picture is not yet complete. Results The gene for the PTGS impaired Arabidopsis mutant sgs3 was recently cloned and was not found to have similarity to any other known protein. By a detailed analysis of the sequence of SGS3 we have defined three new protein domains: the XH domain, the XS domain and the zf-XS domain, that are shared with a large family of uncharacterised plant proteins. This work implicates these plant proteins in PTGS. Conclusion The enigmatic SGS3 protein has been found to contain two predicted domains in common with a family of plant proteins. The other members of this family have been predicted to be transcription factors, however this function seems unlikely based on this analysis. A bioinformatics approach has implicated a new family of plant proteins related to SGS3 as potential candidates for PTGS related functions.

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

Science.gov (United States)

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

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 SARS 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. PMID:16038039

Detection of feline Coronavirus in effusions of cats with and without feline infectious peritonitis using loop-mediated isothermal amplification.

Science.gov (United States)

Günther, Sonja; Felten, Sandra; Wess, Gerhard; Hartmann, Katrin; Weber, Karin

2018-06-01

Feline infectious peritonitis (FIP) is a fatal disease in cats worldwide. The aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect feline Coronavirus (FCoV) in body cavity effusions of cats with and without FIP, in order to minimize the time from sampling to obtaining results. RNA was extracted from body cavity effusion samples of 71 cats, including 34 samples from cats with a definitive diagnosis of FIP, and 37 samples of control cats with similar clinical signs but other confirmed diseases. Two reaction mixtures (Isothermal Mastermix, OptiGene Ltd.and PCRun™ Molecular Detection Mix, Biogal) were tested using the same primers, which were designed to bind to a conserved region of the FCoV membrane protein gene. Both assays were conducted under isothermal conditions (61 °C-62 °C). Using the Isothermal Mastermix of OptiGene Ltd., amplification times ranged from 4 and 39 min with a sensitivity of 35.3% and a specificity of 94.6% for the reported sample group. Using the PCRun™ Molecular Detection Mix of Biogal, amplification times ranged from 18 to 77 min with a sensitivity of 58.8% and a specificity of 97.3%. Although the RT-LAMP assay is less sensitive than real time reverse transcription PCR (RT-PCR), it can be performed without the need of expensive equipment and with less hands-on time. Further modifications of primers might lead to a suitable in-house test and accelerate the diagnosis of FIP. Copyright © 2018 Elsevier B.V. All rights reserved.

Protein: FBA3 [TP Atlas

Lifescience Database Archive (English)

Full Text Available FBA3 Ubiquitination CBLB RNF56 CBLB E3 ubiquitin-protein ligase CBL-B Casitas B-lineage lymphoma pr...oto-oncogene b, RING finger protein 56, SH3-binding protein CBL-B, Signal transduction prote

Vaccines for emerging infectious diseases: Lessons from MERS coronavirus and Zika virus

Science.gov (United States)

Maslow, Joel N.

2017-01-01

ABSTRACT The past decade and a half has been characterized by numerous emerging infectious diseases. With each new threat, there has been a call for rapid vaccine development. Pathogens such as the Middle East Respiratory Syndrome coronavirus (MERS-CoV) and the Zika virus represent either new viral entities or viruses emergent in new geographic locales and characterized by novel complications. Both serve as paradigms for the global spread that can accompany new pathogens. In this paper, we review the epidemiology and pathogenesis of MERS-CoV and Zika virus with respect to vaccine development. The challenges in vaccine development and the approach to clinical trial design to test vaccine candidates for disease entities with a changing epidemiology are discussed. PMID:28846484

HOMOLOGY BETWEEN SEGMENTS OF HUMAN HEMOSTATIC PROTEINS AND PROTEINS OF VIRUSES WHICH CAUSE ACUTE RESPIRATORY INFECTIONS OR DISEASES WITH SIMILAR SYMPTOMS

Directory of Open Access Journals (Sweden)

I. N. Zhilinskaya

2017-01-01

Full Text Available Objectives: To identify homologous segments of human hemostatic and viral proteins and to assess the role of human hemostatic proteins in viral replication. Materials and Methods: The following viruses were chosen for comparison: influenza B (B/Astrakhan/2/2017, coronaviruses (Hcov229E and SARS-Co, type 1 adenovirus (adenoid 71, measles (ICHINOSE-BA and rubella (Therien. The primary structures of viral proteins and 41 human hemostatic proteins were obtained from open–access www.ncbi.nlm.nih. gov and www.nextprot.org databases, respectively. Sequence homology was determined by comparing 12-amino-acid segments. Those sequences identical in ≥ 8 positions were considered homologous. Results: The analysis shows that viral proteins contain segments which mimic a number of human hemostatic proteins. Most of these segments, except those of adenovirus proteins, are homologous with coagulation factors. The increase in viral virulence, as in case of SARS-Co, correlates with an increased number of segments homologous with hemostatic proteins. Conclusion: Hemostasis plays an important role in viral replication and pathogenesis. The conclusion is consistent with the literature data about the relationship of hemostasis and inflammatory response to viral infections.

Identification of NCAM that interacts with the PHE-CoV spike protein.

Science.gov (United States)

Gao, Wei; He, Wenqi; Zhao, Kui; Lu, Huijun; Ren, Wenzhi; Du, Chongtao; Chen, Keyan; Lan, Yungang; Song, Deguang; Gao, Feng

2010-09-24

The spike proteins of coronaviruses associate with cellular molecules to mediate infection of their target cells. The characterization of cellular proteins required for virus infection is essential for understanding viral life cycles and may provide cellular targets for antiviral therapies. We identified Neural Cell Adhesion Molecule (NCAM) as a novel interacting partner of the PHE-CoV S protein. A T7 phage display cDNA library from N2a cells was constructed, and the library was screened with the soluble PHE-CoV S glycoproteins. We used a coimmunoprecipitation assay to show that only the NCAM was a binding partner of spike protein. We found that a soluble form of anti-NCAM antibody blocked association of the PHE-CoV with N2a cells. Furthermore, double-stranded siRNA targeted against NCAM inhibited PHE-CoV infection. A novel interaction was identified between NCAM and spike protein and this association is critical during PHE-CoV infection.

Identification of NCAM that interacts with the PHE-CoV spike protein

Directory of Open Access Journals (Sweden)

Chen Keyan

2010-09-01

Full Text Available Abstract Background The spike proteins of coronaviruses associate with cellular molecules to mediate infection of their target cells. The characterization of cellular proteins required for virus infection is essential for understanding viral life cycles and may provide cellular targets for antiviral therapies. Results We identified Neural Cell Adhesion Molecule (NCAM as a novel interacting partner of the PHE-CoV S protein. A T7 phage display cDNA library from N2a cells was constructed, and the library was screened with the soluble PHE-CoV S glycoproteins. We used a coimmunoprecipitation assay to show that only the NCAM was a binding partner of spike protein. We found that a soluble form of anti-NCAM antibody blocked association of the PHE-CoV with N2a cells. Furthermore, double-stranded siRNA targeted against NCAM inhibited PHE-CoV infection. Conclusions A novel interaction was identified between NCAM and spike protein and this association is critical during PHE-CoV infection.

Molecular characterization of human coronaviruses and their circulation dynamics in Kenya, 2009-2012.

Science.gov (United States)

Sipulwa, Lenata A; Ongus, Juliette R; Coldren, Rodney L; Bulimo, Wallace D

2016-02-01

Human Coronaviruses (HCoV) are a common cause of respiratory illnesses and are responsible for considerable morbidity and hospitalization across all age groups especially in individuals with compromised immunity. There are six known species of HCoV: HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, MERS-CoV and SARS-HCoV. Although studies have shown evidence of global distribution of HCoVs, there is limited information on their presence and distribution in Kenya. HCoV strains that circulated in Kenya were retrospectively diagnosed and molecularly characterized. A total of 417 nasopharyngeal specimens obtained between January 2009 and December 2012 from around Kenya were analyzed by a real time RT-PCR using HCoV-specific primers. HCoV-positive specimens were subsequently inoculated onto monolayers of LL-CMK2 cells. The isolated viruses were characterized by RT-PCR amplification and sequencing of the partial polymerase (pol) gene. The prevalence of HCoV infection was as follows: out of the 417 specimens, 35 (8.4 %) were positive for HCoV, comprising 10 (2.4 %) HCoV-NL63, 12 (2.9 %) HCoV-OC43, 9 (2.1 %) HCoV-HKU1, and 4 (1 %) HCoV-229E. The Kenyan HCoV strains displayed high sequence homology to the prototypes and contemporaneous strains. Evolution analysis showed that the Kenyan HCoV-OC43 and HCoV-NL63 isolates were under purifying selection. Phylogenetic evolutionary analyses confirmed the identities of three HCoV-HKU1, five HCoV-NL63, eight HCoV-OC43 and three HCoV-229E. There were yearly variations in the prevalence and circulation patterns of individual HCoVs in Kenya. This paper reports on the first molecular characterization of human Coronaviruses in Kenya, which play an important role in causing acute respiratory infections among children.

Factors Influencing Emergency Nurses' Burnout During an Outbreak of Middle East Respiratory Syndrome Coronavirus in Korea

OpenAIRE

Kim, Ji Soo; Choi, Jeong Sil

2016-01-01

Purpose: Emergency department (ED) nurses suffer from persistent stress after experiencing the traumatic event of exposure to Middle East respiratory syndrome coronavirus (MERS-CoV), which can subsequently lead to burnout. This study aimed to assess ED nurses' burnout level during an outbreak of MERS-CoV and to identify influencing factors in order to provide basic information for lowering and preventing the level of burnout. Methods: Study participants were ED nurses working in eight hosp...

Ribonucleocapsid Formation of SARS-COV Through Molecular Action of the N-Terminal Domain of N Protein

Energy Technology Data Exchange (ETDEWEB)

Saikatendu, K.S.; Joseph, J.S.; Subramanian, V.; Neuman, B.W.; Buchmeier, M.J.; Stevens, R.C.; Kuhn, P.; /Scripps Res. Inst.

2007-07-12

Conserved amongst all coronaviruses are four structural proteins, the matrix (M), small envelope (E) and spike (S) that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in their lumen. The N terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C-terminus of N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17A (monoclinic) and 1.85 A (cubic) respectively, solved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core and is oriented similar to that in the IBV N-NTD and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggest a common mode of RNA recognition, but probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs hints that they employ different modes of both RNA recognition as well as oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV.

Directory of Open Access Journals (Sweden)

Sanchita Bhadra

Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV, an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU (5 to 50 PFU/ml of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

Prevalence of comorbidities in the Middle East respiratory syndrome coronavirus (MERS-CoV: a systematic review and meta-analysis

Directory of Open Access Journals (Sweden)

Alaa Badawi

2016-08-01

Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV is associated with life-threatening severe illnesses and a mortality rate of approximately 35%, particularly in patients with underlying comorbidities. A systematic analysis of 637 MERS-CoV cases suggests that diabetes and hypertension are equally prevalent in approximately 50% of the patients. Cardiac diseases are present in 30% and obesity in 16% of the cases. These conditions down-regulate the synthesis of proinflammatory cytokines and impair the host's innate and humoral immune systems. In conclusion, protection against MERS-CoV and other respiratory infections can be improved if public health vaccination strategies are tailored to target persons with chronic disorders.

Isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism

NARCIS (Netherlands)

Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker; van der Hoek, Lia

2013-01-01

The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in

BAG3: a multifaceted protein that regulates major cell pathways

Science.gov (United States)

Rosati, A; Graziano, V; De Laurenzi, V; Pascale, M; Turco, M C

2011-01-01

Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression. PMID:21472004

Engineering [Ln(DPA){sub 3}]{sup 3-} binding sites in proteins: a widely applicable method for tagging proteins with lanthanide ions

Energy Technology Data Exchange (ETDEWEB)

Jia Xinying; Yagi, Hiromasa; Su Xuncheng; Stanton-Cook, Mitchell; Huber, Thomas; Otting, Gottfried, E-mail: gottfried.otting@anu.edu.au [Australian National University, Research School of Chemistry (Australia)

2011-08-15

Paramagnetic relaxation enhancements from unpaired electrons observed in nuclear magnetic resonance (NMR) spectra present powerful long-range distance restraints. The most frequently used paramagnetic tags, however, are tethered to the protein via disulfide bonds, requiring proteins with single cysteine residues for covalent attachment. Here we present a straightforward strategy to tag proteins site-specifically with paramagnetic lanthanides without a tether and independent of cysteine residues. It relies on preferential binding of the complex between three dipicolinic acid molecules (DPA) and a lanthanide ion (Ln{sup 3+}), [Ln(DPA){sub 3}]{sup 3-}, to a pair of positively charged amino acids whose charges are not compensated by negatively charged residues nearby. This situation rarely occurs in wild-type proteins, allowing the creation of specific binding sites simply by introduction of positively charged residues that are positioned far from glutamate or aspartate residues. The concept is demonstrated with the hnRNPLL RRM1 domain. In addition, we show that histidine- and arginine-tags present binding sites for [Ln(DPA){sub 3}]{sup 3-}.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Energy Technology Data Exchange (ETDEWEB)

Simarro, Maria [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Gimenez-Cassina, Alfredo [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Kedersha, Nancy [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A. [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Rhee, Kirsten [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Tisdale, Sarah; Danial, Nika [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Benarafa, Charaf [Theodor Kocher Institute, University of Bern, 3012 Bern (Switzerland); Orduna, Anonio [Unidad de Investigacion, Hospital Clinico Universitario de Valladolid, 47005 Valladolid (Spain); Anderson, Paul, E-mail: panderson@rics.bwh.harvard.edu [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States)

2010-10-22

Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

International Nuclear Information System (INIS)

Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

2010-01-01

Research highlights: → Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. → The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. → Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Genetic characterization of Betacoronavirus lineage C viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus HKU5 in Japanese pipistrelle: implications for the origin of the novel Middle East respiratory syndrome coronavirus.

Science.gov (United States)

Lau, Susanna K P; Li, Kenneth S M; Tsang, Alan K L; Lam, Carol S F; Ahmed, Shakeel; Chen, Honglin; Chan, Kwok-Hung; Woo, Patrick C Y; Yuen, Kwok-Yung

2013-08-01

While the novel Middle East respiratory syndrome coronavirus (MERS-CoV) is closely related to Tylonycteris bat CoV HKU4 (Ty-BatCoV HKU4) and Pipistrellus bat CoV HKU5 (Pi-BatCoV HKU5) in bats from Hong Kong, and other potential lineage C betacoronaviruses in bats from Africa, Europe, and America, its animal origin remains obscure. To better understand the role of bats in its origin, we examined the molecular epidemiology and evolution of lineage C betacoronaviruses among bats. Ty-BatCoV HKU4 and Pi-BatCoV HKU5 were detected in 29% and 25% of alimentary samples from lesser bamboo bat (Tylonycteris pachypus) and Japanese pipistrelle (Pipistrellus abramus), respectively. Sequencing of their RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes revealed that MERS-CoV is more closely related to Pi-BatCoV HKU5 in RdRp (92.1% to 92.3% amino acid [aa] identity) but is more closely related to Ty-BatCoV HKU4 in S (66.8% to 67.4% aa identity) and N (71.9% to 72.3% aa identity). Although both viruses were under purifying selection, the S of Pi-BatCoV HKU5 displayed marked sequence polymorphisms and more positively selected sites than that of Ty-BatCoV HKU4, suggesting that Pi-BatCoV HKU5 may generate variants to occupy new ecological niches along with its host in diverse habitats. Molecular clock analysis showed that they diverged from a common ancestor with MERS-CoV at least several centuries ago. Although MERS-CoV may have diverged from potential lineage C betacoronaviruses in European bats more recently, these bat viruses were unlikely to be the direct ancestor of MERS-CoV. Intensive surveillance for lineage C betaCoVs in Pipistrellus and related bats with diverse habitats and other animals in the Middle East may fill the evolutionary gap.

Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

International Nuclear Information System (INIS)

Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

2005-01-01

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

Dynamics of SARS-coronavirus HR2 domain in the prefusion and transition states

Science.gov (United States)

McReynolds, Susanna; Jiang, Shaokai; Rong, Lijun; Caffrey, Michael

2009-12-01

The envelope glycoproteins S1 and S2 of severe acute respiratory syndrome coronavirus (SARS-CoV) mediate viral entry by conformational change from a prefusion state to a postfusion state that enables fusion of the viral and target membranes. In this work we present the characterization of the dynamic properties of the SARS-CoV S2-HR2 domain (residues 1141-1193 of S) in the prefusion and newly discovered transition states by NMR 15N relaxation studies. The dynamic properties of the different states, which are stabilized under different experimental conditions, extend the current model of viral membrane fusion and give insight into the design of structure-based antagonists of SARS-CoV in particular, as well as other enveloped viruses such as HIV.

Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins

Czech Academy of Sciences Publication Activity Database

Palani, Kirubakaran; Pfeiferová, Lucie; Boušová, Kristýna; Bednárová, Lucie; Obšilová, V.; Vondrášek, Jiří

2016-01-01

Roč. 84, č. 10 (2016), s. 1358-1374 ISSN 0887-3585 Institutional support: RVO:61388963 Keywords : protein design * fusion proteins * PDZ3 * SH3 * Trp-cage * two domain proteins Subject RIV: CE - Biochemistry Impact factor: 2.289, year: 2016

Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

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Langella P.

1999-01-01

Full Text Available Lactic acid bacteria (LAB are Gram-positive bacteria and are generally regarded as safe (GRAS organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV epitope-protein fusion (BCV-Nuc. BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

3D complex: a structural classification of protein complexes.

Directory of Open Access Journals (Sweden)

Emmanuel D Levy

2006-11-01

Full Text Available Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.

Crystal Structure of a Monomeric Form of Severe Acute Respiratory Syndrome Coronavirus Endonuclease Nsp15 Suggests a Role for Hexamerization As An Allosteric Switch

Energy Technology Data Exchange (ETDEWEB)

Joseph, J.S.; Saikatendu, K.S.; Subramanian, V.; Neuman, B.W.; Buchmeier, M.J.; Stevens, R.C.; Kuhn, P.; /Scripps Res. Inst.

2007-07-09

Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a novel uridylate-specific Mn{sup 2+}-dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335) determined to a resolution of 2.9 Angstroms. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by {approx}120 degrees into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.

TIM-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine.

Directory of Open Access Journals (Sweden)

Stephanie Jemielity

2013-03-01

Full Text Available Human T-cell Immunoglobulin and Mucin-domain containing proteins (TIM1, 3, and 4 specifically bind phosphatidylserine (PS. TIM1 has been proposed to serve as a cellular receptor for hepatitis A virus and Ebola virus and as an entry factor for dengue virus. Here we show that TIM1 promotes infection of retroviruses and virus-like particles (VLPs pseudotyped with a range of viral entry proteins, in particular those from the filovirus, flavivirus, New World arenavirus and alphavirus families. TIM1 also robustly enhanced the infection of replication-competent viruses from the same families, including dengue, Tacaribe, Sindbis and Ross River viruses. All interactions between TIM1 and pseudoviruses or VLPs were PS-mediated, as demonstrated with liposome blocking and TIM1 mutagenesis experiments. In addition, other PS-binding proteins, such as Axl and TIM4, promoted infection similarly to TIM1. Finally, the blocking of PS receptors on macrophages inhibited the entry of Ebola VLPs, suggesting that PS receptors can contribute to infection in physiologically relevant cells. Notably, infection mediated by the entry proteins of Lassa fever virus, influenza A virus and SARS coronavirus was largely unaffected by TIM1 expression. Taken together our data show that TIM1 and related PS-binding proteins promote infection of diverse families of enveloped viruses, and may therefore be useful targets for broad-spectrum antiviral therapies.

Protein-protein docking using region-based 3D Zernike descriptors.

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Venkatraman, Vishwesh; Yang, Yifeng D; Sael, Lee; Kihara, Daisuke

2009-12-09

Protein-protein interactions are a pivotal component of many biological processes and mediate a variety of functions. Knowing the tertiary structure of a protein complex is therefore essential for understanding the interaction mechanism. However, experimental techniques to solve the structure of the complex are often found to be difficult. To this end, computational protein-protein docking approaches can provide a useful alternative to address this issue. Prediction of docking conformations relies on methods that effectively capture shape features of the participating proteins while giving due consideration to conformational changes that may occur. We present a novel protein docking algorithm based on the use of 3D Zernike descriptors as regional features of molecular shape. The key motivation of using these descriptors is their invariance to transformation, in addition to a compact representation of local surface shape characteristics. Docking decoys are generated using geometric hashing, which are then ranked by a scoring function that incorporates a buried surface area and a novel geometric complementarity term based on normals associated with the 3D Zernike shape description. Our docking algorithm was tested on both bound and unbound cases in the ZDOCK benchmark 2.0 dataset. In 74% of the bound docking predictions, our method was able to find a near-native solution (interface C-alphaRMSD 3D Zernike descriptors are adept in capturing shape complementarity at the protein-protein interface and useful for protein docking prediction. Rigorous benchmark studies show that our docking approach has a superior performance compared to existing methods.

Candidates in Astroviruses, Seadornaviruses, Cytorhabdoviruses and Coronaviruses for +1 frame overlapping genes accessed by leaky scanning

Directory of Open Access Journals (Sweden)

Atkins John F

2010-01-01

Full Text Available Abstract Background Overlapping genes are common in RNA viruses where they serve as a mechanism to optimize the coding potential of compact genomes. However, annotation of overlapping genes can be difficult using conventional gene-finding software. Recently we have been using a number of complementary approaches to systematically identify previously undetected overlapping genes in RNA virus genomes. In this article we gather together a number of promising candidate new overlapping genes that may be of interest to the community. Results Overlapping gene predictions are presented for the astroviruses, seadornaviruses, cytorhabdoviruses and coronaviruses (families Astroviridae, Reoviridae, Rhabdoviridae and Coronaviridae, respectively.

The gut microbiome and mucosal defenses in cats with coronaviruses: a pilot study

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Sara Meazzi

2017-05-01

Full Text Available Feline Infectious Peritonitis (FIP develops from a mutation of enteric feline coronaviruses (FCoVs and an imbalance of the host immune response. The wide polymorphism of FCoVs is associated with the viral replication rate (Licitra et al. 2013.  Changes in the composition of the gut microbiota may induce quali-quantitative modifications in FCoVs and/or different immune profiles (Weese et al., 2015. Few information is available on feline gut microbiome and the association between microbiota and the predisposition to pathological conditions (Ramadan et al., 2014. The aim of this study is to provide preliminary data about the composition of gut microbiota in healthy cats compared with FCoV infected cats (with and without  FIP, in order to evaluate whether changes of gut microbiota may induce changes in FCoV, in its genetic polymorphism and in the mucosal immunity. Screening analyses have been performed on 22 cats: - Routine hematology and biochemistry on EDTA and serum (included electrophoresis and alpha-1-acid glycoprotein measurement for cats suspected with FIP - Nested RT-PCR-3’UTR on frozen faeces - Effusion evaluation - FIV/FeLV serology Due to strict inclusion criteria (cats younger than 2.5 years old, indoor and not assuming antibiotics in the previous two months and based on the results obtained from the complete set of analysis, only 15 cats, specifically 5 cats for each of the following 3 groups: FIP- affected, healthy negative and positive for FCoV, have been recruited to perform the following analyses:  - microbiota analysis through NGS of 16S rRNA gene (V4 region amplicons followed by bioinformatic analysis  -  evaluation of secretory IgA (ELISA kit - phylogenetic analysis of FCoVs S gene sequences Innovative results will be provided on the feline gut microbiota composition. These will be correlated with the presence and genetic polymorphisms of FCoV and mucosal defenses to establish significant correlations between the analysed

Long-term persistence of robust antibody and cytotoxic T cell responses in recovered patients infected with SARS coronavirus.

Directory of Open Access Journals (Sweden)

Taisheng Li

2006-12-01

Full Text Available Most of the individuals infected with SARS coronavirus (SARS-CoV spontaneously recovered without clinical intervention. However, the immunological correlates associated with patients' recovery are currently unknown. In this report, we have sequentially monitored 30 recovered patients over a two-year period to characterize temporal changes in SARS-CoV-specific antibody responses as well as cytotoxic T cell (CTL responses. We have found persistence of robust antibody and CTL responses in all of the study subjects throughout the study period, with a moderate decline one year after the onset of symptoms. We have also identified two potential major CTL epitopes in N proteins based on ELISPOT analysis of pooled peptides. However, despite the potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients have not yet been restored to normal levels. In summary, our study has, for the first time, characterized the temporal and dynamic changes of humoral and CTL responses in the natural history of SARS-recovered individuals, and strongly supports the notion that high and sustainable levels of immune responses correlate strongly with the disease outcome. Our findings have direct implications for future design and development of effective therapeutic agents and vaccines against SARS-CoV infection.

Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins

Czech Academy of Sciences Publication Activity Database

Palani, K.; Pfeiferová, L.; Boušová, Kristýna; Bednárová, L.; Obšilová, Veronika; Vondrášek, J.

2016-01-01

Roč. 84, č. 10 (2016), s. 1358-1374 ISSN 0887-3585 Institutional support: RVO:67985823 Keywords : protein design * fusion proteins * PDZ3 * SH3 * Trp-cage * two domain proteins * molecular dynamics simulation * circular dichroism Subject RIV: BO - Biophysics Impact factor: 2.289, year: 2016

Prevalence of comorbidities in the Middle East respiratory syndrome coronavirus (MERS-CoV): a systematic review and meta-analysis.

Science.gov (United States)

Badawi, Alaa; Ryoo, Seung Gwan

2016-08-01

The Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with life-threatening severe illnesses and a mortality rate of approximately 35%, particularly in patients with underlying comorbidities. A systematic analysis of 637 MERS-CoV cases suggests that diabetes and hypertension are equally prevalent in approximately 50% of the patients. Cardiac diseases are present in 30% and obesity in 16% of the cases. These conditions down-regulate the synthesis of proinflammatory cytokines and impair the host's innate and humoral immune systems. In conclusion, protection against MERS-CoV and other respiratory infections can be improved if public health vaccination strategies are tailored to target persons with chronic disorders. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

SH2/SH3 signaling proteins.

Science.gov (United States)

Schlessinger, J

1994-02-01

SH2 and SH3 domains are small protein modules that mediate protein-protein interactions in signal transduction pathways that are activated by protein tyrosine kinases. SH2 domains bind to short phosphotyrosine-containing sequences in growth factor receptors and other phosphoproteins. SH3 domains bind to target proteins through sequences containing proline and hydrophobic amino acids. SH2 and SH3 domain containing proteins, such as Grb2 and phospholipase C gamma, utilize these modules in order to link receptor and cytoplasmic protein tyrosine kinases to the Ras signaling pathway and to phosphatidylinositol hydrolysis, respectively. The three-dimensional structures of several SH2 and SH3 domains have been determined by NMR and X-ray crystallography, and the molecular basis of their specificity is beginning to be unveiled.

Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic.

Science.gov (United States)

Schmitz, Matthias; Ebert, Elisabeth; Stoeck, Katharina; Karch, André; Collins, Steven; Calero, Miguel; Sklaviadis, Theodor; Laplanche, Jean-Louis; Golanska, Ewa; Baldeiras, Ines; Satoh, Katsuya; Sanchez-Valle, Raquel; Ladogana, Anna; Skinningsrud, Anders; Hammarin, Anna-Lena; Mitrova, Eva; Llorens, Franc; Kim, Yong Sun; Green, Alison; Zerr, Inga

2016-05-01

At present, the testing of 14-3-3 protein in cerebrospinal fluid (CSF) is a standard biomarker test in suspected sporadic Creutzfeldt-Jakob disease (sCJD) diagnosis. Increasing 14-3-3 test referrals in CJD reference laboratories in the last years have led to an urgent need to improve established 14-3-3 test methods. The main result of our study was the validation of a commercially available 14-3-3 ELISA next to the commonly used Western blot method as a high-throughput screening test. Hereby, 14-3-3 protein expression was quantitatively analyzed in CSF of 231 sCJD and 2035 control patients. We obtained excellent sensitivity/specificity values of 88 and 96% that are comparable to the established Western blot method. Since standard protocols and preanalytical sample handling have become more important in routine diagnostic, we investigated in a further step the reproducibility and stability of 14-3-3 as a biomarker for human prion diseases. Ring trial data from 2009 to 2013 revealed an increase of Fleiss' kappa from 0.51 to 0.68 indicating an improving reliability of 14-3-3 protein detection. The stability of 14-3-3 protein under short-term and long-term storage conditions at various temperatures and after repeated freezing/thawing cycles was confirmed. Contamination of CSF samples with blood appears likely to be an important factor at a concentration of more than 2500 erythrocytes/μL. Hemolysis of erythrocytes with significant release of 14-3-3 protein started after 2 days at room temperature. We first define clear standards for the sample handling, short- and long-term storage of CSF samples as well as the handling of blood- contaminated samples which may result in artificially elevated CSF levels of 14-3-3.

Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

DEFF Research Database (Denmark)

Ngo, HT; Pham, Long; Kim, JW

2013-01-01

Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

Middle East respiratory syndrome coronavirus disease is rare in children: An update from Saudi Arabia.

Science.gov (United States)

Al-Tawfiq, Jaffar A; Kattan, Rana F; Memish, Ziad A

2016-11-08

To summarize the reported Middle East respiratory syndrome-coronavirus (MERS-CoV) cases, the associated clinical presentations and the outcomes. We searched the Saudi Ministry of Health website, the World Health Organization website, and the Flutracker website. We also searched MEDLINE and PubMed for the keywords: Middle East respiratory syndrome-coronavirus, MERS-CoV in combination with pediatric, children, childhood, infancy and pregnancy from the initial discovery of the virus in 2012 to 2016. The retrieved articles were also read to further find other articles. Relevant data were placed into an excel sheet and analyzed accordingly. Descriptive analytic statistics were used in the final analysis as deemed necessary. From June 2012 to April 19, 2016, there were a total of 31 pediatric MERS-CoV cases. Of these cases 13 (42%) were asymptomatic and the male to female ratio was 1.7:1. The mean age of patients was 9.8 ± 5.4 years. Twenty-five (80.6%) of the cases were reported from the Kingdom of Saudi Arabia. The most common source of infection was household contact (10 of 15 with reported source) and 5 patients acquired infection within a health care facility. Using real time reverse transcriptase polymerase chain reaction of pediatric patients revealed that 9 out of 552 (1.6%) was positive in the Kingdom of Saudi Arabia. Utilizing serology for MERS-CoV infection in Jordan and Saudi Arabia did not reveal any positive patients. Thus, the number of the pediatric MERS-CoV is low; the exact reason for the low prevalence of the disease in children is not known.

Isolation and molecular characterization of type I and type II feline coronavirus in Malaysia.

Science.gov (United States)

Amer, Alazawy; Siti Suri, Arshad; Abdul Rahman, Omar; Mohd, Hair Bejo; Faruku, Bande; Saeed, Sharif; Tengku Azmi, Tengku Ibrahim

2012-11-21

Feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first time, describes the isolation and biotypes determination of type I and type II FCoV from naturally infected cats in Malaysia. Of the total number of cats sampled, 95% (40/42) were RT-PCR positive for FCoV. Inoculation of clinical samples into Crandell feline kidney cells (CrFK), and Feline catus whole fetus-4 cells (Fcwf-4), show cytopathic effect (CPE) characterized by syncytial cells formation and later cell detachment. Differentiation of FCoV biotypes using RT-PCR assay revealed that, 97.5% and 2.5% of local isolates were type I and type II FCoV, respectively. These isolates had high sequence homology and phylogenetic similarity with several FCoV isolates from Europe, South East Asia and USA. This study reported the successful isolation of local type I and type II FCoV evident with formation of cytopathic effects in two types of cell cultures namely the CrFK and Fcwf-4 , where the later cells being more permissive. However, the RT-PCR assay is more sensitive in detecting the antigen in suspected samples as compared to virus isolation in cell culture. The present study indicated that type I FCoV is more prevalent among cats in Malaysia.

Knowledge and attitude towards the Middle East respiratory syndrome coronavirus among healthcare personnel in the southern region of Saudi Arabia.

Science.gov (United States)

Abbag, Huda F; El-Mekki, Awad Ahmed; Al Bshabshe, Ali Aobaid Ali; Mahfouz, Ahmed A; Al-Dosry, Ahasen A; Mirdad, Rasha T; AlKhttabi, Nora F; Abbag, Lubna F

2018-03-07

Middle East respiratory syndrome coronavirus (MERS-CoV) belongs to the family Coronaviridae, and is named for the crown-like spikes on its surface. The clinical presentation of MERS-CoV infection ranges from asymptomatic to very severe disease, and the classical presentation includes fever, cough chills, sore throat, myalgia, and arthralgia. A cross-sectional study of 339 healthcare personnel was conducted over an 8-month period in the Aseer region of Saudi Arabia using a structured survey that included demographic information and questions testing participant's knowledge. Approximately two-thirds of the respondents properly identified the causative agent of MERS-CoV as an RNA virus (66.4%, n=225) that is enveloped (68.1%, n=231). On the other hand, few respondents identified the proper number of strains or the genus (16.5% and 17.4%, respectively). More than half of the study sample identified the disease as zoonotic (57.2%, n=194). Similarly, 89.1% (n=302) identified that camels and bats are prone to infection with coronaviruses. Only 23.9% (n=81) properly identified March through May as the season with the highest transmission rate. There was a massive lack of adequate knowledge regarding prevalence of antibodies. Only 18.3% (n=62) of respondents identified PCR as the proper diagnostic confirmatory test for MERS-CoV infection. Regarding MERS-CoV clinical features, 76.4% (n=259) recognized the presence of sub-clinical infection, 64.7% (n=218) indicated that cases should be immediately isolated, and 46.9% (n=159) identified the main cause of mortality as respiratory failure. There is limited microbiological and virological knowledge of MERS-CoV infection among healthcare personnel in the southern region of Saudi Arabia, although the clinical aspects are known. Copyright © 2018. Published by Elsevier Ltd.

MSX-3D: a tool to validate 3D protein models using mass spectrometry.

Science.gov (United States)

Heymann, Michaël; Paramelle, David; Subra, Gilles; Forest, Eric; Martinez, Jean; Geourjon, Christophe; Deléage, Gilbert

2008-12-01

The technique of chemical cross-linking followed by mass spectrometry has proven to bring valuable information about the protein structure and interactions between proteic subunits. It is an effective and efficient way to experimentally investigate some aspects of a protein structure when NMR and X-ray crystallography data are lacking. We introduce MSX-3D, a tool specifically geared to validate protein models using mass spectrometry. In addition to classical peptides identifications, it allows an interactive 3D visualization of the distance constraints derived from a cross-linking experiment. Freely available at http://proteomics-pbil.ibcp.fr

Human coronavirus and severe acute respiratory infection in Southern Brazil.

Science.gov (United States)

Trombetta, Hygor; Faggion, Heloisa Z; Leotte, Jaqueline; Nogueira, Meri B; Vidal, Luine R R; Raboni, Sonia M

2016-05-01

Human coronaviruses (HCoVs) are an important cause of respiratory tract infection and are responsible for causing the common cold in the general population. Thus, adequate surveillance of HCoV is essential. This study aimed to analyze the impact of HCoV infections and their relation to severe acute respiratory infection (SARI) in a hospitalized population in Southern Brazil. A cross-sectional study was conducted at a tertiary care hospital, and assessed inpatients under investigation for SARI by the hospital epidemiology department, and all patients who had nasopharyngeal aspirates collected from January 2012 to December 2013 to detect respiratory viruses (RVs). Viral infection was detected by multiplex reverse transcriptase polymerase chain reaction (RT-PCR), with primers specific to the subtypes HCoV-229E/NL63 and OC43/HKU1. The overall positivity rate was 58.8% (444/755), and HCoVs were detected in 7.6% (n = 34) of positive samples. Children below two years of age were most frequently affected (62%). Comorbidities were more likely to be associated with HCoVs than with other RVs. Immunosuppression was an independent risk factor for HCoV infection (OR = 3.5, 95% CI 1.6-7.6). Dyspnea was less frequently associated with HCoV infection (p infected with HCoV (9%) died from respiratory infection. HCoVs are important respiratory pathogens, especially in hospitalized children under 2 years of age and in immunosuppressed patients. They may account for a small proportion of SARI diagnoses, increased need for mechanical ventilation, intensive care unit admission, and death.

Protein: FEA3 [TP Atlas

Lifescience Database Archive (English)

Full Text Available FEA3 AREB pathway: Signaling proteins At4g11890/T26M18_100 At4g11890, Protein kinase family pr...otein, Putative uncharacterized protein At4g11890/T26M18_100 3702 Arabidopsis thaliana 826796 Q8GY82 22225700 ...

Rapid detection of bovine coronavirus by a semi-nested RT-PCR Detecção rápida do Coronavírus Bovino (BCoV por meio de uma semi-nested RT-PCR

Directory of Open Access Journals (Sweden)

Karen M. Asano

2009-11-01

Full Text Available Bovine coronavirus (BCoV is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256 in DEPC treated ultra-pure water, in fetal bovine serum (FBS and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694. The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.O Coronavírus bovino (BCoV pertence ao grupo 2 do gênero Coronavirus (Nidovirales: Coronaviridae e é agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doença

The use of enzyme-linked immunosorbent assay systems for the serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in The Netherlands.

NARCIS (Netherlands)

G.F. Rimmelzwaan (Guus); J. Groen (Jan); H.F. Egberink (Herman); G.H.A. Borst (Gerrit); F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

1991-01-01

textabstractComplex trapping blocking (CTB) enzyme-linked immunosorbent assays (ELISAs) and indirect ELISAs for the detection of antibodies to canine parvovirus (CPV), canine coronavirus (CCV) and rotavirus in sera of dogs were established. Double antibody sandwich ELISAs for the detection of CPV-,

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...

Identification of a noncanonically transcribed subgenomic mRNA of infectious bronchitis virus and other gammacoronaviruses.

Science.gov (United States)

Bentley, Kirsten; Keep, Sarah May; Armesto, Maria; Britton, Paul

2013-02-01

Coronavirus subgenomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving transcription regulatory sequences (TRSs) located in the 5' leader sequence (TRS-L) and upstream of each structural and group-specific gene (TRS-B). Several gammacoronaviruses including infectious bronchitis virus (IBV) contain a putative open reading frame (ORF), localized between the M gene and gene 5, which is controversial due to the perceived absence of a TRS. We have studied the transcription of a novel sgmRNA associated with this potential ORF and found it to be transcribed via a previously unidentified noncanonical TRS-B. Using an IBV reverse genetics system, we demonstrated that the template-switching event during intergenic region (IR) sgmRNA synthesis occurs at the 5' end of the noncanonical TRS-B and recombines between nucleotides 5 and 6 of the 8-nucleotide consensus TRS-L. Introduction of a complete TRS-B showed that higher transcription levels are achieved by increasing the number of nucleotide matches between TRS-L and TRS-B. Translation of a protein from the sgmRNA was demonstrated using enhanced green fluorescent protein, suggesting the translation of a fifth, novel, group-specific protein for IBV. This study has resolved an issue concerning the number of ORFs expressed by members of the Gammacoronavirus genus and proposes the existence of a fifth IBV accessory protein. We confirmed previous reports that coronaviruses can produce sgmRNAs from noncanonical TRS-Bs, which may expand their repertoire of proteins. We also demonstrated that noncanonical TRS-Bs may provide a mechanism by which coronaviruses can control protein expression levels by reducing sgmRNA synthesis.

Spike protein assembly into the coronavirion: exploring the limits of its sequence requirements

International Nuclear Information System (INIS)

Bosch, Berend Jan; Haan, Cornelis A.M. de; Smits, Saskia L.; Rottier, Peter J.M.

2005-01-01

The coronavirus spike (S) protein, required for receptor binding and membrane fusion, is incorporated into the assembling virion by interactions with the viral membrane (M) protein. Earlier we showed that the ectodomain of the S protein is not involved in this process. Here we further defined the requirements of the S protein for virion incorporation. We show that the cytoplasmic domain, not the transmembrane domain, determines the association with the M protein and suffices to effect the incorporation into viral particles of chimeric spikes as well as of foreign viral glycoproteins. The essential sequence was mapped to the membrane-proximal region of the cytoplasmic domain, which is also known to be of critical importance for the fusion function of the S protein. Consistently, only short C-terminal truncations of the S protein were tolerated when introduced into the virus by targeted recombination. The important role of the about 38-residues cytoplasmic domain in the assembly of and membrane fusion by this approximately 1300 amino acids long protein is discussed

A Multigene Approach for Comparing Genealogy of Betacoronavirus from Cattle and Horses

Directory of Open Access Journals (Sweden)

Iracema N. Barros

2013-01-01

Full Text Available Gastroenteritis is one of the leading causes of morbidity and mortality among young and newborn animals and is often caused by multiple intestinal infections, with rotavirus and bovine coronavirus (BCoV being the main viral causes in cattle. Given that BCoV is better studied than equine coronaviruses and given the possibility of interspecies transmission of these viruses, this research was designed to compare the partial sequences of the spike glycoprotein (S, hemagglutinin-esterase protein (HE, and nucleoprotein (N genes from coronaviruses from adult cattle with winter dysentery, calves with neonatal diarrhea, and horses. To achieve this, eleven fecal samples from dairy cows with winter dysentery, three from calves, and two from horses, all from Brazil, were analysed. It could be concluded that the enteric BCoV genealogy from newborn and adult cattle is directly associated with geographic distribution patterns, when S and HE genes are taken into account. A less-resolved genealogy exists for the HE and N genes in cattle, with a trend for an age-related segregation pattern. The coronavirus strains from horses revealed Betacoronavirus sequences indistinguishable from those found in cattle, a fact previously unknown.

An alignment-free method to find similarity among protein sequences via the general form of Chou's pseudo amino acid composition.

Science.gov (United States)

Gupta, M K; Niyogi, R; Misra, M

2013-01-01

In this paper, we propose a method to create the 60-dimensional feature vector for protein sequences via the general form of pseudo amino acid composition. The construction of the feature vector is based on the contents of amino acids, total distance of each amino acid from the first amino acid in the protein sequence and the distribution of 20 amino acids. The obtained cosine distance metric (also called the similarity matrix) is used to construct the phylogenetic tree by the neighbour joining method. In order to show the applicability of our approach, we tested it on three proteins: 1) ND5 protein sequences from nine species, 2) ND6 protein sequences from eight species, and 3) 50 coronavirus spike proteins. The results are in agreement with known history and the output from the multiple sequence alignment program ClustalW, which is widely used. We have also compared our phylogenetic results with six other recently proposed alignment-free methods. These comparisons show that our proposed method gives a more consistent biological relationship than the others. In addition, the time complexity is linear and space required is less as compared with other alignment-free methods that use graphical representation. It should be noted that the multiple sequence alignment method has exponential time complexity.

Isolation and molecular characterization of type I and type II feline coronavirus in Malaysia

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Amer Alazawy

2012-11-01

Full Text Available Abstract Background Feline infectious peritonitis virus (FIPV and feline enteric coronavirus (FECV are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first time, describes the isolation and biotypes determination of type I and type II FCoV from naturally infected cats in Malaysia. Findings Of the total number of cats sampled, 95% (40/42 were RT-PCR positive for FCoV. Inoculation of clinical samples into Crandell feline kidney cells (CrFK, and Feline catus whole fetus-4 cells (Fcwf-4, show cytopathic effect (CPE characterized by syncytial cells formation and later cell detachment. Differentiation of FCoV biotypes using RT-PCR assay revealed that, 97.5% and 2.5% of local isolates were type I and type II FCoV, respectively. These isolates had high sequence homology and phylogenetic similarity with several FCoV isolates from Europe, South East Asia and USA. Conclusions This study reported the successful isolation of local type I and type II FCoV evident with formation of cytopathic effects in two types of cell cultures namely the CrFK and Fcwf-4 , where the later cells being more permissive. However, the RT-PCR assay is more sensitive in detecting the antigen in suspected samples as compared to virus isolation in cell culture. The present study indicated that type I FCoV is more prevalent among cats in Malaysia.

Protein-protein docking using region-based 3D Zernike descriptors

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Sael Lee

2009-12-01

Full Text Available Abstract Background Protein-protein interactions are a pivotal component of many biological processes and mediate a variety of functions. Knowing the tertiary structure of a protein complex is therefore essential for understanding the interaction mechanism. However, experimental techniques to solve the structure of the complex are often found to be difficult. To this end, computational protein-protein docking approaches can provide a useful alternative to address this issue. Prediction of docking conformations relies on methods that effectively capture shape features of the participating proteins while giving due consideration to conformational changes that may occur. Results We present a novel protein docking algorithm based on the use of 3D Zernike descriptors as regional features of molecular shape. The key motivation of using these descriptors is their invariance to transformation, in addition to a compact representation of local surface shape characteristics. Docking decoys are generated using geometric hashing, which are then ranked by a scoring function that incorporates a buried surface area and a novel geometric complementarity term based on normals associated with the 3D Zernike shape description. Our docking algorithm was tested on both bound and unbound cases in the ZDOCK benchmark 2.0 dataset. In 74% of the bound docking predictions, our method was able to find a near-native solution (interface C-αRMSD ≤ 2.5 Å within the top 1000 ranks. For unbound docking, among the 60 complexes for which our algorithm returned at least one hit, 60% of the cases were ranked within the top 2000. Comparison with existing shape-based docking algorithms shows that our method has a better performance than the others in unbound docking while remaining competitive for bound docking cases. Conclusion We show for the first time that the 3D Zernike descriptors are adept in capturing shape complementarity at the protein-protein interface and useful for

Molecular and phylogenetic characterization of bovine coronavirus virus isolated from dairy cattle in Central Region, Thailand.

Science.gov (United States)

Singasa, Kanokwan; Songserm, Taweesak; Lertwatcharasarakul, Preeda; Arunvipas, Pipat

2017-10-01

Bovine coronavirus (BCoV) is involved mainly in enteric infections in cattle. This study reports the first molecular detection of BCoV in a diarrhea outbreak in dairy cows in the Central Region, Thailand. BCoV was molecularly detected from bloody diarrheic cattle feces by using nested PCR. Agarose gel electrophoresis of three diarrheic fecal samples yielded from the 25 samples desired amplicons that were 488 base pairs and sequencing substantiated that have BCoV. The sequence alignment indicated that nucleotide and amino acid sequences, the three TWD isolated in Thailand, were more quite homologous to each other (amino acid at position 39 of TWD1, TWD3 was proline, but TWD2 was serine) and closely related to OK-0514-3strain (virulent respiratory strain; RBCoV).The amino acid sequencing identities among TWD1, TWD2,TWD3, and OK-0514-3 strain were 96.0 to 96.6%, those at which T3I, H65N, D87G, H127Y, andQ136R were changed. In addition, the phylogenetic tree of the hypervariable region S1subunit spike glycoprotein BCoV gene was composed of three major clades by using the 54 sequences generated and showed that the evolutionally distance, TWD1, TWD2, and TWD3 were the isolated group together and most similar to OK-0514-3 strain (98.2 to 98.5% similarity). Further study will develop ELISA assay for serologic detection of winter dysentery disease.

3DSwap: Curated knowledgebase of proteins involved in 3D domain swapping

KAUST Repository

Shameer, Khader

2011-09-29

Three-dimensional domain swapping is a unique protein structural phenomenon where two or more protein chains in a protein oligomer share a common structural segment between individual chains. This phenomenon is observed in an array of protein structures in oligomeric conformation. Protein structures in swapped conformations perform diverse functional roles and are also associated with deposition diseases in humans. We have performed in-depth literature curation and structural bioinformatics analyses to develop an integrated knowledgebase of proteins involved in 3D domain swapping. The hallmark of 3D domain swapping is the presence of distinct structural segments such as the hinge and swapped regions. We have curated the literature to delineate the boundaries of these regions. In addition, we have defined several new concepts like \\'secondary major interface\\' to represent the interface properties arising as a result of 3D domain swapping, and a new quantitative measure for the \\'extent of swapping\\' in structures. The catalog of proteins reported in 3DSwap knowledgebase has been generated using an integrated structural bioinformatics workflow of database searches, literature curation, by structure visualization and sequence-structure-function analyses. The current version of the 3DSwap knowledgebase reports 293 protein structures, the analysis of such a compendium of protein structures will further the understanding molecular factors driving 3D domain swapping. The Author(s) 2011.

14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

DEFF Research Database (Denmark)

Andersen, Sofie Dabros; Keijzers, Guido; Rampakakis, Emmanouil

2012-01-01

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins...... are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding...... and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate...

Detection of Avian coronavirus infectious bronchitis virus type QX infection in Switzerland.

Science.gov (United States)

Sigrist, Brigitte; Tobler, Kurt; Schybli, Martina; Konrad, Leonie; Stöckli, René; Cattoli, Giovanni; Lüschow, Dörte; Hafez, Hafez M; Britton, Paul; Hoop, Richard K; Vögtlin, Andrea

2012-11-01

Infectious bronchitis, a disease of chickens caused by Avian coronavirus infectious bronchitis virus (IBV), leads to severe economic losses for the poultry industry worldwide. Various attempts to control the virus based on vaccination strategies are performed. However, due to the emergence of novel genotypes, an effective control of the virus is hindered. In 1996, a novel viral genotype named IBV-QX was reported for the first time in Qingdao, Shandong province, China. The first appearance of an IBV-QX isolate in Europe was reported between 2003 and 2004 in The Netherlands. Subsequently, infections with this genotype were found in several other European countries such as France, Italy, Germany, United Kingdom, Slovenia, and Sweden. The present report describes the use of a new set of degenerate primers that amplify a 636-bp fragment within the S1 gene by reverse transcription polymerase chain reaction to detect the occurrence of IBV-QX infection in Switzerland.

Tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the Middle East respiratory coronavirus (MERS-CoV receptor-binding domain as an antigen.

Directory of Open Access Journals (Sweden)

Jiaming Lan

Full Text Available The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV pandemic. Some studies have indicated the receptor-binding domain (RBD protein of MERS-CoV spike (S is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m. with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum alone, alum and polyriboinosinic acid (poly I:C or alum and cysteine-phosphate-guanine (CpG oligodeoxynucleotides (ODN. The immune responses of mice vaccinated with RBD, incomplete Freund's adjuvant (IFA and CpG ODN by a subcutaneous (s.c. route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production. Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting

Transient oligomerization of the SARS-CoV N protein--implication for virus ribonucleoprotein packaging.

Science.gov (United States)

Chang, Chung-ke; Chen, Chia-Min Michael; Chiang, Ming-hui; Hsu, Yen-lan; Huang, Tai-huang

2013-01-01

The nucleocapsid (N) phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genome into a helical ribonucleocapsid and plays a fundamental role during viral self-assembly. The N protein consists of two structural domains interspersed between intrinsically disordered regions and dimerizes through the C-terminal structural domain (CTD). A key activity of the protein is the ability to oligomerize during capsid formation by utilizing the dimer as a building block, but the structural and mechanistic bases of this activity are not well understood. By disulfide trapping technique we measured the amount of transient oligomers of N protein mutants with strategically located cysteine residues and showed that CTD acts as a primary transient oligomerization domain in solution. The data is consistent with the helical oligomer packing model of N protein observed in crystal. A systematic study of the oligomerization behavior revealed that altering the intermolecular electrostatic repulsion through changes in solution salt concentration or phosphorylation-mimicking mutations affects oligomerization propensity. We propose a biophysical mechanism where electrostatic repulsion acts as a switch to regulate N protein oligomerization.

14-3-3 Proteins in Plant Hormone Signaling: Doing Several Things at Once

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Lorenzo Camoni

2018-03-01

Full Text Available In this review we highlight the advances achieved in the investigation of the role of 14-3-3 proteins in hormone signaling, biosynthesis, and transport. 14-3-3 proteins are a family of conserved molecules that target a number of protein clients through their ability to recognize well-defined phosphorylated motifs. As a result, they regulate several cellular processes, ranging from metabolism to transport, growth, development, and stress response. High-throughput proteomic data and two-hybrid screen demonstrate that 14-3-3 proteins physically interact with many protein clients involved in the biosynthesis or signaling pathways of the main plant hormones, while increasing functional evidence indicates that 14-3-3-target interactions play pivotal regulatory roles. These advances provide a framework of our understanding of plant hormone action, suggesting that 14-3-3 proteins act as hubs of a cellular web encompassing different signaling pathways, transducing and integrating diverse hormone signals in the regulation of physiological processes.

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity.

Science.gov (United States)

Chu, Daniel K W; Hui, Kenrie P Y; Perera, Ranawaka A P M; Miguel, Eve; Niemeyer, Daniela; Zhao, Jincun; Channappanavar, Rudragouda; Dudas, Gytis; Oladipo, Jamiu O; Traoré, Amadou; Fassi-Fihri, Ouafaa; Ali, Abraham; Demissié, Getnet F; Muth, Doreen; Chan, Michael C W; Nicholls, John M; Meyerholz, David K; Kuranga, Sulyman A; Mamo, Gezahegne; Zhou, Ziqi; So, Ray T Y; Hemida, Maged G; Webby, Richard J; Roger, Francois; Rambaut, Andrew; Poon, Leo L M; Perlman, Stanley; Drosten, Christian; Chevalier, Veronique; Peiris, Malik

2018-03-20

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal-human interface. Copyright © 2018 the Author(s). Published by PNAS.

Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation.

Directory of Open Access Journals (Sweden)

Pooja Singhmar

Full Text Available Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

A retrospective clinical and epidemiological study on feline coronavirus (FCoV) in cats in Istanbul, Turkey.

Science.gov (United States)

Tekelioglu, B K; Berriatua, E; Turan, N; Helps, C R; Kocak, M; Yilmaz, H

2015-04-01

The presence of antibodies to feline coronavirus (FCoV) and feline immunodeficiency virus (FIV), together with feline leukemia virus (FeLV) antigen was investigated in 169 ill household and stray cats attending a veterinary surgery in Istanbul in 2009-14. The estimated FCoV and FIV seroprevalence (95% confidence intervals) were 37% (30-45%) and 11% (6-16%), respectively and FeLV prevalence was 1% (0-3%). FCoV seroprevalence increased until 2 years of age, was highest in 2014 and among household cats living with other cats and with outdoor access, and was lower in FIV seropositive compared to seronegative cats. Symptoms typically associated with wet feline infectious peritonitis (FIP) including ascites, abdominal distention or pleural effusion, coupled in many cases with non-antibiotic responsive fever, were observed in 19% (32/169) of cats, and 75% (24/32) of these cats were FCoV seropositive. FCoV seropositivity was also associated with a high white blood cell count, high plasma globulin, low plasma albumin and low blood urea nitrogen. The percentage of FCoV seropositive and seronegative cats that died in spite of supportive veterinary treatment was 33% (21/63) and 12% (13/106), respectively. These results indicate that FCoV is widespread and has a severe clinical impact in cats from Istanbul. Moreover, the incidence of FCoV infections could be rising, and in the absence of effective vaccination cat owners need to be made aware of ways to minimize the spread of this virus. Copyright © 2015 Elsevier B.V. All rights reserved.

Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

International Nuclear Information System (INIS)

Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

1987-01-01

3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3

Differentially expressed proteins on postoperative 3

Directory of Open Access Journals (Sweden)

Jialili Ainuer

2011-04-01

Full Text Available 【Abstract】Objectives: Surgical repair of Achilles tendon (AT rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Early kinesitherapy leads to reduced rehabilitation period. However, an insight into the detailed mechanism of this process has not been gained. Proteomic technique can be used to separate and purify the proteins by differential expression profile which is related to the function of different proteins, but research in the area of proteomic analysis of AT 3 days after repair has not been studied so far. Methods: Forty-seven New Zealand white rabbits were randomized into 3 groups. Group A (immobilization group, n=16 received postoperative cast immobilization; Group B (early motion group, n=16 received early active motion treatments immediately following the repair of AT rupture from tenotomy. Another 15 rabbits served as control group (Group C. The AT samples were prepared 3 days following the microsurgery. The proteins were separated employing twodimensional polyacrylamide gel electrophoresis (2D-PAGE. PDQuest software version 8.0 was used to identify differentially expressed proteins, followed by peptide mass fingerprint (PMF and tandem mass spectrum analysis, using the National Center for Biotechnology Information (NCBI protein database retrieval and then for bioinformatics analysis. Results: A mean of 446.33, 436.33 and 462.67 protein spots on Achilles tendon samples of 13 rabbits in Group A, 14 rabbits in Group B and 13 rabbits in Group C were successfully detected in the 2D-PAGE. There were 40, 36 and 79 unique proteins in Groups A, B and C respectively. Some differentially expressed proteins were enzyme with the gel, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS. We successfully identified 9 and 11 different proteins in Groups A and B, such as GAPDH, phosphoglycerate kinase 1

Evaluation of a multiplex immunoassay for bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk against two indirect ELISAs using latent class analysis

DEFF Research Database (Denmark)

Toftaker, Ingrid; Toft, Nils; Stokstad, Maria

2018-01-01

Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCV) are responsible for respiratory disease and diarrhea in cattle worldwide. The Norwegian control program against these infections is based on herd-level diagnosis using a new multiplex immunoassay. The objective of this study...... was to estimate sensitivity and specificity across different cut-off values for the MVD-Enferplex BCV/BRSV multiplex, by comparing them to a commercially available ELISA, the SVANOVIR® BCV-Ab and SVANOVIR® BRSV-Ab, respectively. We analyzed bulk tank milk samples from 360 herds in a low- and 360 herds in a high...

Tunable paramagnetic relaxation enhancements by [Gd(DPA)3]3- for protein structure analysis

International Nuclear Information System (INIS)

Yagi, Hiromasa; Loscha, Karin V.; Su, Xun-Cheng; Stanton-Cook, Mitchell; Huber, Thomas; Otting, Gottfried

2010-01-01

Paramagnetic relaxation enhancements (PRE) present a powerful source of structural information in nuclear magnetic resonance (NMR) studies of proteins and protein-ligand complexes. In contrast to conventional PRE reagents that are covalently attached to the protein, the complex between gadolinium and three dipicolinic acid (DPA) molecules, [Gd(DPA) 3 ] 3- , can bind to proteins in a non-covalent yet site-specific manner. This offers straightforward access to PREs that can be scaled by using different ratios of [Gd(DPA) 3 ] 3- to protein, allowing quantitative distance measurements for nuclear spins within about 15 A of the Gd 3+ ion. Such data accurately define the metal position relative to the protein, greatly enhancing the interpretation of pseudocontact shifts induced by [Ln(DPA) 3 ] 3- complexes of paramagnetic lanthanide (Ln 3+ ) ions other than gadolinium. As an example we studied the quaternary structure of the homodimeric GCN4 leucine zipper.